ENZYME

Abstract: A method of altering the substrate specificity of phosphoinositide-dependent protein kinase 1 (PDK1) wherein the said PDK1 is exposed to a polypeptide which comprises the amino acid sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr wherein Zaa represents a negatively charged amino acid residue. The PDK1 with altered substrate specificity is capable of phosphorylating the underlined residue in a polypeptide with an amino acid sequence corresponding to the consensus sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-<u>Ser/Thr</u>-Phe/Tyr. The PDK1 with altered specificity may be useful in screening assays and for phosphorylating substrates having the above consensus sequence.

Description: ENZYME The present invention relates to enzymes, in particular to phosphoinositide-dependent protein kinase 1 (PDKl). Protein kinase B (PKB) is activated by a phosphoinositide 3 -kinase dependent pathway when cells are stimulated with insulin or growth factors. The mechanism involves the phosphorylation of Thr308 located in the kinase domain of PKB and Ser 473 which is situated in a hydrophobic motif -Phe-Xaa-Xaa-Phe-Ser-Phe- located close to its C- terminus. Thr308 is phosphorylated by PDKl in vitro, but the identity of the kinase that phosphorylates Ser473 (provisionally termed PDK2) is not known. The first steps in insulin signal transduction lead to the activation of phosphoinositide (PI) 3-kinase and the formation of PtdIns(3,4,5)P 3 at the plasma membrane [1-3] , which can then be converted to PtdIns(3,4)P 2 [4] or to PtdIns(4,5)P 2 [5,6] by specific phosphatases. The inhibition of PI 3- kinase suppresses nearly all the metabolic actions of insulin whereas expression of constitutively active forms of these enzymes mimics these responses in the absence of insulin. These findings demonstrate that PtdIns(3,4,5)P 3 (and/or PtdIns(3,4)P 2 ) are key second messengers for insulin signalling [reviewed in 1-3] . Protein kinase B (PKB) is activated very rapidly in response to insulin, its activation is prevented by inhibitors of PI 3-kinase [7], and further studies have revealed that PKB is likely to mediate many of the effects of insulin in cells including stimulation of glycogen synthesis in skeletal muscle [8] , the stimulation of glycolysis in cardiac muscle [9] and some of the effects that insulin has on gene transcription [10,11] and translation [12]. PKB contains an N-terminal pleckstrin-homology (PH) domain followed by a kinase catalytic domain and then a C-terminal tail. The catalytic domain of PKB belongs to the AGC subfamily of protein kinases and was originally identified by its similarity to that of protein kinase C (PKC) isoforms and protein kinase A (PKA) [13]. Recent work has led to a greater understanding of the mechanism by which PKB is activated. PKB (through its PH domain) interacts with PtdIns(3,4,5)P 3 the product of PI 3- kinase activation. This results in the translocation of PKB to the plasma membrane and a conformational change that allows it to be phosphorylated, but does not directly stimulate PKB activity [2, 3 and 13] . Instead, PKB is activated at the membrane by phosphorylation of two residues, namely Thr308 and Ser473. Both of these residues need to be phosphorylated for maximal activation of PKB and their phosphorylation in vivo is prevented by inhibitors of Ptdlns 3-kinase [14] . Thr 308 is located between subdomains VII and VIII of the kinase domain, a region in which many kinases activated by phosphorylation are phosphorylated. Ser473 is located C-terminal to the catalytic domain, in a region that nevertheless displays high homology between different AGC family members. Interestingly, other members of the AGC subfamily of protein kinases, including p70 S6K [15] and PKC isoforms [16] also possess residues lying in equivalent sequences to Thr308 and Ser473 of PKB and phosphorylation of these residues is necessary for activation of these kinases in vivo. The residues equivalent to Thr308 lie in a Thr-Phe-Cys- Gly-Thr-Xaa-Glu-Leu consensus motif (where the underlined Thr corresponds to Thr308 and Xaa is a variable residue). The residues surrounding Ser473 lie in a Phe-Xaa-Xaa-Phe-Ser/Thr-Phe/Tyr consensus motif. We [17, 18] and others [19,20] have identified a protein kinase termed 3-phosphoinositide dependent protein kinase-1 (PDKl) which, in the presence of PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 , phosphorylates PKB at Thr308. Subsequent work has also shown that PDKl phosphorylates the equivalent residues on PKC isoforms [21-23], p70 S6 kinase [24,25] and PKA [26] . The finding that PDKl does not phosphorylate PKB significantly at Ser473, in vitro or in co-transfection experiments, suggested that a distinct protein kinase which provisionally has been termed PDK2 may catalyse this reaction [17]. Here we present findings that indicate that, surprisingly, this may not be the case. In particular we demonstrate that PDKl can be converted to a form that phosphorylates both Ser473 and Thr308 by interaction with a small peptide, for example a peptide corresponding to the C-terminal region of protein kinase C-related protein kinase-2 (PRK2). These observations suggest that PDKl and PDK2 may be the same enzyme and that the specificity of PDKl towards Thr308 and Ser473 may be regulated through its interaction with other cellular components. A first aspect of the invention provides a method of altering the substrate specificity of phosphoinositide-dependent protein kinase 1 (PDKl) wherein the said PDKl is exposed to a polypeptide which comprises the amino acid sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr wherein Zaa represents a negatively charged amino acid residue. The negatively charged amino acid residue Zaa may be, for example, an aspartate, glutamate, phosphorylated serine (phosphoserine), phosphorylated threonine (phosphothreonine) or phosphorylated tyrosine (phospho tyrosine) residue, or a negatively charged non-naturally occuring residue. It is preferred that Zaa is an aspartate, glutamate, phosphoserine or phosphothreonine residue, still more preferably an aspartate or glutamate residue. It is preferred that the first residue in the sequence corresponding to the above consensus sequence is a phenylalanine residue. Phenylalanine is found in this position in naturally occuring polypeptides in which the consensus sequence has been identified. It may also be preferred that the residue preceding the Zaa residue is a phenylalanine residue. Phenylalanine and tyrosine are both (separately) found in this position in naturally occuring polypeptides in which the consensus sequence has been identified. It will be appreciated that the term PDKl as used herein includes a polypeptide (a PDKl polypeptide) comprising the amino acid sequence shown in Figure 10 or identified as PDKl in Alessi D.R et al (1997) Characterisation of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase B . Curr. Biol. 7: 261-269, Alessi D.R et al (1997) 3-phosphoinositide-dependent protein kinase- 1 (PDKl): structural and functional homology with the Drosophila DSTPK61 kinase. Curr. Biol. 7: 776-789, Stokoe D et al (1997) Dual role of phosphatidyinositiol-3,4,5-trisphosphate in the activation of protein kinase B. Science 277: 567-570 or Stephens L et al (1998) Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent activation of protein kinase B. Science 279: 710-714, or a variant, fragment, fusion or derivative thereof, or a fusion of a said variant or fragment or derivative, for example as described in W098/41638. It is preferred that the said PDKl polypeptide is a protein kinase. It is preferred that the said PDKl polypeptide is a protein kinase that is capable of phosphorylating a threonine residue that lies in a Thr-Phe-Cys-Gly-Thr- Xaa-Glu-Leu consensus motif (where the underlined Thr corresponds to the threonine that is phosphorylated by PDKl and Xaa is a variable residue), and preferably that is capable of phosphorylating PKB, for example PKBα, at residue Thr308. The rate at which the said PDKl polypeptide is capable of phosphorylating a threonine residue as described above may be increased in the presence of PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 . The said polypeptide may be capable of phosphorylating the equivalent residues to Thr308 of PKBα on PKC isoforms [21-23], p70 S6 kinase [24,25], SGK (sequence given in Webster, M.K. , Goya, Ge, Y. , Malyar, A.C. and Firestone, G.L. (1993) Mol. Cell. Biol. 13, 1031- 2040; equivalent residues identified in US application no 112217 filed on 14 December 1998) and PKA [26] . It may further be preferred that the substrate specificity and/or other characteristics of the said polypeptide in vitro may be substantially as reported in Alessi D.R et al (1997) Curr. Biol. 7: 261-269, Alessi D.R et al (1997) Curr. Biol. 1: 776-789, Stokoe D et al (1997) Science 277: 567-570 or Stephens L et al (1998) Science 279: 710-714. It is particularly preferred, although not essential, that the variant or fragment or derivative or fusion of the PDKl , or the fusion of the variant or fragment or derivative has at least 30% of the enzyme activity of full- length human PDKl with respect to the phosphorylation of full-length human PKBα on residue Thr308 in either the presence or absence of PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 . It is more preferred if the variant or fragment or derivative or fusion of the said protein kinase, or the fusion of the variant or fragment or derivative has at least 50 % , preferably at least 70% and more preferably at least 90% of the enzyme activity of PDKl with respect to the phosphorylation of PKBα. However, it will be appreciated that variants or fusions or derivatives or fragments which are devoid of enzymatic activity may nevertheless be useful, for example by interacting with another polypeptide. Thus, variants or fusions or derivatives or fragments which are devoid of enzymatic activity may be useful in a binding assay, which may be used, for example, in a method of the invention in which an interaction of PDKl (as defined above) with a polypeptide comprising the amino acid sequence motif Phe/Tyr-Xaa-Xaa- Phe/Tyr-Zaa-Phe/Tyr, for example Phe/Tyr-Xaa-Xaa-Phe/Tyr-Asp/Glu- Phe/Tyr or Phe/Tyr-Xaa-Xaa-Phe/Tyr-PhosphoSer/PhosphoThr-Phe/Tyr is measured. By "variants" of a polypeptide we include insertions, deletions and substitutions, either conservative or non-conservative. In particular we include variants of the polypeptide where such changes do not substantially alter the activity of the said polypeptide, for example the protein kinase activity of PDKl , as described above. By "conservative substimtions" is intended combinations such as Gly, Ala; Val, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr. It is particularly preferred if the PDKl variant has an amino acid sequence which has at least 65 % identity with the amino acid sequence of PDKl shown in Figure 10, more preferably at least 70% , 71 % , 72% , 73 % or 74% , still more preferably at least 75% , yet still more preferably at least 80% , in further preference at least 85 % , in still further preference at least 90% and most preferably at least 95 % or 97% identity with the amino acid sequence defined above. It is still further preferred if the PDKl variant has an amino acid sequence which has at least 65 % identity with the amino acid sequence of the catalytic domain of PDKl shown in Figure 10, more preferably at least 70% , 71 % , 72% . 73 % or 74% , still more preferably at least 75 % , yet still more preferably at least 80% , in further preference at least 83 or 85 % , in still further preference at least 90% and most preferably at least 95 % or 97 % identity with the amino acid sequence defined above. It will be appreciated that the catalytic domain of a protein kinase-related polypeptide may be readily identified by a person skilled in the art, for example using sequence comparisons as described below. The percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally. The alignment may alternatively be carried out using the Clustal W program (Thompson et al (1994) Nucl Acid Res 22, 4673-4680). The parameters used may be as follows: Fast pairwise alignment parameters: K-tuple(word) size; 1 , window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent. Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05. Scoring matrix: BLOSUM. It is preferred that the PDKl is a polypeptide which consists of the amino acid sequence of the protein kinase PDKl as shown in Figure 10 or naturally occurring allelic variants thereof. The PDKl may also be a polypeptide with the amino acid sequence of residues 51 to 404 of full- length human PDKl ; this may comprise the protein kinase domain of PDKl , as described in Example 1. The PDKl may also be a Myc epitope-tagged PDKl or His-tagged PDKl , as described in Example 1. It is preferred that the PDKl is a polypeptide that is capable of binding to a polypeptide comprising the amino acid sequence motif Phe/Tyr-Xaa- Xaa-Phe/Tyr-Zaa-Phe/Tyr, for example Phe/Tyr-Xaa-Xaa-Phe/Tyr- Asp/Glu-Phe/Tyr or Phe/Tyr-Xaa-Xaa-Phe/Tyr-PhosphoSer/PhosphoThr- Phe/Tyr. The capability of the said PDKl polypeptide with regard to binding a polypeptide comprising the amino acid sequence motif Phe/Tyr-Xaa-Xaa- Phe/Tyr-Zaa-Phe/Tyr may be measured by any method of detecting/measuring a protein/protein interaction, as discussed further below. Suitable methods include methods analagous to those discussed above and described in Example 1 , for example yeast two-hybrid interactions, co-purification, ELISA or co-immunoprecipitation methods. Thus, the said PDKl may be considered capable of binding a polypeptide comprising the amino acid sequence motif Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa- Phe/Tyr, for example Phe/Tyr-Xaa-Xaa-Phe/Tyr-Asp/Glu-Phe/Tyr or Phe/Tyr-Xaa-Xaa-Phe/Tyr-PhosphoSer/PhosphoThr-Phe/Tyr (the interacting polypeptide) if an interaction may be detected between the said PDKl polypeptide and the said interacting polypeptide by ELISA, co- immunoprecipitation methods or by a yeast two-hybrid interaction or copurification method, for example as described in Example 1. It is preferred that the interacting polypeptide comprising the amino acid sequence motif Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr is a polypeptide that is capable of binding PDKl and changing its activity towards Ser473 of PKBα in substantially the same way as a polypeptide with the amino acid sequence REPRILSEEEQEMFRDFDYIADWC, as described in Example 1. The three-letter amino acid code of the IUPAC-IUB Biochemical Nomenclature Commission is used herein, with the exception of the symbol Zaa, defined above. In particular, Xaa represents any amino acid. It is preferred that Xaa and Zaa represent a naturally occuring amino acid. It is preferred that at least the amino acids corresponding to the consensus sequence are L-amino acids. It is preferred that after the said exposure the PDKl is capable of phosphorylating the underlined residue in a polypeptide with an amino acid sequence corresponding to the consensus sequence Phe-Xaa-Xaa-Phe- Ser/Thr-Phe/Tyr. It is further preferred that after said exposure the PDKl is capable of phosphorylating residue Ser473 of PKBα. It is still further preferred that after said exposure the protein kinase is also capable of phosphorylating the underlined residue in a polypeptide with an amino acid sequence corresponding to the consensus sequence Thr/Ser-Phe-Cys- Gly-Thr-Xaa-Glu-Leu. It is still further preferred that after said exposure the protein kinase is capable of phosphorylating PKBα on Thr308 and Ser473. Either said capability of PDKl may be altered, preferably increased, in the presence of PtdIns(3,4,5)P 3 (PIP 3 ) or PtdIns(3,4,)P 2 dependent manner. The requirement for PtdIns(3,4,5)P 3 or PtdIns(3,4,)P 2 may be extremely specific, because only the D-enantiomers of PtdIns(3,4,5)P 3 appear to be effective, and many other Ptdlns phospholipids including PtdIns(4,5)P 2 appear to be ineffective, as discussed in Example 1. Synthetic sn-1-stearoyl, 2-arachidonoyl D-PtdIns(3,4,5,)P 3 (the predominant form of PtdIns(3,4,5)P 3 which occurs naturally, lipid 8 in Fig 9), synthetic ^n-2-stearoyl, 3-arachidonoyl D-PtdIns(3,4,5,)P 3 (lipid 10 in Fig 9) may be capable of enhancing the phosphorylation by PDKl (after said exposure) of GST-PKBα at Ser473 (Fig 9B) and the phosphorylation of Thr308 (measured by the activation of GST-S473D-PKBα), indicating that the enantiomeric configuration of the glycerol moiety is not an important determinant of specificity. The L-enantiomers of these lipids (Lipids 9 and 11 , Fig 9) may not induce a significant phosphorylation of PKB at Ser473 or activation of GST-S473D-PKBα. rac-l ,2-dilinoleoyl phosphatidyl D/L-myo-inositol 3,4,5 trisphosphate (linoleic acid is C18:2, Lipid 7 Fig 9), src-l ,2-dipalmitoyl D-PtdIns(3,4,5)P 3 , (Lipid 6, Fig 6) as well as 72-1 ,2-dipalmitoyl PtdIns(3,4)P 2 (Lipid 3 Fig 9) may also be effective at inducing the PDKl (after said exposure) to phosphorylate PKBα at Ser473 and Thr308. However, Ptdlns-3P (lipid 2, Fig 9), PtdIns(3,5)P 2 (lipid 4 Fig 9) and PtdIns(4,5)P 2 (lipid 5 Fig 9), did not induce the PDKl (after said exposure) to phosphorylate PKBα at Ser473 or Thr308. In the absence of GST-PIF none of the Ptdlns derivatives tested induced any phosphorylation of Ser473 or activation of GST- S473D-PKBα (data not shown). The said interacting polypeptide may be derivable from PRK2 or PKCς, preferably from the C-terminal portion of PRK2 or PKCς. The said interacting polypeptide may be derivable from PRK2 by proteolytic cleavage, for example by Caspase 3, as described in Example 1. Thus, the interacting polypeptide may comprise or consist essentially of the amino acid sequence from residue 701 to the C-terminus of PRK2. This may correspond to the C-terminal 77 amino acids of PRK2. The C- terminal 77 amino acids of PRK2 may be termed the PDKl -Interacting Fragment (PIF). The PIF region of PRK2 may lie immediately C-terminal to the kinase catalytic domain of PRK2. The polypeptide may comprise or consist essentially of the amino acid sequence of residues 960 to 984 of PRK2 (termed Region B, as described in Example 1) or the equivalent region of PRK1 , PRK1, PKBα, p70S6 kinase, SGK, a PKC isoform, for example PKCς or PKCα, or PKAβ as shown in Figure IE. PKC isoforms are described, for example, in Mellor & Parker (1998) The extended protein kinase C superfamily Biochem J 332, 281-292. The PIF region and Region B of PRK2 comprise an amino acid sequence corresponding to the consensus sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr or Phe/Tyr-Xaa-Xaa-Phe/Tyr-Asp/Glu-Phe/Tyr. The C-terminal region of PKCς comprises the amino acid sequence Phe-Glu-Gly-Phe-Glu-Tyr, which corresponds to the consensus sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr- Zaa-Phe/Tyr or Phe/Tyr-Xaa-Xaa-Phe/Tyr-Asp/Glu-Phe/Tyr. It will be appreciated that a polypeptide that comprises an amino acid sequence that corresponds to the consensus sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr- Ser/Thr-Phe/Tyr may interact with PDKl when the serine or threonine residue is phosphorylated, such that the polypeptide comprises an amino acid sequence that corresponds to the consensus sequence Phe/Tyr-Xaa- Xaa-Phe/Tyr-PhosphoSer/PhosphoThr-Phe/Tyr, or if the serine or threonine residue is replaced by an aspartate or glutamate residue. Region B of PRK2 may have the amino acid sequence REPRILSEEEQEMFRDFDYIADWC. Thus, the said interacting polypeptide to which the said PDKl may be exposed may comprise or consist essentially of the sequence REPRILSEEEQEMFRDFDYIADWC or REPRILSEEEQEMARDFDYIADWC or REPRILSEEEQEMFGDFDYIADWC . Region A of PRK2 may have the amino acid sequence EDVKKHPFFRLIDWSALMDKKVKPPFIPTIRGREDVSNFDDEFTSEA PILTPP. Thus, the said interacting polypeptide may further comprise the sequence EDVKKHPFFRLIDWSALMDKKVKPPFIPTIRGREDVSNFDDEFTSEA PILTPP or variants thereof. The region of PKCς equivalent to Region B of PRK2 may have the amino acid sequence DEDAIKRIDQSEFEGFEYINPLL, as shown in Figure 1. Thus, the said interacting polypeptide may comprise the sequence DEDAIKRIDQSEFEGFEYINPLL or variants thereof. The said interacting polypeptide may comprise a GST portion, as described in Example 1. This may be useful in purifying and/or detecting the said interacting polypeptide. The said PDKl and said interacting polypeptide may be exposed to each other in a cell in which the said PDKl and the said interacting polypeptide are both expressed, as described in Example 1. The PDKl may be endogenous PDKl or it may be PDKl expressed from a recombinant construct. Similarly, the said interacting polypeptide may be endogenous or it may be expressed from a recombinant construct, as described in Example 1. It is preferred that the said PDKl and the said interacting polypeptide are not exposed to each other in a cell in which the said PDKl and the said interacting polypeptide are both naturally expressed. It is preferred that the said PDKl and the said interacting polypeptide are not both endogenous polypeptides to the cell in which the said PDKl and the said interacting polypeptide are exposed to each other. The PDKl and said polypeptide may form a complex, which may be detected by BiaCore measurements, as described in Example 1. The estimated equilibrium dissociation constant of the association between GST-PIF and His-tagged PDKl may be 600nM. The estimated dissociation constant I between His-PDKl and an immobilised and biotinylated 24 residue synthetic peptide corresponding to Region B above detected using Surface Plasmon Resonance measurements was 800 nM, or 1.5μM. A complex may also be detected by coimmunoprecipitation or copurification experiments, for example in material from cells in which PDKl (as defined above) and the said polypeptide are coexpressed, as described in Example 1. The complex between PDKl (for example full- length wild type PDKl) and the said polypeptide (for example, GST-PIF) may be a very strong interaction, as indicated by not being dissociated by up to 2M LiBr (a strong chaotrophic agent) or by incubation with 1 % (by vol) Triton X100. A further aspect of the invention provides a form of PDKl derivable by the method of the first aspect of the invention wherein the said PDKl has altered substrate specificity. As described above, the said PDKl with altered substrate specificity may be capable of phosphorylating the underlined residue in a polypeptide with an amino acid sequence corresponding to the consensus sequence Phe-Xaa-Xaa-Phe-Ser/Thr- Phe/Tyr. The said PDKl may be substantially free of polypeptides with which PDKl is present or associated in a cell in which it is naturally found other than a said interacting polypeptide or a substrate for PDKl . A further aspect of the invention provides a method of making a preparation that comprises PDKl and a polypeptide which comprises the amino acid sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr wherein substantially pure PDKl (as defined above) is mixed with a substantially pure polypeptide which comprises the amino acid sequence Phe/Tyr-Xaa- Xaa-Phe/Tyr-Zaa-Phe/Tyr. It will be appreciated that the method may further comprise the step of adding further components to the preparation, for example a stabilising component such as bovine serum albumin or a substrate of the said PDKl , for example PKBα. A further aspect of the invention provides a preparation derivable by the above method of the invention. By "substantially pure" we mean that the PDKl or interacting polypeptide is substantially free of other proteins. Thus, we include any composition that includes at least 30% of the protein content by weight as the said PDKl or interacting polypeptide, preferably at least 50% , more preferably at least 70% , still more preferably at least 90% and most preferably at least 95 % of the protein content is the said PDKl or interacting polypeptide. Thus the substantially pure PDKl or interacting polypeptide may include a contaminant wherein the contaminant comprises less than 70% of the composition by weight, preferably less than 50% of the composition, more preferably less than 30% of the composition, still more preferably less than 10% of the composition and most preferably less than 5 % of the composition by weight. The substantially pure said PDKl or interacting polypeptide may be combined with other components ex vivo, said other components not being all of the components found in the cell in which said PDKl or interacting polypeptide is naturally found. A further aspect of the invention provides a preparation comprising PDKl and a polypeptide which comprises the amino acid sequence Phe/Tyr-Xaa- Xaa-Phe/Tyr-Zaa-Phe/Tyr, for example Phe/Tyr-Xaa-Xaa-Phe/Tyr- Asp/Glu-Phe/Tyr or Phe/Tyr-Xaa-Xaa-Phe/Tyr-PhosphoSer/PhosphoThr- Phe/Tyr (the interacting polypeptide), wherein the said preparation does not comprise all of the components found in a cell in which said PDKl is naturally found. The said preparation may not comprise components found in a cell, for example a rat brain cell, that are (1) not capable of binding to a Q-Sepharose column equilibrated in buffer solution containing 50 mMTris/HCl pH7.5, ImM EDTA, ImM EGTA, 0.27M sucrose and 0.1 % (by vol) β-mercaptoethanol, or (2) capable of binding to a Q- Sepharose column equilibrated in buffer solution containing 50 mMTris/HCl pH7.5, ImM EDTA, ImM EGTA, 0.27M sucrose and 0.1 % (by vol) β-mercaptoethanol but are not eluted by the said buffer solution further containing 0.3M NaCl. The said preparation may further not comprise components found in a cell that are (1) not capable of binding to a heparin-Sepharose column equilibrated in buffer solution containing 50 mMTris/HCl pH7.5, ImM EDTA, ImM EGTA, 0.27M sucrose, 0.1 % (by vol) β-mercaptoethanol and 0.2M NaCl or (2) capable of binding to a heparin-Sepharose column equilibrated in buffer solution containing 50 mMTris/HCl pH7.5, ImM EDTA, ImM EGTA, 0.27M sucrose, 0.1 % (by vol) β-mercaptoethanol and 0.2M NaCl but are eluted by the said buffer solution containing up to 0.75M NaCl. Thus, the preparation may be substantially free of polypeptides with which PDKl is present or associated in a cell other than a said interacting polypeptide or a substrate of PDKl , for example PKBα. The said preparation may not be obtainable by (1) homogenising rat tissue (fore example brain or thymus) in 20mM Tris pH7.5, ImM EDTA, 25mM NaF, ImM dithiothreitol (DTT), 1 mM NaVn, leupeptin (10 μg/ml), soybean trypsin inhibitor (10 μg/ml), aprotinin (10 μg/ml) and 100 μM pefabloc, (2) centrifuging at 20,000g for 30 min, (3) loading extracts (for example, 20mg) onto a Mono Q column (Pharmacia) and (4) eluting the bound proteins with up to 250mM NaCl, as described in Stokoe et al (1997) Science 211, 567-570. Thus, we include any composition that includes at least 30% of the protein content by weight as the said PDKl or interacting polypeptide (ie in combination), preferably at least 50% , more preferably at least 70%, still more preferably at least 90% and most preferably at least 95 % of the protein content is the said PDKl or interacting polypeptide. Thus, the invention also includes preparations comprising the said PDKl and the said interacting polypeptide and a contaminant wherein the contaminant comprises less than 70% of the composition by weight, preferably less than 50% of the composition, more preferably less than 30% of the composition, still more preferably less than 10% of the composition and most preferably less than 5 % of the composition by weight. The invention also includes a preparation comprising the said PDKl and the said interacting polypeptide when combined with other components ex vivo, said other components not being all of the components found in the cell in which said PDKl and/or interacting polypeptide is naturally found. Alternatives and preferences for PDKl and the said interacting polypeptide are as described in relation to the first aspect of the invention. A further aspect of the invention provides a preparation capable of phosphorylating the underlined residue in a polypeptide with an amino acid sequence corresponding to the consensus sequence Phe-Xaa-Xaa-Phe- Ser/Thr-Phe/Tyr, wherein said preparation comprises two, three, four, five, six or up to ten species of polypeptide and substantially (by mass) no other polypeptides. One said polypeptide species is PDKl and a second said polypeptide species (the interacting polypeptide) comprises the amino acid sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr, for example Phe/Tyr-Xaa-Xaa-Phe/Tyr-Asp/Glu-Phe/Tyr or Phe/Tyr-Xaa-Xaa- Phe/Tyr-PhosphoSer/PhosphoThr-Phe/Tyr. Alternatives and preferences for PDKl and the said interacting polypeptide are as described in relation to the first aspect of the invention. A third polypeptide species may be a substrate for PDKl . A fourth polypeptide species may be a polypeptide that stabilises the preparation, for example bovine serum albumin or gelatin. It is preferred that the said preparation is capable of phosphorylating a residue corresponding to the underlined residue in a polypeptide with an amino acid sequence corresponding to the consensus sequence Thr/Ser- Phe-Cys-Gly-Thr-Xaa-Glu-Leu. It will be appreciated that the two, three, four, five, six or up to ten species of polypeptide may be capable of forming an isolatable complex, as known to those skilled in the art and discussed in Example 1 in relation to PDKl and a polypeptide comprising the amino acid sequence Phe/Tyr- Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr . A further aspect of the invention provides a method of phosphorylating a residue corresponding to the underlined residue in a substrate polypeptide with an amino acid sequence corresponding to the consensus sequence Phe-Xaa-Xaa-Phe-Ser/Thr-Phe/Tyr (PDK2 consensus sequence) wherein a PDKl derivable by the method of the first aspect of the invention, characterised in that the said PDKl has altered substrate specificity, or a preparation according to either of the preceding aspects of the invention is used. The substrate polypeptide may be PKB, for example PKBα, SGK, p70S6 kinase, PKA or a PKC isoform. It is preferred that the substrate polypeptide is also phosphorylated on a residue corresponding to the underlined residue in the consensus sequence Thr/Ser-Phe-Cys-Gly-Thr- Xaa-Glu-Leu. It will be appreciated that the method may be carried out in the presence of a phosphoinositide, for example PIP 2 or PtdIns(3,4,5)P 3 (PIP 3 ). The said PIP 2 or PIP 3 may increase the rate or extent of phosphorylation of the underlined residue in a substrate polypeptide with an amino acid sequence corresponding to the consensus sequence Phe-Xaa-Xaa-Phe-Ser/Thr- Phe/Tyr PDK2 consensus sequence and/or of the residue corresponding to the underlined residue in the consensus sequence Thr/Ser-Phe-Cys-Gly- Thr-Xaa-Glu-Leu. A further aspect of the invention provides a method of phosphorylating PRK2 wherein the said PRK2 is exposed to PDKl . PRK2 may be phosphorylated on the residue corresponding to Thr807 of full-length PRK2. A further aspect of the invention provides the use of PDKl in a method of phosphorylating PRK2. A further aspect of the invention provides a method of identifying a compound that modulates the activation and/or phosphorylation of PRK2 by PDKl wherein the activation and/or phosphorylation of PRK2 by PDKl is measured in the presence of more than one concentration (for example in the presence or absence) of the compound. A still further aspect of the invention provides a preparation comprising PDKl and PRK2 in the substantial absence of other proteins or cellular components of a cell in which said PDKl and/or PRK2 are naturally found. Preferences for this preparation of the invention are analogous to the preferences described above for the preparation of the invention comprising PDKl and a polypeptide which comprises the amino acid sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr wherein the said preparation does not comprise all of the components found in a cell in which said PDKl and/or interacting polypeptide is naturally found. By PRK2 is included a polypeptide with the amino acid sequence shown in Figure 11 or given in Palmer et al (1994) Identification of multiple, novel, protein kinase C-related gene products FEBS Lett 356(1), 5-8, and fragments, variants, derivatives and fusions thereof. A further aspect of the invention provides a method of identifying a compound that modulates the activity of PDKl wherein the said PDKl is exposed to the said compound in the presence of a polypeptide comprising the amino acid sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr, for example Phe/Tyr-Xaa-Xaa-Phe/Tyr-Asp/Glu-Phe/Tyr or Phe/Tyr-Xaa- Xaa-Phe/Tyr-PhosphoSer/PhosphoThr-Phe/Tyr. By activity of PDKl is included the capability of phosphorylating the underlined residue in a substrate polypeptide with an amino acid sequence corresponding to the consensus sequence Phe-Xaa-Xaa-Phe-Ser/Thr-Phe/Tyr (ie PDK2 activity) and/or the capability of phosphorylating the residue corresponding to the underlined residue in the consensus sequence Thr/Ser-Phe-Cys-Gly-Thr- Xaa-Glu-Leu (ie PDKl activity). The method of identifying a compound that modulates the activity of PDKl may comprise the step of measuring the activity of the said PDKl in the presence of more than one concentration of the compound (for example, in the presence of the compound and in the presence of substantially none of the compound) wherein the said PDKl is or has been exposed to a polypeptide which comprises the amino acid sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr, for example Phe/Tyr-Xaa-Xaa- Phe/Tyr-Asp/Glu-Phe/Tyr or Phe/Tyr-Xaa-Xaa-Phe/Tyr- PhosphoSer/PhosphoThr-Phe/Tyr. The said compound may be capable of modulating the interaction between the said polypeptide and PDKl . It will be appreciated that the said compound may interact with PDKl or with the said polypeptide or with both. A further aspect of the invention provides a method of identifying a compound that is capable of altering the substrate specificity of PDKl wherein the ability of the said PDKl to phosphorylate a residue corresponding to the underlined residue in a polypeptide with an amino acid sequence corresponding to the consensus sequence Phe-Xaa-Xaa-Phe- Ser/Thr-Phe/Tyr is measured, and is increased in the presence of the said compound. It will be appreciated that the said ability of PDKl may be measured in the presence of a phosphoinositide, for example PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 . A further aspect of the invention is a compound identified or identifiable by the said method. The compound may be, for example, a compound selected on the basis of, or designed to have, as well known to those skilled in the art, a three- dimensional conformation that may be similar to that of a polypeptide with or comprising an amino acid sequence corresponding to the consensus sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr, for example Phe/Tyr- Xaa-Xaa-Phe/Tyr-Asp/Glu-Phe/Tyr, in particular a polypeptide with the sequence FRDFDY or REPRILSEEEQEMFRDFDYIADWC. A further aspect of the invention is a method of altering the substrate specificity of phosphoinositide-dependent protein kinase 1 (PDKl) wherein the said PDKl is exposed to a compound identified or identifiable by the above method of the invention. A further aspect of the invention is a PDKl derivable by the method of the above aspect of the invention wherein the said PDKl has altered substrate specificity such that the said PDKl is capable of phosphorylating a residue corresponding to the underlined residue in a polypeptide having an amino acid sequence corresponding to the consensus sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr- Ser/Thr-Phe/Tyr. A further aspect of the invention provides a method of identifying a compound that is capable of mimicking the effect of a 3 -phosphoinositide, for example PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 , on the PDKl or PDK2 activity of a PDKl which has altered substrate specificity as defined above, the method comprising deteπnining whether said compound activates a said PDKl so that it can phosphorylate a suitable substrate, the activation by said compound being in the absence of a 3-phosphoinositide. The suitable substrate may be a polypeptide comprising an amino acid sequence corresponding to the consensus sequence Phe-Xaa-Xaa-Phe- Ser/Thr-Phe/Tyr (for measuring PDK2 activity) or a polypeptide comprising an amino acid sequence corresponding to the consensus sequence Thr/Ser-Phe-Cys-Gly-Thr-Xaa-Glu-Leu (for measuring PDKl activity). A further aspect of the invention provides a protein kinase derivable from mammalian, for example rat, brain wherein said protein kinase is capable of phosphorylating a residue corresponding to the underlined residue in a polypeptide with an amino acid sequence corresponding to the consensus sequence Phe-Xaa-Xaa-Phe-Ser/Thr-Phe/Tyr, for example Ser473 of PKBα in the presence of PtdIns(3,4,5)P 3 , wherein the said protein kinase may be eluted from Heparin-Sepharose by at least 0.75M NaCl at pH 7.5 and is capable of binding to an antibody reactive with PDKl . Further details of a method of purifying the said protein kinase are given in Example 1. It will be appreciated that the said protein kinase may comprise more than one polypeptide chain, for example in a non- covalently bound complex. A further aspect of the invention provides a polypeptide which comprises the amino acid sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr, for example Phe/Tyr-Xaa-Xaa-Phe/Tyr-Asp/Glu-Phe/Tyr, wherein said polypeptide is not full-length PRK2, PRK1 or PKCς and wherein Zaa is not phosphoserine or phosphothreonine. The sequence of PRK2 is shown in Figure 11 and in Palmer et al (1994) Identification of multiple, novel, protein kinase C-related gene products FEBS Lett 356(1), 5-8. The sequence of PRK1 is shown in Figure 14 and in Palmer et al (1995) Cloning and expression patterns of two members of a novel protein- kinase-C-related kinase family Eur J Biochem 227(1-2), 344-351. The sequence of PKCς is shown in Figure 15 and in Kochs et al (1993) Activation and substrate specificity of the human protein kinase C alpha and zeta isoenzymes Eur J Biochem 216(2), 597-606. Thus, the polypeptide may comprise or consist essentially of the amino acid sequence from residue 701 to the C-terminus of PRK2. This may correspond to the C-terminal 77 amino acids of PRK2. The C-terminal 77 amino acids of PRK2 may be termed the PDKl -Interacting Fragment (PIF). The PIF region of PRK2 may lie immediately C-terminal to the kinase catalytic domain of PRK2. 21. The polypeptide may comprise or consist essentially of the amino acid sequence of residues 960 to 984 of PRK2 (termed Region B, as described in Example 1). The PIF region and Region B of PRK2 comprise an amino acid sequence corresponding to the consensus sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Asp/Glu-Phe/Tyr. Region A may have the amino acid sequence REPRILSEEEQEMFRDFDYIADWC. Thus, the said polypeptide of the invention may comprise or consist essentially of the sequence REPRILSEEEQEMFRDFDYIADWC or REPRILSEEEQEMARDFDYIADWC or REPRILSEEEQEMFGDFDYIADWC. Region B may have the amino acid sequence EDVKKHPFFRLIDWSALMDKKVKPPFIPTIRGREDVSNFDDEFTSEA PILTPP. Thus, the said polypeptide may further comprise the sequence EDVKKHPFFRLIDWSALMDKKVKPPFIPTIRGREDVSNFDDEFTSEA PILTPP or variants thereof. The said polypeptide of the invention may comprise a GST portion, as described in Example 1. This may be useful in purifying and/or detecting the said polypeptide. A further aspect of the invention provides a polypeptide consisting essentially of residues 51 to 404 of PDKl or a fusion of a polypeptide consisting essentially of residues 51 to 404 of PDKl . A further aspect of the invention provides a polynucleotide encoding a polypeptide of the invention. A still further aspect of the invention provides a recombinant polynucleotide suitable for expressing a polypeptide of the invention. A yet further aspect of the invention provides host cell comprising a polynucleotide of the invention. A further aspect of the invention provides a method of making a polypeptide of the invention, the method comprising culturing a host cell of the invention which expresses said polypeptide and isolating said polypeptide. It will be appreciated that the said polypeptide of the invention that comprises the amino acid sequence Phe/Tyr-Xaa-Xaa- Phe/Tyr-Asp/Glu-Phe/Tyr may be isolated as a complex with endogenous PDKl expressed in the cell or with recombinant PDKl expressed in the cell. It will be appreciated that the said polypeptide of the invention that consists essentially of residues 51 to 404 of PDKl or a fusion of a polypeptide consisting essentially of residues 51 to 404 of PDKl may be isolated as a complex with endogenous PRK2 or a fragment thereof, as described above and in Example 1 , or with recombinant PRK2 or a fragment, derivative or fusion thereof as described in Example 1 expressed in the cell. A further aspect of the invention provides a polypeptide obtainable by the above method. A further aspect of the invention provides a polypeptide of the invention for use in medicine. A still further aspect of the invention provides the use of a polypeptide of the invention in the manufacture of a medicament for the treatment of a patient in need of modulation of the insulin signalling pathway and/or PDK1/PDK2/PRK2 signalling. The polypeptide of the invention or interacting polypeptide which comprises the amino acid sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa- Phe/Tyr, for example Phe/Tyr-Xaa-Xaa-Phe/Tyr-Asp/Glu-Phe/Tyr or Phe/Tyr-Xaa-Xaa-Phe/Tyr-PhosphoSer/PhosphoThr-Phe/Tyr as defined above may be up to about 950, 900, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, 50, 40, 30, 20, 18, 16, 15, 14, 12, 10, 8 or 7 amino acids in length. The polypeptide may consist of or comprise contiguous residues derivable from PRK2, PKCς or PDKl (as appropriate), for example rat or human PRK2, PKCς or PDKl . The polypeptide may be capable of reducing, preferably substantially preventing, an interaction between full length PDKl , for example full length human PDKl and a polypeptide comprising the amino acid sequence Phe/Tyr-Xaa-Xaa- Phe/Tyr-Zaa-Phe/Tyr, for example Phe/Tyr-Xaa-Xaa-Phe/Tyr-Asp/Glu- Phe/Tyr or Phe/Tyr-Xaa-Xaa-Phe/Tyr-PhosphoThr/PhosphoSer-Phe/Tyr, measured, for example, as described in Example 1. It will be appreciated that the peptide may comprise a covalent modification, for example it may be modified by biotinylation ie comprise a biotin group. Such a peptide may be useful in altering the enzymic activity of PDKl in vitro or in vivo. The above polypeptides or peptide may be made by methods well known in the art and as described below and in Example 1 , for example using molecular biology methods or automated chemical peptide synthesis methods. It will be appreciated that peptidomimetic compounds may also be useful. Thus, by "polypeptide" or "peptide" we include not only molecules in which amino acid residues are joined by peptide (-CO-NH-) linkages but also molecules in which the peptide bond is reversed. Such retro-inverso peptidomimetics may be made using methods known in the art, for example such as those described in Meziere et al (1997) J. Immunol. 159, 3230-3237, incorporated herein by reference. This approach involves making pseudopeptides containing changes involving the backbone, and not the orientation of side chains. Meziere et al (1997) show that, at least for MHC class II and T helper cell responses, these pseudopeptides are useful. Retro-inverse peptides, which contain NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis. Similarly, the peptide bond may be dispensed with altogether provided that an appropriate linker moiety which retains the spacing between the Cα atoms of the amino acid residues is used; it is particularly preferred if the linker moiety has substantially the same charge distribution and substantially the same planarity as a peptide bond. It will be appreciated that the peptide may conveniently be blocked at its N- or C-terminus so as to help reduce susceptibility to exoproteolytic digestion. Thus, it will be appreciated that the polypeptide which comprises the amino acid sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr, for example Phe/Tyr-Xaa-Xaa-Phe/Tyr-Asp/Glu-Phe/Tyr or Phe/Tyr-Xaa-Xaa- Phe/Tyr-PhosphoSer/PhosphoThr-Phe/Tyr to which PDKl may be exposed may be a peptidomimetic compound, as described above. A further aspect of the invention is a cell containing a recombinant nucleic acid suitable for expressing PDKl and a recombinant nucleic acid suitable for expressing a polypeptide comprising the amino acid sequence Phe/Tyr- Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr. Recombinant polynucleotides suitable for expressing a given polypeptide are well known to those skilled in the art, and examples are described in Example 1. It will be appreciated that a recombinant nucleic acid molecule may be suitable for expressing PDKl and a polypeptide comprising the amino acid sequence Phe/Tyr-Xaa-Xaa- Phe/Tyr-Zaa-Phe/Tyr. The cell is preferably a mammalian or insect cell. A further aspect of the invention is a method of altering the substrate specificity of PDKl according to the first aspect of the invention wherein PDKl is exposed to an interacting polypeptide comprising the amino acid sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr in a cell as defined in the above aspect of the invention, by co-expression of PDKl and the said interacting polypeptide. A further aspect of the invention is a method of making a preparation comprising PDKl and an interacting polypeptide comprising the amino acid sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa- Phe/Tyr wherein PDKl and the said interacting polypeptide are co- expressed in a cell as defined in the above aspect of the invention. The PDKl and the said interacting polypeptide may be separated from other cellular components, for example using methods discussed above or in Example 1. A further aspect of the invention is a preparation obtainable by the above method of the invention. An antibody reactive towards a polypeptide with the amino acid sequence REPRILSEEEQEMFRDFDYIADWC or REPRILSEEEQEMARDFDYIADWC or REPRILSEEEQEMFGDFDYIADWC is described in example 1. An antibody reactive towards PKBα or a fragment or fusion thereof that is phosphorylated on the residue equivalent to Ser473 but is not reactive with PKBα or a fragment or fusion thereof that is not phosphorylated on the residue equivalent to Ser473 is described in Example 1. The antibody may react with the peptide Pro-His-Phe-Pro-Gln-Phe-PhosphoSer-Tyr-Ser- Ala-Ser (corresponding to residues 467 to 477 of PKBα). Methods of preparing such antibodies are given in Example 1. Antibodies reactive towards the said polypeptides may be made by methods well known in the art. In particular, the antibodies may be polyclonal or monoclonal. Suitable monoclonal antibodies which are reactive towards the said polypeptide may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques" , H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications" , SGR Hurrell (CRC Press, 1982). Techniques for preparing antibodies are well known to those skilled in the art, for example as described in Harlow, ED & Lane, D "Antibodies: a laboratory manual" (1988) New York Cold Spring Harbor Laboratory. The enhancement or disruption of the interaction between PDKl and an interacting polypeptide can be measured in vitro using methods well known in the art of biochemistry and include any methods which can be used to assess protein-protein interactions. It will be appreciated that the methods described may be performed in cells. In a further embodiment the yeast two-hybrid system may be used. It will be appreciated that the invention provides screening assays for drugs which may be useful in modulating, for example either enhancing or inhibiting, the PDKl or PDK2 activity (as discussed above) of PDKl , for example PDKl that has been exposed to a polypeptide comprising the amino acid sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr, for example Phe/Tyr-Xaa-Xaa-Phe/Tyr-Asp/Glu-Phe/Tyr or Phe/Tyr-Xaa-Xaa- Phe/Tyr-PhosphoSer/PhosphoThr-Phe/Tyr, or the interaction of PDKl with a phosphoinositide, for example PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 , or the interaction of PDKl with a polypeptide comprising the amino acid sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr, for example Phe/Tyr- Xaa-Xaa-Phe/Tyr-Asp/Glu-Phe/Tyr or Phe/Tyr-Xaa-Xaa-Phe/Tyr- PhosphoSer/PhosphoThr-Phe/Tyr. The compounds identified in the methods may themselves be useful as a drug or they may represent lead compounds for the design and synthesis of more efficacious compounds. The compound may be a drug-like compound or lead compound for the development of a drug-like compound for each of the above methods of identifying a compound. It will be appreciated that the said methods may be useful as screening assays in the development of pharmaceutical compounds or drugs, as well known to those skilled in the art. The term "drug-like compound" is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament. Thus, for example, a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 daltons. A drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate cellular membranes, but it will be appreciated that these features are not essential. The term "lead compound" is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non-selective in its action, unstable, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics. A further aspect of the invention is a kit of parts useful in carrying out a method, for example a screening method, of the invention. Such a kit may comprise PDKl and a polypeptide comprising the amino acid sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr, for example Phe/Tyr- Xaa-Xaa-Phe/Tyr-Asp/Glu-Phe/Tyr or Phe/Tyr-Xaa-Xaa-Phe/Tyr- PhosphoSer/PhosphoThr-Phe/Tyr. It may further comprise a 3- phosphoinositide, for example PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 . It will be understood that it will be desirable to identify compounds that may modulate the activity of the PDKl polypeptide in vivo. Thus it will be understood that reagents and conditions used in the method may be chosen such that the interactions between, for example, the said PDKl and the interacting polypeptide comprising the amino acid sequence Phe/Tyr- Xaa-Xaa-Phe/Tyr-Asp/Glu-Phe/Tyr or Phe/Tyr-Xaa-Xaa-Phe/Tyr- PhosphoSer/PhosphoThr-Phe/Tyr, are substantially the same as between human PDKl and a naturally occuring interacting polypeptide comprising the said amino acid sequence. It will be appreciated that the compound may bind to the PDKl , or may bind to the interacting polypeptide comprising the said amino acid sequence, for example a polypeptide derivable from PRK2. A further aspect of the invention is a compound identifiable or identified by the said screening method. It will be appreciated that such a compound may be a modulator, for example an inhibitor of the PDKl or PDK2 protein kinase activity of the PDKl used in the screen and that the intention of the screen may be to identify compounds that act as modulators, for example inhibitors of the PDKl or PDK2 protein kinase activity, even if the screen makes use of a binding assay rather than an enzymic activity assay. It will be appreciated that the modulatory, for example inhibitory action of a compound found to bind the protein kinase may be confirmed by performing an assay of enzymic activity (ie PDKl and/or PDK2 protein kinase activity) in the presence of the compound. A still further aspect of the invention is a compound (or polypeptide or polynucleotide) of the invention for use in medicine. A still further aspect of the invention is a polypeptide which comprises the amino acid sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr, for example Phe/Tyr-Xaa-Xaa- Phe/Tyr-Asp/Glu-Phe/Tyr or Phe/Tyr-Xaa-Xaa-Phe/Tyr- PhosphoSer/PhosphoThr-Phe/Tyr, or a polypeptide consisting essentially of residues 51 to 404 of PDKl or a fusion of a polypeptide consisting essentially of residues 51 to 404 of PDKl wherein Zaa represents a negatively charged amino acid residue for use in medicine. Relevant preferences, for example concerning length, for the said polypeptides are as described above for polypeptides of the invention. The compound (or polypeptide or polynucleotide) may be administered in any suitable way, usually parenterally, for example intravenously, intraperitoneally or intravesically, in standard sterile, non-pyrogenic formulations of diluents and carriers. The compound (or polypeptide or polynucleotide) may also be administered topically, which may be of particular benefit for treatment of surface wounds. The compound (or polypeptide or polynucleotide) may also be administered in a localised manner, for example by injection. A further aspect of the invention is the use of a compound (or polypeptide or polynucleotide) as defined above in the manufacture of a medicament for the treatment of a patient in need of modulation of the insulin signalling pathway and/or signalling by PDKl, PDK2 or PRK2. A compound that is capable of reducing the activity (ie the PDKl and/or the PDK2 activity) of PDKl may be useful in treating cancer. PDKl , for example via PKB and/or SGK, may be capable of providing a survival signal that protects cells from apoptosis induced in a variety of ways (reviewed in [8, 13]). Thus, such compounds may aid apoptosis. Reduction of the activity of PDKl may promote apoptosis and may therefore be useful in treating cancer. Conditions in which aiding apoptosis may be of benefit may also include resolution of inflammation. A compound is capable of increasing the activity of PDKl may be useful in treating diabetes or obesity, or may be useful in inhibiting apoptosis. Increased activity of PDKl may lead to increased levels of leptin, as discussed above, which may lead to weight loss; thus such compounds may lead to weight loss. For example, such compounds may suppress apoptosis, which may aid cell survival during or following cell damaging processes. It is believed that such compounds are useful in treating disease in which apoptosis is involved. Examples of such diseases include, but are not limited to, mechanical (including heat) tissue injury or ischaemic disease, for example stroke and myocardial infarction, neural injury and myocardial infarction. Thus the patient in need of modulation of the activity of PDKl may be a patient with cancer or with diabetes, or a patient in need of inhibition of apoptosis, for example a patient suffering from tissue injury or ischaemic injury, including stroke. Thus, a further aspect of the invention provides a method of treating a patient with an ischaemic disease the method comprising administering to the patient an effective amount of a compound identifiable by the screening methods of the invention. A still further invention provides a use of a compound identifiable by the screening methods of the invention in the manufacture of a medicament for treating an ischaemic disease in a patient. Thus, a further aspect of the invention provides a method of treating a patient with an ischaemic disease the method comprising administering to the patient an effective amount of a compound identifiable by the screening methods of the invention. If the patient is a patient in need of promotion of apoptosis, for example a patient with cancer, it is preferred that the compound of the invention that is used in the preparation of the medicament is capable of reducing the activity of PDKl . If the patient is a patient with diabetes or a patient in need of inhibition of apoptosis, for example a patient with ischaemic disease, it is preferred that the compound of the invention that is used in the preparation of the medicament is capable of increasing the activity of SGK. The invention will now be described by reference to the following Examples and Figures: Figure 1. Two hybrid interaction of PIF with the kinase domain of PDKl. Yeast were transformed with a pGADIO plasmid (LEU2) harbouring the C-terminal 77 amino acids of PRK2 (termed PIF) in the presence of either a pAS2-l plasmid (TRP1) coding for the expression of the wild type PDKl , the kinase domain of PDKl (residues 1 to 404) or the PH domain of PDKl (residues 428 to 556) or the empty pAS2-l vector as a control (denoted by - (A). The position on the plate of each yeast strain is indicated in (A). In (B) the yeast are grown on SD-Leu and Trp deficient media in which all the strains of yeast are capable of growing independently of whether PIF is interacting with PDKl . An interaction between PIF and PDKl leads to the expression of reporter genes coding for URA3 and H/S7 that will enable growth on SD-Leu-Trp-Ura-Ηis + 3- AT medium (C) as well as the induction of β-galactosidase gene expression (D). Alignment of the amino acid sequence of the C-terminal 77 amino acids of PRK2 with the equivalent region of AGC subfamily kinases indicated (E). Identical residues are denoted by white-on-black letters, and similar residues by grey boxes. Figure 2. Specific interaction of GST-PIF with PDKl in 293 cells. (A+B) 293 cells were transiently transfected with DNA constructs expressing GST or GST-PIF together with either wild type Myc-PDKl, kinase dead Myc-PDKl (Ki l l A, D223A) or the kinase domain of Myc- PDKl (residues 51 to 404). 36 h post transfection the cells were lysed and either the GST or GST-PIF purified from 350 μg of lysate by affinity chromatography of glutathione-Sepharose beads (A) or the Myc-PDKl proteins immunoprecipitated using Myc antibodies covalently attached to Protein G-Sepharose (B). The total protein from each purification was electrophoresed on a 10 % SDS/polyacrylamide gel and stained with Coomassie blue. The position of the molecular mass markers, glycogen phosphorylase (97 kDa), bovine serum albumin (67 kDa) and ovalbumin (43 kDa) are indicated. (C) 293 cells were transiently transfected with DNA constructs expressing either GST-PIF, GST alone, GST-p90RSKl , GST-MSK1 , GST-p70 S6 kinase lacking the C-terminal 104 residues, and GST-PKBα. 36 hours post transfection the cells were lysed and the GST fusion proteins purified by affinity chromatography on glutathione-Sepharose beads. Each GST-fusion protein was incubated for 30 min at 30°C with GST-S473D-PKBα and MgATP in the presence or absence of phospholipid vesicles containing 100 μM PtdCho, 100 μM PtdSer, 10 μM sn-l-stearoyl-2-arachidonoyl-D- PtdIns(3,4,5)P 3 , and the increase in specific activity of GST-S473D-PKBα was determined relative to a control incubation in which the GST-S473D- PKBα fusion protein was omitted (average for 6 determinations, three independent experiments). The basal activity of GST-S473D-PKBα was 3.5 U/mg. 2 μg of each protein was electrophoresed on a 10 % SDS/polyacrylamide gel and immunoblotted using a PDKl antibody raised against the PDKl protein (used at 0.2 μg/ml) to detect any endogenous PDKl associated with the glutathione-Sepharose pull downs. Figure 3. Mapping the residues on PIF that are required for the interaction with PDKl. (A) 293 cells were transiently transfected with DNA constructs expressing either the wild type GST-PIF or the indicated point mutations of this protein. The cells were lysed and the GST fusion proteins purified by affinity chromatography on glutathione-Sepharose beads. Each GST-fusion protein was incubated for 30 min at 30°C with GST-S473D-PKBα and MgATP in the presence or absence of phospholipid vesicles containing 100 μM PtdCho, 100 μM PtdSer, 10 μM 5 , /2-l-stearoyl-2-arachidonoyl-D-PtdIns(3,4,5)P 3 , and the increase in specific activity of GST-S473D-PKBα was determined relative to a control incubation in which the GST fusion protein was omitted. The basal activity of GST-S473D-PKBα was 3.5 U/mg. 2 μg of each protein was electrophoresed on a 10% SDS/polyacrylamide gel and either immunoblotted using a PDKl antibody raised against whole protein to detect any endogenous PDKl , or stained with Coomassie blue to establish the level of expression of the GST-PIF protein. Similar results were obtained in 5 separate experiments using 2 or more preparations of the wild type and mutant GST-PIF proteins. (B) Yeast harbouring the pAS2-l wild type PDKl vector ("PDKl-Vector") or the empty pAS2-l vector ("Vector") were transformed with pGADIO plasmid coding for the expression of the wild type PIF or the indicated mutants of PIF. The resulting yeast strain was grown on SD-Leu-Trp-Ura-His + 3-AT medium which only permits growth of yeast constructs in which GST-PIF and PDKl are capable of interacting. Figure 4. Incubation of PIF with PKBα induces phosphorylation of PKBα at both Thr308 and Ser473. GST-PKBα (A) or GST-S473D- PKBα (B) was incubated at 30°C for lh with MgATP in the presence of either His-PDKl (0.1 μg) or GST-PIF purified from 293 cells which is associated with endogenous PDKl (1 μg termed GST-PIF/PDK1) in the presence or absence of phospholipid vesicles containing 100 μM PtdCho, 100 μM PtdSer, and 10 μM ,572-l-stearoyl-2-arachidonoyl-D- PtdIns(3,4,5)P 3 . The increase in specific activity of GST-PKBα was determined, relative to a control incubation in which His-PDKl or GST- PIF was omitted (average for 6 determinations, two independent experiments). The basal activity of GST-PKBα was 1.0 U/mg, GST- S473D-PKBα was 3.5 U/mg. Under the conditions used, a maximal activation of GST-PKBα (A) and GST-S473D-PKBα (B) was observed which could not be increased by prolonging the incubation period or increasing the concentration of His-PDKl or GST-PIF added to the assay. (C) GST-PKBα was incubated with GST-PIF/PDK1 (1.0 μg) as in (A). The reactions were terminated by making the solutions 1 % in SDS, the samples were subjected to SDS/polyacylamide gel electrophoresis and immunoblotted using the P-Ser473 antibody that was incubated in the presence or absence of the following synthetic peptides (all at 10 μg/ml) corresponding to residues 467 to 477 of PKBα phosphorylated at the residue equivalent to Ser473 (phospho- 473 peptide), the same peptide that was not phosphorylated (de-phospho-473 peptide), or a peptide corresponding to residues 301 to 313 of PKBα phosphorylated at the residue equivalent to Thr308 (phospho-308 peptide). (D) as in (A) except radioactive Mg[γ 32 P]ATP (10 6 cpm per nmol) was employed, the samples alleviated with 4-vinylpyridine. electrophoresed on a 10% polyacrylamide gel. The phosphorylated GST-PKBα was excised from the gel, digested with trypsin and applied to a Vydac 218TP54 C 18 column (Separations Group, Hesperia, CA) equilibrated in 0.1 % (v/v) trifluoroacetic acid (TFA), as described previously [17] . The column was developed with a linear acetonitrile gradient (diagonal line) at a flow rate of 0.8 rnl/min and fractions of 0.4 ml were collected. Between 50-70% of the radioactivity applied to the column was recovered in the major 32 P-containing peptides eluting at 24% and 26% acetonitrile, which were identified (see results) as the PKBα tryptic peptides phosphorylated at Ser473 and Thr308 respectively. Figure 5. The ability of GST-PIF to phosphorylate PKBα at Ser473 is dependent upon the presence of PDKl. GST-PIF purified from 293 cells, Fig 2C (20 μg in 50 μl) was incubated with Protein G-Sepharose (10 μl) conjugated with either no antibody (control), MSK1 antibody A [40] (5 μg, non specific antibody), a PDKl antibody raised against the whole protein (5 μg) or a PDKl peptide antibody raised against the 16 C- terminal amino acid residues of PDKl (5 μg, peptide antibody) in the presence or absence of the PDKl C-terminal peptide immunogen (0.2 mM) to which this antibody was raised. After incubation on a shaking platform for 60 min at 4°C, the suspension was centrifuged and GST-PIF in the supernatant (2.0 μg) was incubated with GST-PKBα and MgATP for 60 min at 30°C in the presence or absence of phospholipid vesicles containing 100 μM PtdCho, 100 μM PtdSer, and 10 μM «-l-stearoyl-2- arachidonoyl-D-PtdIns(3,4,5)P 3 (l ,2-SAD-PtdIns(3,4,5)P 3 ), or sn-1 ,2 di- palmitoyl-D-PtdIns(3,4,5)P 3 (C 16 -PtdIns(3,4,5)P 3 ) or sn-1 ,2 di-palmitoyl- D-PtdIns(3,4)P 2 (C 16 -PtdIns(3,4)P 2 ). The reactions were terminated by making the solutions 1 % in SDS, and the samples were subjected to SDS/polyacylamide gel electrophoresis and immunoblotted using the P- Ser473 antibody. Identical results were obtained in 2 separate experiments (B) The wild type and mutant GST-PIF proteins (2.0 μg) used in Fig 3 were incubated with GST-PKBα and MgATP for 60 min at 30°C in the presence or absence of lipid vesicles containing 10 μM 572-l-stearoyl-2- arachidonoyl-D-PtdIns(3,4,5)P 3 and the degree of phosphorylation of PKBα at Ser473 was established by immunoblotting. Similar results were obtained in 4 separate experiments using 2 different preparations of the wild type and mutant GST-PIF proteins. Figure 6. Conversion of PDKl into an enzyme that can phosphorylate both Thr308 and Ser473 of PKB by addition of a synthetic peptide.(A) His-PDKl (20 ng) was incubated in the presence or absence of the following peptide (all at 20 M) "Region B peptide" (residues 969 to 984 of PIF), "D978A Region B peptide" (residues 919 to 945 of PIF in which Asp 978 is mutated to Ala) or "Region A peptide" (residues 927 to 951 of PIF). After allowing to stand for 1 h at 4 °C GST-PKBα, MgATP and the indicated phospholipid vesicles containing 100 μM PtdCho, 100 μM PtdSer, and either 10 μM 5 , n-l-stearoyl-2-arachidonoyl-D-PtdIns(3,4,5)P 3 (l ,2-SAD-PtdIns(3,4,5)P 3 ), or sn-1 ,2 di-palmitoyl-D-PtdIns(3,4,5)P 3 (C 16 - PtdIns(3,4,5)P 3 ) or sn-1 ,2 di-palmitoyl-D-PtdIns(3,4)P 2 (C 16 - PtdIns(3,4)P 2 ) were added. After 1 h at 30°C, the reactions were terminated by making the solutions 1 % in SDS, the samples were subjected to SDS/polyacylamide gel electrophoresis and immunoblotted using P-Ser473 antibody. Identical results were obtained in 2 separate experiments. (B to D) His-PDKl was incubated for 1 h on ice as above in the presence of "Region B peptide" (B), "D978A-Region B peptide" (C) or no peptide (D) and then incubated with Mg[γ 32 P]ATP and phospholipid vesicles containing 100 μM PtdCho, 100 μM PtdSer, and 10 μM sn-1- stearoyl-2-arachidonoyl-D-PtdIns(3,4,5)P 3 . After 1 h at 30°C the reactions were terminated and the residues on PKB that became phosphorylated under these conditions were established as described in the legend to Fig 4. Figure 7. Specificity of phosphoinositide-dependent PIF/PDK1- induced phosphorylation of Thr308 and Ser473 of PKBα. GST-PKBα was incubated for 60 min at 30°C with GST-PIF purified from 293 cells (which is associated with endogenous PDKl see Fig 3) (1.0 μg), MgATP and phospholipid vesicles containing 100 μM PtdCho, 100 μM PtdSer, and various Ptdlns lipids (numbered 1-11 , see below) all at a final concentration of 10 μM in the assay. In panel A the reactions were terminated by the addition of Triton XI 00 and the increase in specific activity of GST-S473D-PKBα (U/mg), determined, relative to a control incubation in which GST-PIF was omitted (_+ SEM for 6 determinations, two independent experiments). The basal activity of GST-S473D-PKBα was 3.5 U/mg. In Panel B the reactions were terminated by making the solutions 1 % in SDS, the samples were subjected to SDS/polyacylamide gel electrophoresis and the extent of PKBα phosphorylation was established using the phospho-specific antibody that recognises PKBα phosphorylated at Ser 473. In B the results of 2 separate experiments with different preparations of lipid are shown. Lipid 1 , Buffer control; Lipid 2, Ptdlns 3P; Lipid 3, n -1 ,2-dipalmitoyl PtdIns(3,4)P 2 ; Lipid 4, PtdIns(3,5)P 2 ; Lipid 5, PtdIns(4,5)P 2 ; Lipid 6, sn- 1,2-dipalmitoyl D- PtdIns(3,4,5)P 3 ; Lipid 7, rac-l ,2-dilinoleoyl phosphatidyl D/L-myo- inositol 3,4,5trisphosphate; Lipid 8, .svz-l-stearoyl, 2-arachidonoyl D- PtdIns(3,4,5,)P 3 ; Lipid 9, sn-1-stearoyl, 2-arachidonoyl L- PtdIns(3,4,5,)P 3 ; Lipid 10, 5/2-2-stearoyl, 3-arachidonoyl D- PtdIns(3,4,5,)P 3 ; Lipid 11 , «-2-stearoyl, 3-arachidonoyl L- PtdIns(3,4,5,)P 3 . Figure 8. Maximal activation of PKB by PDK1/PIF requires PtdIns(3,4,5)P 3 to interact with both PKB and PDKl. GST-ΔPH-PKBα was incubated at 30°C for lh with MgATP in the presence of either buffer (control), His-PDKl (0.1 μg), or His-PDKl (0.1 μg) + GST-PIF* (1.0 μg in which all the PDKl had been removed by immuno-depletion), or GST-PIF* alone (1.0 μg) or GST-PIF purified from 293 cells which is associated with endogenous PDKl (2 μg termed GST-PIF/PDK1) in the presence or absence of phospholipid vesicles containing 100 μM PtdCho, 100 μM PtdSer, and 10 μM sft-l-stearoyl-2-arachidonoyl-D- PtdIns(3,4,5)P 3 . The increase in specific activity of GST-ΔPH-PKBα was determined, relative to a control incubation in which His-PDKl or GST- PIF was omitted (average for three determinations in one experiment are shown, similar results were obtained in three other separate experiments). The basal activity of GST-ΔPH-PKBα was 3.0 U/mg . Figure 9. Fractionation of rat brain extract. (A) Brain extract was fractionated by batchwise chromatography on Q-Sepharose followed by gradient elution from Heparin-Sepharose (see methods). The column fractions were assayed for their ability to phosphorylate PKBα at Thr308 (measured by the increase in GST-S473D-PKBα activity) and Ser473 (using the P-473 antibody) in the presence (open squares) or absence (closed squares) of 10μM l ,2-SAD-PtdIns(3,4,5)P 3 . The broken line indicates the salt gradient, (b) Frations 7 and 8 of the Heparin-Sepharose column were pooled and aliquots (0.1ml) incubated with Protein G_Sepharose (10 μl) conjugated to an MSK1 antibody A [41] (5 μg, nonspecific antibody), a PDKl antibody raised against the whole protein (5μg), or no antibody (control). After incubation on a shaking platform for 60 min at 4 °C, the suspensionwas centrifuged, and an aliquot of the supernatant (5μl) incubated for 60 min at 30°C ith either GST-S473D- PKBα (PDKl assay) or GST-PKBα (PDK2 assay) and MgATP in the presence or absence of 10 μM l ,2-SAD-PtdIns(3,4,5)P 3 or C 16 - PtdIns(3,4)P 2 . The phosphorylation of PKBα at Ser473 and Thr308 was then measured as in A. Similar results were obtained in experiments from two separate purifications. Figure 10. PDKl sequence Figure 11. PRK2 sequence Figure 12. Quantitative analysis of the binding of PDKl to PIF. Surface Plasmon Resonance measurements were carried out on a BiaCore instrument as described in the methods section. His-PDKl was injected at the indicated concentrations over (A) 2000Rus of GST-PIF, which was immobilised by amine coupling to a CM5 Sensorchip, or (B) 740 Rus of biotinylated wild type PIF, which was immobilised on a Sensorchip SA. The responses at steady state binding were recorded. (C) shows the inhibition of the steady- state response when 500 nM HisPDKl was co- injected with the indicated concentrations of the wild type and D393A peptides. All data are single determinations from a representative experiment that was repeated at least 3 times with similar results. Figure 13. Conversion of PDKl into an enzyme that can phosphorylate both Thr308 and Ser473 of PKB by addition of PIF. (A) GST-PIF in which all the PDKl has been removed by immuno- depletion as described in Fig 5 (1.0 μg, termed GST-PIF*) or the equivalent volume of Buffer was added to 20 ng of wild type GST-PDK1 or kinase dead-GST-PDKl (D223A mutant [18]). After allowing to stand for 1 h at 4°C GST-PKBα, MgATP and the indicated phospholipid vesicles containing 100 μM PtdCho, 100 μM PtdSer, and either 10 μM sn- 1 -stearoyl-2-arachidonoyl-D-PtdIns(3 ,4 , 5)P 3 (1 ,2-S AD- PtdIns(3,4,5)P 3 ), or sn-1 ,2 di-palmitoyl-D-PtdIns(3,4,5)P 3 (C 16 - PtdIns(3,4,5)P 3 ) or sn-1 ,2 di-palmitoyl-D-PtdIns(3,4)P 2 (C 16 - PtdIns(3,4)P 2 ) were added. After 1 h at 30°C, the reactions were terminated by making the solutions 1 % in SDS, the samples were subjected to SDS/polyacylamide gel electrophoresis and immunoblotted using the P-473 antibody. Identical results were obtained in 2 separate experiments. (B and C) wild type GST-PDKl (20 ng) was incubated in the absence (B) or presence of GST-PIF* for 1 h on ice and then incubated with Mg[γ 32 P]ATP and phospholipid vesicles containing 100 μM PtdCho, 100 μM PtdSer, and either 10 μM ^n-l-stearoyl-2-arachidonoyl-D- PtdIns(3,4,5)P 3 . After 1 h at 30°C the reactions were terminated and the residues on PKB that became phosphorylated under these conditions were determined as described in the legend to Fig 5. Figure 14: PRK1 sequence Figure 15: PKCς sequence Example 1: PDKl and PDK2 may be the same enzyme; PDKl displays PDK2 activity in the presence of a synthetic peptide. Abbreviations: PKB, Protein kinase B; Ptdlns, Phosphatidylinositol; PI 3- kinase, Phosphoinositide 3-kinase; PtdCho, Phosphatidylcholine; PtdSer, Phosphatidylserine; PH, pleckstrin homology; RSK, Ribosomal S6 kinase; MSK, Mitogen and Stress Stimulated kinase. Results. Using the yeast two hybrid system we observed that the kinase domain of PDKl interacted with a region of the Protein Kinase C-Related Kinase-2 (PRK2), termed the PDKl Interacting Fragment (PIF). PIF is situated C-terminal to the kinase domain of PRK2, and contains a consensus motif for phosphorylation by PDK2, similar to that found in PKBα, except that the residue equivalent to Ser473 is changed to Asp. Mutation of any of the conserved aromatic residues in the PDK2 motif of PIF or mutation of the Asp residue to either Ala or Ser prevented the interaction of PIF with PDKl . Remarkably, the interaction of PDKl with PIF or a 24 residue synthetic peptide whose sequence encompasses the PDK2 consensus sequence of PIF, converted PDKl from an enzyme that could only phosphorylate Thr308 of PKBα to a kinase that phosphorylates both Thr308 and Ser473 of PKBα in a PtdIns(3,4,5)P 3 dependent manner. Furthermore, the interaction of PIF with PDKl , converted the latter from a form which is not activated by PtdIns(3,4,5)P 3 directly to a form which is activated 3 to 4-fold by PtdIns(3,4,5)P 3 in vitro. We have partially purified a kinase from brain extract that phosphorylates Ser 473 of PKBα in a PtdIns(3,4,5)P 3 dependent manner which is immunoprecipitated with a PDKl antibody. Conclusions. PDKl possesses the intrinsic ability to phosphorylate Ser473 as well as Thr308 of PKBα in a 3-phosphoinositide dependent manner when complexed through its kinase domain to PIF. These finding raise the possibility that PDKl and PDK2 are the same enzyme, and that the substrate specificity and activity of PDKl is regulated through its interaction with another protein(s) in vivo. PRK2 may be a substrate for PDKl . The C-terminal region of PRK2 interacts specifically with PDKl. A yeast two-hybrid screen was carried out to identify proteins expressed in human skeletal muscle that interact with PDKl . We identified one clone corresponding to the C-terminal 77 amino acids of PRK2 that yielded a positive interaction with full length PDKl as well as with the isolated kinase domain (residues 51-404), but not with the PH domain of PDKl (Fig 1A to ID). We have termed this region of PRK2 the "PDK1- Interacting Fragment" (PIF). PRK2 is a Rho and lipid dependent protein kinase [27,28] that, like PDKl and PKB belongs to the AGC subfamily of protein kinases. The PIF region in PRK2 lies immediately C-terminal to the kinase catalytic domain, in a region where there is high sequence homology between AGC subfamily kinases (Fig IE). Most notably the C- terminus of PIF contains the consensus sequence for PDK2 phosphorylation (Phe/Tyr-Xaa-Xaa-Phe/Tyr-Ser/Thr-Phe/Tyr), except that in PRK2 the Ser/Thr residue equivalent to Ser473 of PKB is replaced with a negatively charged Asp residue (Asp978), perhaps mimicking a phosphorylated state. In order to confirm that PDKl did indeed interact with PIF, we expressed PIF as a glutathione-S-transferase (GST) fusion protein in 293 cells together with Myc epitope-tagged PDKl . The cells were lysed and the GST-PIF affinity purified by chromatography on glutathione-Sepharose (Fig 2 A) or the Myc-PDKl isolated by immunoprecipitation using a Myc antibody (Fig 2B) . The samples were made 1 % in SDS, then electrophoresed on an SDS-polyacrylamide gel and the proteins visualised by staining with Coomassie blue (Fig 2). A near-stoichiometric complex was observed between GST-PIF and wild type PDKl , with the isolated kinase of PDKl or with a catalytically-inactive mutant. The complex formed between PDKl and GST-PIF could not be dissociated at concentrations of LiBr up to 2 M (a strong chaotrophic agent) or by incubation with 1 % (by vol) Triton X100 (data not shown), indicating a very strong interaction. No interaction was observed between either GST and PDKl (Fig 2) or between GST-PIF and epitope tagged PKB, p70 S6 kinase, or 6-phosphofructo-2-kinase when each of these were coexpressed with PIF in 293 cells (data not shown). Analysis of the interaction using Surface Plasmon Resonance confirmed that GST-PIF was capable of associating directly with purified His-tagged PDKl with an estimated equilibrium dissociation constant of 600 nM (Figure 12). No association between GST-PIF and His-PKBα was detected using this method. PIF binds specifically to the endogenous PDKl in 293 cells. GST-PIF purified from 293 cells was found to be associated with the endogenous PDKl as judged by Western blotting. In 293 cells, two PDKl immunoreactive bands are observed running at 63 kDa and 66 kDa, and both were observed to co-purify with GST-PIF (Fig 2C). The PDKl associated with GST-PIF (0.5μg) was active because in the presence of MgATP and Ptdlns(3,4,5) P 3 it greatly activated a GST-A473D-PKBα mutant (Fig 2C). Thus, PDKl bound to PIF can phosphorylate PKB at Thr308 and the interaction between PIF and the kinase domain of PDKl does not interfere with the activation of PKB. In contrast PKB, p70 S6 kinase, p90 RSK1 or MSK1 , when expressed in 293 cells as GST fusion proteins, were not associated with any endogenous PDKl after their precipitation on glutathione-Sepharose, as judged by immunoblotting and failure to activate GST-S473DPKBα in the presence of MgATP and PtdIns(3,4,5)P 3 (Fig 2C). The "PDK2 substrate motif" mediates the interaction between PIF and PDKl. We have termed the N-terminal 53 amino acids of PIF "Region A" and the C-terminal 24 amino acids "Region B" (Fig IE). In order to identify the amino acid residues in PIF that were required for the interaction of this fragment with the kinase domain of PDKl , we mutated many of the residues in PIF that are conserved between the AGC subfamily of protein kinases to Ala. The mutant GST-PIF proteins were expressed in 293 cells at similar levels, and the amount of endogenous PDKl activity and PDKl protein associated with each of the purified constructs was determined (Fig 3A). Mutation of the conserved residues in Region A did not prevent the association of PDKl with GST-PIF. Remarkably, however, the mutation to Ala of any of the conserved aromatic residues in the PDK2 phosphorylation site motif in Region B (F974, F977 and Y979) completely abolished the association of endogenous PDKl with these GST-PIF mutants (Fig 3A). Furthermore, mutation of Asp978 (the residue that is equivalent to Ser473 in the PKB phosphorylation site) to either Ala or Ser also abolished the association of PDKl with PIF. In contrast, mutation of the residues located 2 and 3 amino acids N-terminal to Asp978 (e.g.Asp976, Arg975), which are not conserved between the AGC subfamily members) did not affect the association of PDKl with PIF (Fig 3A). A yeast 2 hybrid screen was also used to verify these findings (Fig 3B). A strong interaction (Kj 800 nM) between His-PDKl and an immobilised and biotinylated 24 residue synthetic peptide corresponding to the "Region B peptide" was detected using Surface Plasmon Resonance measurements. This interaction was competed by the addition of free peptide, and analysis of this competition revealed that the K^ of this interaction was 1.5 μM. In contrast, a mutant peptide in which the residue corresponding to Asp978 was changed to Ala (termed "D978A-Region B peptide") interacted with PDKl > fold 10-less strongly (Fig 13). Incubation of purified GST-PIF expressed in 293 cells with the Region B peptide (100 μM) resulted in the dissociation of the endogenous PDKl from GST-PIF, whereas a Region B peptide in which Asp978 was mutated to Ala was incapable of inducing the dissociation of PDKl from PIF (data not shown). GST-PIF converts PDKl to a form that is able to phosphorylate Ser473 as well as Thr308 in PKBα. When the purified GST-PIF which is associated with the endogenous PDKl of 293 cells (Fig 2C) was incubated with GST-PKBα in the presence of MgATP and PtdIns(3,4,5)P 3 we noticed that GST-PKBα could be activated to a 3-fold higher maximum specific activity ( " 90 U/mg) than is achievable by incubation of GST- PKBα with very high concentrations of His-PDKl alone ( " 30 U/mg, Fig 4A). In contrast the mutant form of PKBα in which the Ser473 phosphorylation site is mutated to Asp (GST-S473D-PKBα) was activated to the same maximum specific activity by either PDKl of PIF/PDK1 (Fig 4B). These findings raised the possibility that GST-PIF/PDK1 had induced phosphorylation of PKBα at Ser473 as well as Thr308. In order to investigate this possibility we prepared phospho-specific antibodies that only recognise PKB that has been phosphorylated at Ser473 (termed P-473 antibody). This antibody did not recognise GST-PKBα that had been incubated with GST-PIF and MgATP in the absence of PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 , nor did PKBα become phosphorylated under these conditions (Fig 4C). Following the addition of PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 , GST-PKBα became strongly phosphorylated and recognised by the P-473 antibody. Incubation of the P-473 antibody with the phosphorylated Ser473 peptide immunogen used to raise the antibody (but not with the dephosphorylated peptide or a phosphopeptide corresponding to the sequence surrounding Thr308) abolished its recognition of GST- PKBα (Fig 4C). The phosphorylation at Ser473 observed in these experiments was dependent upon the presence of MgATP in the reaction and was abolished if the GST-PIF was heated for 2 min at 65 °C (data not shown). A much higher concentration of His-PDKl that was not bound to PIF induced no phosphorylation of Ser473 in the presence or absence of PtdIns(3,4,5)P 3 . In order to identify all the residue(s) in PKBα phosphorylated by the GST- PIF/PDK1 complex the 32 P-labelled GST-PKBα was digested with trypsin and chromatographed on a C 18 column [14] . Two major 32 P-labelled peptides were observed that eluted at 24% and 26% acetonitrile (Fig 4D). These peptides coeluted with the 32 P-labelled tryptic peptide containing Ser473 and Thr308, respectively [14, 18] and the identity of these peptides and the location of the phosphorylated residues (Thr308 and Ser473) was established by MALDI-TOF mass spectrometry and solid phase amino acid sequencing (data not shown). The peptide eluting at 24% acetonitrile contained phosphoserine, and when subjected to solid phase sequencing, 32 P-radioactivity was released after the eighth cycle of Edman degradation (data not shown). Its identity was established by MALDI-TOF mass spectrometry and revealed that the molecular weight of the peptide (1732.8) was identical to that expected for the tryptic phosphopeptide comprising residues 466-480 and phosphorylated at Ser473. The peptide eluting at 26% acetonitrile contained phosphothreonine and when subjected to solid phase sequencing, 32 P-radioactivity was released after the first cycle of Edman degradation (data not shown). MALDI-TOF mass spectrometry revealed its molecular weight (2573.3) was identical to that expected for the tryptic phosphopeptide comprising residues 308-328 and phosphorylated at Thr308 (calculated mass 2573.1). The PtdIns(3,4,5)P 3 dependent phosphorylation of PKBα at Ser473 is mediated by PDKl. The results presented in Fig 4 could be explained in two different ways. Firstly, the interaction of PIF with PDKl might have allowed the latter to phosphorylate Ser473 as well as Thr308. Secondly, purified GST-PIF might be complexed to two different protein kinases, namely PDKl and PDK2. In order to distinguish between these possibilities we immuno-depleted the PDKl from GST-PIF using 2 different antibodies. We then tested the ability of the supernatant to phosphorylate PKBα at Ser473. Strikingly, both PDKl antibodies completely removed all the Ser473 kinase activity associated with GST- PIF (Fig 5 A) as well as the Thr308 kinase activity (data not shown). A peptide immunogen to which one of the antibodies was raised prevented immuno-depletion of the PDK2 activity associated with PIF (Fig 5A). These results indicated that the PtdIns(3,4,5)P 3 dependent Ser473 kinase activity associated with GST-PIF was catalysed by PDKl rather than a separate enzyme. Consistent with this notion, all the mutant GST-PIF proteins purified from 293 cells that were able to interact with the endogenous PDKl (Fig 3) were able to induce the phosphorylation of PKBα at Ser473 (Fig 5B). In contrast, the point mutations in GST-PIF that abolish its association with PDKl also prevented the phosphorylation of PKBα at Ser473 (Fig 5B). Conversion of PDKl into a kinase that phosphorylates Thr308 and Ser473 of PKBα. It could still be argued that the phosphorylation of Ser473 was catalysed by a protein kinase distinct from PDKl , but that this kinase was tightly complexed to PDKl and not dissociated by chaotrophic reagents, detergents and therefore also removed from GST-PIF by immuno-depletion of PDKl . In order to address this point we removed PDKl from purified GST-PIF by immuno-depletion and added the PDK1- depleted PIF (termed GST-PIF*) to purified wild type GST-PDKl (Fig 14) or His-PDKl (data not shown). Initial rate studies demonstrated that, under the conditions used, the GST-PIF*/PDK1 complex phosphorylates Thr308 of PKBα at twice the rate of Ser473 (data not shown). Moreover, the ability of GST-PIF* to convert PDKl into a Ser473 PKBα kinase was dependent upon PDKl catalytic activity, since a catalytically inactive GST-PDKl mutant was not capable of phosphorylating PKBα at Ser473 in the presence of GST-PIF* (Figure 13). The above experiments do not exclude the possibility that another protein associated with PIF (rather than PIF itself) is capable of interacting with PDKl and altering its substrate specificity. This possibility was eliminated by the demonstration that a 24 residue synthetic peptide corresponding to "Region B" of PIF was capable of inducing His-PDKl to phosphorylate PKBα at Ser473 in a 3-phosphoinositide dependent manner (Fig 6). In contrast, a Region B peptide in which Asp978 was mutated to Ala was far less effective at inducing this alteration in the specificity of PDKl . The region B peptide converts the wild type GST-PDKl, but not the kinase dead GST-PDKl , into a kinase which can phosphorylate Ser473 (data not shown). Lipid specificity of Ser473 phosphorylation. We compared the ability of a panel of Ptdlns derivatives to stimulate the phosphorylation of Thr308 and Ser473 by the GST-PIF/PDKI complex in the presence of a vesicle background containing PtdCho/PtdSer [17]. Synthetic 5/2-l-stearoyl, 2- arachidonoyl D-PtdIns(3,4,5,)P 3 (l ,2-SAD-PtdIns(3,4,5)P 3 the predominant form of Ptdlns (3, 4, 5 )P 3 which occurs naturally, lipid 8 in Fig 7) and synthetic 2, 3-SAD-PtdIns(3,4,5,)P 3 (lipid 10 in Fig 7) were highly effective in inducing both the phosphorylation of GST-PKBα at Ser473 (Fig 7B) and the phosphorylation of Thr308 (measured by the activation of GST-S473D-PKBα), indicating that the enantiomeric configuration of the glycerol moiety is not an important determinant of specificity. The L- enantiomers of these lipids (Lipids 9 and 11 , Fig 7) failed to induce a significant phosphorylation of PKB at Ser473 or activation of GST-S473D- PKBα. rac-l ,2-dilinoleoyl phosphatidyl D/L-myo-inositol 3,4,5- trisphosphate (linoleic acid is C18:2, Lipid 7 Fig 7), sn- 1 ,2-dipalmitoyl D- PtdIns(3,4,5)P 3 , (Lipid 6, Fig 7) as well as sn- 1 ,2-dipalmitoyl PtdIns(3,4)P 2 (Lipid 3 Fig 7) were also effective at inducing the PIF/PDKl complex to phosphorylate PKBα at Ser473 and Thr308. However, Ptdlns-3P (lipid 2, Fig 7), PtdIns(3,5)P 2 (lipid 4 Fig 7) and PtdIns(4,5)P 2 (lipid 5 Fig 7), did not induce the PIF/PDKl complex to phosphorylate PKBα at Ser473 or Thr308. In the absence of the PIF/PDKl complex none of the Ptdlns derivatives tested induced any phosphorylation of Ser473 or activation of GST-S473D-PKBα (data not shown) . Maximal activation of PKB by PDK1/PIF requires PtdIns(3,4,5)P 3 to interact with PDKl. Although PDKl binds to PtdIns(3,4,5)P 3 with high affinity through its PH domain, this interaction does not influence the ability to activate a PKB mutant lacking the PH domain [18,29]. This is confirmed in Fig 8 which shows that the rate at which His-PDKl phosphorylates and activates a mutant form of PKB lacking the PH domain (GST-ΔPH-PKBα) is the same in the presence or absence of PtdIns(3,4,5)P 3 (Fig 8A). Intriguingly, the addition of GST-PIF* to His- PDKl halves the rate at which PDKl activates GST-ΔPH-PKBα in the absence of PtdIns(3,4,5)P 3 . However the addition of PtdIns(3,4,5)P 3 results in a 3 to 4-fold enhancement of the rate at which His-PDKl /GST- PIF* can activate GST-ΔPH-PKBα (Fig 8A). Furthermore, the GST- PIF/PDKI complex purified from 293 cells also activates GST-ΔPH- PKBα at a " 4-fold higher rate in the presence of PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 than in its absence. PtdIns(4,5)P 2 or PtdIns(3)P do not increase the rate at which the GST-PIF/PDKI complex phosphorylates GST-ΔPH-PKBα (Fig 8B). Identification of a Ptdlns (3, 4, 5) P 3 dependent Ser473 kinase activity as PDKl. We fractionated rat brain extracts by batchwise chromatography on Q-Sepharose, followed by gradient elution from Heparin-Sepharose (see methods). PDKl (assayed as a PtdIns(3,4,5)P 3 dependent enzyme that activates GST-S473D-PKBα) was eluted from Heparin-Sepharose as a broad peak at 0.75M NaCl (Fig 9A, upper panel). A PtdIns(3,4,5)P 3 dependent PDK2 activity that phosphorylated PKBα at Ser473 was also identified in the Heparin-Sepharose eluate and was present in the tailing half of the PDKl activity (Fig 9, lower panel). The peak containing the PDK2 activity (fractions 7 and 8) was pooled, PDKl removed by immunoprecipitation and the supernatant reassayed for PDKl and PDK2 activity. Strikingly, the antibody completely removed not only the PDKl (Thr308 kinase) activity but also the PDK2 (Ser473 kinase) activity (Fig 9B). This result indicates that a fraction of the PDKl in the Heparin- Sepharose eluate possesses PDK2 activity. Discussion PDKl has been shown to phosphorylate and activate several of the AGC subfamily of protein kinases in a conserved motif between subdomains VII and VIII of the kinase catalytic domain ([3, 13] see introduction). Most of the PDKl substrates identified to date possess a PDK2 consensus motif " 160 residues C-terminal to the PDKl phosphorylation site, apart from PKA whose sequence actually terminates at the second Phe of the PDK2 phosphorylation motif (see Fig IE). In PKA it is possible that the C- terminal carboxylate group may mimic the effect of phosphorylation at the PDK2 site, thereby generating a fully active enzyme. Phosphorylation of both the PDKl and PDK2 sites is required for maximal activation of PKB and p70 S6 kinase and/or stability for PKC isoforms [30] . In this Example, we demonstrate that the kinase domain of PDKl is capable of interacting specifically and with high affinity with a modified PDK2 phosphorylation consensus motif present at the C-terminus of PRK2, in which the phosphorylated Ser/Thr residue is replaced by Asp (Asp 978) instead of a Ser/Thr residue at the site of phosphorylation. The Asp residue may mimick a phosphorylated PDK2 motif. Mutation of any of the conserved aromatic residues in the PDK2 consensus sequence to Ala or mutation of Asp978 to Ala or Ser completely abolished the interaction of PDKl with PIF (Fig 3). PRK2 itself is likely to be a substrate for PDKl as it possesses a perfect PDKl consensus sequence between sub-domain VII and VIII of the kinase domain (TFCGTPEFL, where the underlined Thr residue corresponds to Thr816, the putative site of PDKl phosphorylation). In the case of PRK2, it is possible that the role of the PIF domain is to interact directly with PDKl ensuring that Thr816 is phosphorylated efficiently by PDKl . As PRK2 [28] (and its close relative PRK1[31 ,32] ) may be dependent on Rho for activity, it is possible that the interaction between PRK2 and Rho complexed to GTP is also required for the phosphorylation of PRK2 by PDKl to take place. One scenario is that the C-terminus of PRK2 is exposed upon the formation of a complex between PRK2 and Rho-GTP. This may then enable interaction with and phosphorylation by PDKl leading to the activation of PRK2. However, PRK2 is known to become proteolysed during apoptosis as a result of cleavage by caspase-3 immediately C-terminal to Aspl l7 and Asp700 [33] . PRK2 is highly susceptible to proteolysis when purified or overexpressed in cells [34] . Therefore, a possibility is that a C-terminal fragment of PRK2 devoid of any catalytic activity, is present in cells that interacts with PDKl . It is also possible that a fragment of PRK2 may be generated by an alternative splicing mechanism or the use of alternative promoters. Future work will need to address whether PDKl is complexed with PRK2 or fragments of this enzyme in intact cells. SGK and the p70 S6 kinase are far better substrates for PDKl if the PDK2 phosphorylation site (Thr389) is changed to an acidic residue, for example Glu [24,25]. Thus PDKl may be able to interact directly with SGK or the p70S6K when the PDK2 site is phosphorylated enabling PDKl to phosphorylate these enzymes at the PDKl site in the activation loop. PDKl complexed to another protein may be capable of inducing phosphorylation of the PDK2 site of these enzymes. PKCς which is also activated by PDKl [21 ,22] , possesses an acidic residue (Glu 579) rather than Ser/Thr in its PDK2 consensus motif (FEGFEY). Furthermore, PKCς, like PIF, may interact directly with the kinase domain of PDKl . Activation of PKBα in cells is mediated by phosphorylation of both Thr308 and Ser473. Phosphorylation of either residue only partially activates PKBα and the full activation requires the phosphorylation of Ser473 as well as Thr308 [14] . A catalytically inactive mutant of PKBα becomes phosphorylated at Ser473 in response to insulin, while PKBα phosphorylated at Thr308 alone or in which Thr308 is mutated to Asp does not become phophorylated at Ser473 in vitro upon incubation with MgATP in the presence of PtdIns(3,4,5)P 3 [14, 17]. These observations indicate that phosphorylation of PKBα at Ser473 is unlikely to be an autophosphorylation event, catalysed by PKBα itself. We have reported previously [14] that Ser473 is phosphorylated in vitro by MAP kinase- activated protein kinase-2 [14], but this enzyme does not mediate the phosphorylation of Ser473 in vivo in response to insulin, IGF1 , heat shock or hydrogen peroxide [35] . Furthermore, the phosphorylation of PKBα at Ser473 by MAPKAPK-2 in vitro is not dependent upon the presence of PtdIns(3,4,5)P 3 [14]. It has also been claimed that the integrin-linked kinase (ILK) is capable of phosphorylating Ser473 of PKBα in vitro [36]. We have not been able to demonstrate that ILK phosphorylates Ser473 of PKBα, and it is also uncertain as to whether ILK is actually a protein kinase as it lacks certain motifs found in every other protein kinase. It has also been claimed that mTOR can phosphorylate the PDK2 site of p70S6 kinase [37]; however, we were unable to reproduce this finding and furthermore were unable to phosphorylate PKBα at Ser473 with mTOR. Since the insulin/IGF 1 -induced phosphorylation of Ser473, like that of Thr308, is prevented by inhibitors of PI 3-kinase, it is likely that phosphorylation of Ser473 is also catalysed by a PtdIns(3,4,5)P 3 dependent protein kinase. In this smdy we have made the surprising observation that the interaction of PIF with the kinase domain of PDKl converts it from an enzyme that only phosphorylates PKBα at Thr308 to a form that phosphorylates both Thr308 and Ser473 in a PtdIns(3,4,5)P 3 and PtdIns(3,4,)P 2 dependent manner. The requirement for PtdIns(3,4,5)P 3 or PtdIns(3,4,)P 2 is extremely specific, because only the D-enantiomers of PtdIns(3,4,5)P 3 are effective, and many other Ptdlns phospholipids including PtdIns(4,5)P 2 are ineffective. The residues surrounding Thr308 and Ser473 are distinct, the only common feature being an aromatic residue at the residue immediately C-terminal to the phosphorylation site. This suggests that PDKl possesses two distinct specificities when complexed to PIF. If a mutant of PKBα lacking the PH domain (which cannot interact with PtdIns(3,4,5)P 3 ) is expressed in cells, it nevertheless undergoes a wortmannin-sensitive activation and phosphorylation at Thr308 and Ser473 [38]. This implies that PDKl is activated by PtdIns(3,4,5)P 3 in vivo. However, PDKl isolated from either control or insulin stimulated cells possessed similar activity towards GST-ΔPH-PKBα [18,25]. Furthermore, in vitro experiments demonstrated that despite binding PtdIns(3,4,5)P 3 /PtdIns(3,4)P 2 through its PH domain [20, 39], these 3- phosphoinositides have no influence on the rate at which PDKl phosphorylates ΔPH-PKBα, p70S6 kinase or SGK substrates which do not interact with 3-phosphoinositides [3, 43, 44]. Importantly, the interaction with PIF confers on PDKl the ability to be activated by PtdIns(3,4,5)P 3 /PtdIns(3,4)P 2 in vitro (Fig 8). This does not happen if the PH domain of PDKl is deleted (data not shown). Thus PDKl complexed to PIF activates GST-ΔPH-PKBα at a " 3 to 4-fold higher rate in the presence of PtdIns(3,4,5)P 3 than in its absence. Our data suggest that the interaction of PDKl with PIF leads to a " 2-fold inhibition of the catalytic activity of PDKl which is relieved and further activated in the presence of PtdIns(3,4,5)P 3 . The results presented in this paper raise the strong possibility that PDKl itself may be the enzyme responsible for mediating the phosphorylation of PKBα at both Thr308 and Ser473. It is possible that PRK2 (or a proteolytic fragment of PRK2) or a related peptide/protein(s) may interact with the kinase domain of PDKl , converting it into an enzyme that can phosphorylate both Ser473 and Thr308 of PKBα, as well as permitting the direct activation of PDKl by PtdIns(3,4,5)P 3 . Consistent with this hypothesis we have identified a PtdIns(3,4,5)P 3 dependent Ser473 kinase activity that is immunoprecipitated with a PDKl-specific antibody (Fig 9). This form of PDKl may be complexed to PRK2 or its C-terminal fragment or to another peptide/protein with similar properties. The overexpression of PDKl in cells has been shown to potentiate the phosphorylation of PKBα at Thr308 only [18], which might be explained if the putative PDKl regulatory subunits are present in limiting amounts. In summary, the present work provides the first evidence that PDKl and PDK2 may be the same enzyme and that its specificity for its two different phosphorylation substrate sites may be modulated by the interaction with one or more proteins. Our results also suggest that when PDKl interacts with the PH domain of PDKl with Ptdlns (3,4,5)P 3 /PtdIns(3,4)P 2 . Materials and Methods Materials. Peptides were synthesised by Dr G. Blomberg (University of Bristol, U.K.). Protein G-Sepharose, glutathione Sepharose and CHX- Sepharose were purchased from Pharmacia (Milton Keynes, UK), alkylated trypsin from Promega (Southampton, UK), tissue cultore reagents, microcystin-LR, Life Technologies Inc. (Paisley, UK), the pCR 2.1-TOPO cloning vector from Invitrogen (Leek, Netherlands), sensorChips CM5 and SA were from BiaCore AB and biotinylated reagent was from Pierce. The yeast two hybrid human skeletal muscle (in pGADIO vector) and the pAS2-l vector were purchased from Clontech (Basingstoke, UK). Expression constructs for GST-PKBα [17], GST- PDKl , Myc-PDKl and other mutants of PDKl [18] , GST-90 RSK1 [39], GST-MSK1 [39] and GST-p70 S6 kinase lacking the C-terminal 104 residues [24] were generated as described previously. Synthesis of 1-[(1- O-Stearoyl-2-0-arachidonoyl-sn-glycer-3-yl)-phosphoryl]-D-myo-inositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] and its stereoisomers is described in [40] , all lipids were obtained from sources described previously [17]. All the experiments using 3-phosphoinositides were carried out in the presence of phospholipids containing lOOμM Phosphatidylcholine and lOOμM Phosphatidylserine . Antibodies. The PDKl whole protein antibody was raised in sheep against GST-PDKl expressed and purified from 293 cells. The antibody was affinity-purified on CH-Sepharose columns to which His-PDKl had been covalently coupled. The peptide PDKl antibody was raised in sheep against the peptide RQRYQSHPDAAVQ (corresponding to residues 544 to 556 of PDKl) and affinity-purified on CH-Sepharose columns to which this peptide was coupled covalently. The phospho-specific antibody recognising PKB phosphorylated at Ser 473 was raised against the peptide PHFPQFSYSAS in which the underlined serine is phosphorylated (corresponding to residues 467 to 477 of PKBα) and affinity purified on CH-Sephaose covalently coupled to the phosphorylated peptides. The antibodies were then passed through a column coupled to the non- phosphorylated peptide and the antibodies that did not bind to this column were selected. The above antibodies are available commercially from UBI (Lake Placid, USA). A monoclonal antibody recognising the Myc epitope was purchased from Boehringer Mannheim (Lewes, UK). Buffer solutions. Buffer A - 50 mM Tris-HCl pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 % (by mass) Triton-X 100, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M sucrose, 1 μM microcystin-LR, 0.1 % (by vol) β-mercaptoethanol and 'complete' proteinase inhibitor cocktail (one tablet per 50 ml; Boehringer Mannheim, Lewes, UK). Buffer B - 50 mM Tris/HCl pH 7.5, 0.1 mM EGTA, 10 mM β-mercaptoethanol. Buffer C - 50 mM Tris/HCl pH 7.5, ImM EDTA, 1 mM EGTA, 0.27M sucrose and 0.1 % (by vol) β- mercaptoethanol. General methods. E. coli cells (typically DH5 ) were grown at 37°C in Luria-Bertani medium containing (when needed) 50 μg/ml ampicillin for plasmid selection. Restriction enzyme digests, DNA ligations and other recombinant DNA procedures were performed as using standard protocols. Transformation of bacterial cells was achieved using electroporation. Site-directed mutagenesis was performed using the QuikChange Kit (Strategene) following instructions provided by the manufacturer. All DNA constructs were verified by automatic DNA sequencing using an automated DNA Sequencer (Model 373; Applied Biosy stems). DNA constructs were used in this study were purified from bacteria using Qiagen plasmid Mega kit according to the manufacturer's protocol. 293 cells were cultured to confluence on 10 cm diameter dishes in Dulbecco's Modified Eagle's Medium containing 10% (by vol) foetal bovine serum. Ptd/Cho and Ptd/Ser phospholipid vesicles containing the indicated phosphoinositide lipids were prepared as described previously [17]. Yeast two hybrid screen. Myc-tagged human PDKl , kinase domain of PDKl (residues 1-404) and the PH domain of PDKl (428 to 556) were subcloned into the EcoRI/Sα/I of the yeast pAS2-l vector. A yeast two hybrid screen was carried out using a human skeletal muscle library subcloned into the pGADIO vector (Clontech) transformed into the yeast strain Y166. 4 x 10 6 yeast cells were transformed with pGADIO library constructs and PDKl constructs in the pAS2-l vector. In order to select positive colonies these yeast were plated onto synthetic complete (SC) media lacking tryptophan, leucine, histidine and uracil (SC-Trp-Leu-His-Ura), containing 25 mM 3-amino- 1 ,2,4-triazole (3-AT) (Sigma). In this system interacting clones from the library are able to grow in medium lacking histidine and uracil and express β-galactosidase which was detected using the Clontech protocol. pGADIO plasmids from interacting clones were isolated transferred to E. coli, and sequenced. One positive clone termed by us TH110, contained a 2.4 kbp fragment which possessed in the 5' region a 25 nucleotide sequence from a rRNA, followed by the 77 C-terminal amino acids of PRK2 (termed PIF) in frame with the GAL4 protein. In order to eliminate the 25 nucleotide rRNA sequence from this construct, a PCR reaction was carried out using, as a template, the TH110 clone and the oligonucleotides 5 '-CGGGATCCGAGGATGTAAAAAAGCACCC-3 '(incorporating a BamRl site which is underlined) and an oligonucleotide corresponding to the polylinker region at the 3' end of the pGADIO vector. The resulting fragment contains an internal BamRl site and the PCR product was digested with BamHl to obtain a 1.2 kb fragment encoding PIF which was ligated into the pGADIO plasmid. This construct was used in the experiments in Fig 1 to demonstrate that PIF interacts with the kinase domain of PDKl in the yeast two hybrid system. Preparation of mammalian GST-PIF expression constructs. A ligation was set up to generate a GST-PIF expression construct by subcloning the PIF cDNA from the pGADIO vector as the BamRl fragment into the BamRl site of the pEBG2T [41] expression vector. Expression of His-PDKl in insect cells. The cDNA for Myc-PDKl was subcloned from pEBG2T vector [41] as a BglRl/Kpnl fragment into pFASTBAC HTb vector and this vector was used to generate recombinant baculovirus using the Bac-to-Bac system (Life Technologies, Paisley, UK). The resulting virus encodes Myc-PDKl with an N-temiinal hexahistidine sequence, and was used to infect Sf21 cells (1.5 x 10 6 /ml) at a multiplicity of infection of 5. The infected cells were harvested 72 h post-infection and the His-PDKl purified by Ni 2+ /NTA-agarose chromatography as described previously for His-PKBβ [42], and dialysed into Buffer B containing 0.27 M sucrose, 0.03 % (by vol) Brij-35, 1 mM benzamidine and 0. 2 mM phenylmethylsulphonyl fluoride. The PDKl (4 mg/ml) was snap frozen in aliquots and stored at -80°C. Purified His- PDKl was recovered with a yield of 4 mg/ litre of infected Sf21 cells and was > 90% homogeneous as judged by polyacrylamide gel electrophoresis followed by Coomassie Blue staining. Expression of GST-fusion proteins in 293 cells. Up to twenty 10 cm diameter dishes of 293 cells were cultured and each dish transfected with 20 μg of pEBG2T DNA encoding the desired GST-fusion protein using a modified calcium phosphate method [14]. In experiments described in Fig 1 in which Myc-PDKl constructs (pCMV5 vector) were coexpressed with GST-PIF (pEBG2T vector) or GST alone (empty pEBG2T vector) 10 μg of each construct was used. 36 h after transfection the cells were lysed in 1 ml of ice-cold Buffer A without any serum starvation. The ly sates were pooled, centrifuged at 4°C for 10 min at 13, 000 x g and the supernatant incubated for 60 min on a rotating platform with 1 ml of glutathione- Sepharose (for a 20 dishes experiment) previously equilibrated in Buffer A. The suspension was centrifuged for 1 min at 3000 x g, the beads washed three times with 10 ml of Buffer A containing 0.5 M NaCl, and then a further ten times with 10 ml of Buffer B containing 0.27 M sucrose. GST-PIF was eluted from the resin at ambient temperature with three volumes (equivalent to the GST-Sepharose volume) of Buffer B containing 20 mM glutathione and 0.27 M Sucrose. The combined eluates were divided into aliquots, snap frozen in liquid nitrogen, and stored at - 80°C. Measurement of PKBα activation after incubation with GST-PIF. This assay was carried out in 2 stages, as described previously [17]; briefly, in the first, GST-PKBα was incubated with GST-PIF (or other indicated PDKl proteins in the presence or absence of peptides) in the presence of MgATP and phospholipid vesicles. In the second stage, the solution was made 0.5 % (by vol) in Triton XI 00 (which completely inhibits phosphorylation of GST-PKBα without affecting GST-PKBα activity [17]) together with Mg[γ 32 P]ATP, and the specific PKBα substrate peptide substrate RPRTAAF [43]. In Stage 1 , an 15 μl reaction mixture was set up containing 66.5 mM Tris/HCl pH 7.5, 0.13 mM EGTA, 0.13 % (by vol) 2-mercaptoethanol, 3.3 μM PKI, 1.3 μM microcystin-LR, 13.3 mM Mg(Ac) 2 , 133 μM unlabelled ATP, 0.4 μM GST-PKBα, 133 μM PtdSer, 133 μM PtdCho in the presence or absence of 13.3 μM PtdIns(3,4,5)P 3 . The assay was initiated by the addition of 5 μl of 0.2 mg/ml GST-PIF (or other indicated proteins or peptides), after incubation for 30 min at 30°C, stage 2 of the assay was initiated by the addition of 30 μl of a mixture made up of Buffer B containing 2.5 μM PKI, 1 μM microcystin-LR, 10 mM Mg(Ac) 2 , 100 μM [γ 32 P]ATP (200-400 cpm/pmol), 100 μM of the peptide RPRTAAF and 1.25 % (by vol) Triton X-100. After 10 min at 30°C, the reactions were terminated by spotting the reaction mixture on to P81 phosphocellulose paper. The papers were washed in 75 mM phosphoric acid and analysed as described previously [44] . A control reaction in which GST-PKBα was omitted was taken as the blank and was always less than 5 % of the activity measured in the presence of GST-PKBα. The basal GST-PKBα activity is the activity measured in the absence of PDKl . 1 Unit of GST-PKBα activity was that amount of enzyme required to catalyse the phosphorylation of 1 nmol of the peptide RPRTAAF in 1 min. The assays were linear with time up to a final concentration of 3 U/ml of PDKl activity in the assay. Phosphorylation of GST-PKBα by incubation with GST-PIF. In experiments in which the phosphorylation of PKBα was determined using the phospho-specific Ser473 PKBα antibodies, the incubations were identical to the Stage 1 described above except that the reactions were terminated by the addition of 1 % (by mass) SDS and immunoblotting is canied out as described below. In experiments in which the sites of phosphorylation on PKBα are determined the incubations were identical to the Stage 1 described above except [γ 32 P] ATP (1000-2000 cpm/pmol) was used instead of unlabelled ATP. The reactions were stopped by the addition of SDS and 2-mercaptoethanol to final concentrations of 1 % (by mass) and 1 % (by vol) respectively, and heated for 5 min at 95°C. After cooling to ambient temperature, 4-vinylpyridine was added to a concentration of 2.5 % (by vol) and the sample left on a shaking platform for 1 h at 30°C to alkylate cysteine residues. The sample was then electrophoresed on a 7.5 % SDS poiyacrylamide gel, the 32 P-labelled GST- PKBα was eluted from the gel and digested with alkylated trypsin as described previously [14, 17] . Immunoblotting for PKBα phosphorylated at Ser473. Reaction mixtures containing GST-PKBα (0.2 μg) were subjected to SDS/polyacylamide gel electrophoresis and subsequently transferred to nitrocellulose and immunoblotted using the phospho-specific antibody recognizing PKBα phosphorylated on Ser 473 at a concentration of antibody of 0. 1 mg/ml in 50 mM Tris pH7.5, 0.15M NaCl, 0.5 % (by vol) Tween and 10 % (by mass) skimmed milk. Detection of phosphorylated PKBα was performed using the enhanced chemiluminescence reagent (Amersham). BiaCore measurements. GST-PIF was coupled through amine groups to the SensorChip CM5 according to the manufacturer's instructions. His- PDKl (0-2.0 μM) was injected over this surface and steady state binding determined. The 24 residue peptide overlapping Region B was biotinylated through its C-terminal Cys and bound to Avidin coated SensorChip SA. His-PDKl (0.5 μM) was mixed with region B peptide (0-3 μM) and the PDKl /peptide mixtures injected over the immobilised peptides. The decrease in steady state binding was used to determine the Kd of interaction between PDKl and the peptide, according to the Cheng- Prescott relationship. The measure of response in our experiments is termed RU; 1000 RU = 1 ng/mm 2 of protein bound to the surface. Partial purification of PDK2 from rat brain. Ten rats were killed with C0 2 , the brains rapidly extracted and homogenised in 50ml of ice-cold Buffer A. 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Claims: CLAIMS 1. A method of altering the substrate specificity of phosphoinositide- dependent protein kinase 1 (PDKl) wherein the said PDKl is exposed to an interacting polypeptide which comprises the amino acid sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr wherein Zaa represents a negatively charged amino acid residue. 2. A preparation comprising PDKl and an interacting polypeptide which comprises the amino acid sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa- Phe/Tyr wherein Zaa represents a negatively charged amino acid residue characterised in that the said preparation is substantially free of polypeptides with which PDKl is present or associated in a cell in which it is naturally found other than a said interacting polypeptide or a substrate for PDKl . 3. PDKl derivable by the method of claim 1 wherein the said PDKl has altered substrate specificity and is substantially free of polypeptides with which PDKl is present or associated in a cell in which it is naturally found other than a said interacting polypeptide or a substrate for PDKl . 4. A method of phosphorylating a residue corresponding to the underlined residue in a substrate polypeptide with an amino acid sequence corresponding to the consensus sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr- Ser/Thr-Phe/Tyr wherein (1) a preparation comprising PDKl and a polypeptide which comprises the amino acid sequence Phe/Tyr-Xaa-Xaa- Phe/Tyr-Zaa-Phe/Tyr or (2) PDKl derivable by a method of altering the substrate specificity of phosphoinositide-dependent protein kinase 1 (PDKl) wherein the said PDKl is exposed to a polypeptide which comprises the amino acid sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa- Phe/Tyr is used wherein Zaa represents a negatively charged amino acid residue. 5. A method of phosphorylating PRK2 wherein the said PRK2 is exposed to PDKl . 6. A preparation comprising PDKl and PRK2 in the substantial absence of other proteins or cellular components. 7. A method of identifying a compound that modulates the activation and/or phosphorylation of PRK2 by PDKl wherein the activation and/or phosphorylation of PRK2 by PDKl is measured in the presence of more than one concentration of the compound. 8. A method of identifying a compound that modulates the activity of PDKl wherein the said PDKl is exposed to the said compound in the presence of a polypeptide comprising the amino acid sequence Phe/Tyr- Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr wherein Zaa represents a negatively charged amino acid residue. 9. A method according to claim 8 comprising the step of measuring the activity of the said PDKl in the presence of more than one concentration of the compound wherein the said PDKl is or has been exposed to a polypeptide which comprises the amino acid sequence Phe/Tyr-Xaa-Xaa- Phe/Tyr-Zaa-Phe/Tyr . 10. A method according to claim 8 or 9 wherein the said compound is capable of modulating the interaction between a polypeptide which comprises the amino acid sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa- Phe/Tyr and PDKl . 11. A method of identifying a compound that is capable of altering the substrate specificity of PDKl wherein the ability of the said PDKl to phosphorylate a residue corresponding to the underlined residue in a polypeptide with an amino acid sequence conesponding to the consensus sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Ser/Thr-Phe/Tyr is measured, and is increased in the presence of the said compound 12. A method of altering the substrate specificity of phosphoinositide- dependent protein kinase 1 (PDKl) wherein the said PDKl is exposed to a compound identified or identifiable by the method of claim 11. 13. A method of identifying a compound that is capable of mimicking the effect of a 3-phosphoinositide, for example PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 , on the PDKl or PDK2 activity of (1) a PDKl which has altered substrate specificity derivable by the method of claim 1 or 12 or (2) a preparation according to claim 2, 24 or 25, the method comprising determining whether said compound activates a said PDKl or preparation so that it can phosphorylate a suitable substrate, the activation by said compound being in the absence of a 3-phosphoinositide. 14. A protein kinase derivable from mammalian brain wherein said protein kinase is capable of phosphorylating a residue corresponding to the underlined residue in a polypeptide with an amino acid sequence corresponding to the consensus sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr- Ser/Thr-Phe/Tyr, for example Ser473 of PKBα in the presence of PtdIns(3,4,5)P 3 , wherein the said protein kinase is eluted from Heparin- Sepharose by at least 0.75M NaCl at pH 7.5 and is capable of binding to an antibody reactive with PDKl . 15. A polypeptide which comprises the amino acid sequence Phe/Tyr- Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr wherein said polypeptide is not full-length PRK2, PRK1 or PKCς and wherein Zaa is a negatively charged amino acid that is not phosphoserine or phosphothreonine. 16. A polypeptide consisting essentially of residues 51 to 404 of PDKl or a fusion of a polypeptide consisting essentially of residues 51 to 404 of PDKl . 17. A polynucleotide encoding a polypeptide as defined in claim 15 or 16. 18. A recombinant polynucleotide suitable for expressing a polypeptide as defined in claim 15 or 16. 19. A host cell comprising a polynucleotide as defined in claim 18. 20. A method of making a polypeptide as defined in claim 15 or 16 the method comprising culturing a host cell as defined in Claim 19 which expresses said polypeptide and isolating said polypeptide. 21. A polypeptide obtainable by the method of Claim 20. 22. A cell containing a recombinant nucleic acid suitable for expressing PDKl and a recombinant nucleic acid suitable for expressing a polypeptide comprising the amino acid sequence Phe/Tyr-Xaa-Xaa- Phe/Tyr-Zaa-Phe/Tyr. 23. A method according to claim 1 wherein the said PDKl is exposed to the said interacting polypeptide in a cell as defined in claim 22. 24. A method of making a preparation comprising PDKl and an interacting polypeptide comprising the amino acid sequence Phe/Tyr-Xaa- Xaa-Phe/Tyr-Zaa-Phe/Tyr wherein PDKl and the said interacting polypeptide are co-expressed in a cell as defined in claim 22. 25. A method according to claim 24 wherein the said PDKl and the said interacting polypeptide are separated from other components of the said cell. 26. A preparation obtainable by the method of claim 24 or 25. 27. A compound identifiable or identified by the method of any one of claims 8 to 13. 28. A compound as defined in claim 27 for use in medicine. 29. Use of a compound as defined in claim 27 in the manufacture of a medicament for the treatment of a patient in need of modulation of the insulin signalling pathway and/or PDKl , PDK2 or PRK2 signalling. 30. A polypeptide which comprises the amino acid sequence Phe/Tyr- Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr or a polypeptide consisting essentially of residues 51 to 404 of PDKl or a fusion of a polypeptide consisting essentially of residues 51 to 404 of PDKl wherein Zaa represents a negatively charged amino acid residue for use in medicine. 31. Use of a polypeptide as defined in claim 30 in the manufacture of a medicament for the treatment of a patient in need of modulation of the insulin signalling pathway and/or PDKl , PDK2 or PRK2 signalling. 32. A kit of parts useful in carrying out a screening method according to any one of claims 8 to 13 wherein the kit comprises PDKl and a polypeptide comprising the amino acid sequence Phe/Tyr-Xaa-Xaa- Phe/Tyr-Zaa-Phe/Tyr wherein Zaa represents a negatively charged amino acid residue. 33. A method of making a preparation that comprises PDKl and a polypeptide which comprises the amino acid sequence Phe/Tyr-Xaa-Xaa- Phe/Tyr-Zaa-Phe/Tyr wherein substantially pure PDKl is mixed with a substantially pure polypeptide which comprises the amino acid sequence Phe/Tyr-Xaa-Xaa-Phe/Tyr-Zaa-Phe/Tyr.

Inventors: ALESSI DARIO, BALENDRAN ANUDHARAN, CASAMAYOR ANTONIO, CURRIE RICHARD, DEAK MARIA, DOWNES PETER, ALESSI, DARIO, BALENDRAN, ANUDHARAN, CASAMAYOR, ANTONIO, CURRIE, RICHARD, DEAK, MARIA, DOWNES, PETER
Applicants: ALESSI DARIO, BALENDRAN ANUDHARAN, CASAMAYOR ANTONIO, CURRIE RICHARD, DEAK MARIA, DOWNES PETER, UNIV DUNDEE, ALESSI, DARIO, BALENDRAN, ANUDHARAN, CASAMAYOR, ANTONIO, CURRIE, RICHARD, DEAK, MARIA, DOWNES, PETER, UNIVERSITY OF DUNDEE
Application year: 2000
Main classification: C12N9
IPC codes: A61K38, A61K45, A61P5, A61P43, C12N1, C12N5, C12N9, C12N15, C12Q1, G01N33