BACKGROUND
the phosphorylation of proteins is one of the most prevalent covalent modifications known, affecting essentially every aspect of cellular function  <cit> .
the protein kinases and phosphatases responsible are highly conserved across species and, with few exceptions, the kinases belong to one large gene family while the phosphatase complement is more complex and can be divided into three broad groups based on protein sequence, catalytic signature and substrate preference  <cit> .
the action of protein phosphatases is tightly controlled with cellular targeting being an important means of regulation.
most phospho-serine and threonine dephosphorylation can be attributed to the ppm family and the more diverse ppp family, which includes pp <dig>  pp2a, pp2b, and pp <dig> through to pp <dig>  pp <dig> is thought to not exist as a free catalytic subunit in the cell, but to reside in complexes with a large array of targeting or regulatory subunits that define its function.
numerous pp <dig> docking proteins have been identified, but they most likely represent only a small fraction of the total number in the cell.
the microcystins are a group of cyclic peptides that bind with remarkable specificity and affinity to the type one, 2a and several recently identified protein phosphatases of the ppp family .
microcystin covalently couples to a conserved cysteine residue of ppp family members through its methyl-dehydroalanine residue  <cit> .
nishiwaki et al  <cit>  first used microcystin-sepharose to purify pp2a.
we exploited a different synthetic approach whereby the carbon-carbon double bond of methyl-dehydroalanine in microcystin couples the latter to aminoethanethiol, which is then linked to a sepharose bead.
this generates a high affinity binding matrix for the microcystin-sensitive protein phosphatases that does not covalently couple the phosphatase  <cit> .
microcystin-sepharose has proved to be a powerful tool to purify these protein phosphatases and their associated regulatory subunits from a variety of tissues and cell types  <cit> .
with only a few characterized exceptions, pp <dig> interacting proteins bind pp <dig> through their primary docking sequence called the rvxf/w motif  <cit> .
their molecular interaction with pp <dig> has been visualized via pp1-peptide and pp <dig> regulatory subunit structures  <cit> .
it has also emerged that additional or secondary interaction sites often play a role in binding pp <dig> and likely contribute to pp <dig> isoform specificity recognition, substrate docking and modulation of pp <dig> activity  <cit> .
based on a compilation of demonstrated rvxf/w interaction motifs  <cit> , the panning of a random peptide library  <cit> , and mutagenesis and modeling studies  <cit>  we noted preferences for particular amino acids within and adjacent to the rvxf/w motif.
this led us to speculate that a pp <dig> interaction motif peptide, based on this comparison, could be a unique means to specifically disrupt pp1-targetting or regulatory subunit interactions  <cit> .
slight variation in the rvxf/w-motif combined with the now recognized additional, secondary pp <dig> interaction sites provide sufficient interaction specificity which may allow the development of drugs or peptide mimetics to abolish specific pp <dig> binding protein interactions in vivo.
the idea of targeting protein-protein interaction domains with drugs has historically not been favored by the pharmaceutical industry yet, due to improved understanding of the underlying molecular mechanisms, it is now a concept that is growing in popularity  <cit> .
this idea has been explored with pka anchoring proteins  where optimal ri and rii subunit binding peptides were derived from parent peptides and used to target pka in vivo and displace it from its normal anchoring site  <cit> .
in addition to in vivo targeting, disrupting pp1-regulatory subunit interactions with a peptide would be an effective means to aid in identifying proteins in pp <dig> complexes and thus uncover new cellular processes regulated by this protein phosphatase.
RESULTS
we initiated our pp <dig> peptide displacement study selecting rvxf/w containing peptides from the pp <dig> targeting subunits nipp <dig>  <cit>  and zap   <cit> .
they were synthesized and tested for their ability to displace pp <dig> binding proteins from complexes retained on the microcystin matrix.
in brief, we isolated rat liver nuclei, extracted proteins and incubated extracts with microcystin-sepharose to bind the microcystin-sensitive protein phosphatases  <cit> .
after extensive column washing, we incubated the matrix with the nipp <dig> or zap peptides  to selectively displace proteins retained on the matrix through pp <dig>  this implies that pp <dig> itself and other microcystin-sensitive phosphatase complexes should be retained on the matrix and can subsequently be eluted with the chaotrophic agent sodium thiocyanate , which will also displace other bound proteins  <cit>  including some that may be retained by binding non-specifically to the sepharose bead.
blotting for pp <dig> and the pp2a regulatory subunit pr <dig> show this to be true  with no retention of these phosphatase subunits on a control matrix coupled with tris alone.
the nipp <dig> peptide  readily displaced pp <dig> regulatory subunits , yet we chose to continue working with the zap peptide  because it is small, readily soluble and an excellent match to the optimal pp <dig> binding peptide discovered through panning a random peptide library  <cit> .
indeed, the zap peptide rvrw motif and the additional c-terminal amino acids adl and n-terminal basic amino acids  were most frequently obtained in the random peptide library screen .
we thus predict that the zap sequence gkkrvrwadle is the most suitable, high affinity peptide that will compete and displace most other proteins docked to pp <dig> through variations of the rvxf/w-motif.
we acknowledge that even with an 'optimal binding peptide' there are certainly other pp <dig> interacting proteins on the matrix that may not be displaced because of completely novel pp <dig> interaction sites and/or additional interaction sites outside of the rvxf/w-motif  <cit>  that could maintain the interaction with pp <dig> even if the rvxf/w site is displaced.
thus, the pp <dig> binding partners uncovered here still most likely only represent a sub-population of the total nuclear pp <dig> binding partners.
rvrw peptide eluted samples were blotted for the known nuclear pp <dig> binding proteins zap, p <dig>  and nipp <dig> .
this revealed that these proteins were readily displaced from the matrix by the peptide with essentially no further regulatory subunit being eluted with nascn.
a direct comparison of elution conditions with the gkkrvrwadle peptide showed that displacement was much more effective in a higher ionic strength buffer .
having established that the gkkrvrwadle peptide is an excellent tool to displace pp <dig> regulatory subunits, we tried several peptide concentrations to optimize displacement from the matrix .
this demonstrated that  <dig>  mm peptide was sufficient to displace zap and p <dig>  but  <dig>  to  <dig> mm peptide was needed to remove all of the more abundant and perhaps higher affinity binding nipp <dig> protein.
at the  <dig> mm peptide concentration we noted that displacement was in fact not as effective for zap or p <dig>  in subsequent experiments we eluted by first incubating with  <dig>  mm, then  <dig> mm peptide to ensure all pp <dig> complex proteins were displaced.
to ensure the specificity of the method, we included  <dig> independent negative controls.
nuclear phosphatases bound to the microcystin matrix were equally divided into  <dig> parts and eluted with either the gkkrvrwadle peptide, the gkkrvrwadle peptide where the key pp <dig> interacting amino acids  were replaced by alanine , or a scrambled zap peptide .
these fractions were blotted for zap, p <dig> and nipp <dig>  confirming the specificity of the displacement technique.
to extend this method to pp <dig> complexes outside the nucleus, we isolated glycogen particles from skeletal muscle where the well characterized pp <dig> regulatory subunit gm resides and functions to target pp <dig> to glycogen  <cit> .
as shown in figure 1g, the peptide gkkrvrwadle effectively displaced gm from the microcystin-matrix.
in addition, prior to the rvrw displacement, we performed an incubation with the gkkraraadle peptide and, as predicted, due to the absence of the key v and w residues in the docking motif, this peptide did not displace the gm protein from the matrix which supports the idea that the peptide elution is specific for pp <dig> complexes.
all subsequent experiments included the gkkraraadle peptide elution and buffer wash step prior to gkkrvrwadle peptide displacement.
having developed this methodology, we scaled it up to specifically release pp <dig> interacting proteins from the matrix with the aim of identifying novel, less abundant nuclear pp <dig> regulatory subunits and/or proteins that reside in pp <dig> complexes.
we predict many more unrecognized pp <dig> regulatory subunits exist whose identification may have been masked in previous studies when all microcystin-sensitive phosphatases  and non-specifically bound proteins were released from the matrix using nascn alone  <cit> .
furthermore, peptides may not be detected during mass spectrometry in the presence of more abundant peptides.
using an extract prepared from isolated rat liver nuclei we could show that gkkrvrwadle peptide displacement gave a banding pattern on sds-page that was clearly distinct from the subsequent nascn elution .
individual gkkrvrwadle eluted bands were excised from the gel and proteins identified by mass spectrometry .
many previously characterized and new potential pp <dig> binding proteins were identified.
to validate our findings in different organisms, we then switched our attention to the human hela cell line.
here the gkkraraadle, gkkrvrwadle and nascn column elutions again show distinct protein patterns .
mass spectrometry identification of individual bands from the gkkrvrwadle displacement is shown in additional file  <dig> and file  <dig>  for many of the identified proteins we were able to obtain antibodies and blotting of the individual elution steps confirmed that the gkkraraadle peptide did not elute any of the known or putative new pp <dig> interactors, supporting the idea that all of the gkkrvrwadle released proteins reside in pp <dig> complexes and that many are direct pp <dig> binding proteins .
of note is the abundant presence of putative pp <dig> docking sites within the sequences of identified proteins .
although we predict only pp <dig> binding/complex proteins should elute with docking site peptide, mass spectrometry reveals that some pp <dig> catalytic and pp2a components did release with gkkrvrwadle elution, while blotting showed that the vast majority of the total pool of these subunits were retained on the matrix .
this is not surprising given that some complexes likely contain both pp <dig> and pp2a  <cit>  and other proteins may contain more than one pp <dig> dock site.
the lack of displacement with the gkkraraadle peptide, and subsequent elution with gkkrvrwadle provide compelling evidence that pp <dig> directly docks or is in these protein complexes.
to explore this further we performed immunoprecipitations with antibodies we obtained for many of these proteins and in every instance tested we could demonstrate the interaction with pp <dig> .
these data again support the idea that we specifically eluted pp <dig> complex proteins from the matrix.
one reason for using the rvrw peptide elution was to specifically displace proteins and not release any polypeptides that associate non-specifically.
it is known that some proteins bind non-specifically to sepharose beads and thus we sought to determine if the proteins discovered here were retained on sepharose alone and eluted with the gkkrvrwadle peptide.
therefore, as an additional control, we passed a hela nuclear extract over sepharose beads alone, washed as usual, then eluted with the gkkraraadle and gkkrvrwadle peptides and nascn, as described for the microcystin-matrix.
these samples were then western blotted for a selection of interactors that were previously retained on the microcystin-sepharose and eluted with the gkkrvrwadle peptide .
when the entire gkkraraadle, gkkrvrwadle and nascn fractions were individually concentrated and run on a single lane and blotted versus 1/ <dig> of the input  we did weakly detect several of these proteins in the nascn elution , but only noted signals for filamin a and psf in the gkkrvrwadle peptide elution .
given that this is in comparison to 1/ <dig> of the input, this represents a very minor background displacement and functions to remind us that, as in any exploratory work, additional experiments are needed to confirm true interactors.
interestingly, psf has previously been defined as a pp <dig> docking protein  <cit> .
it is notable from the mass spectrometry data that we identified many nuclear processes or complexes not previously linked to pp <dig>  for instance, we found numerous tip <dig>  complex proteins  <cit> .
immunoprecipitation of the tip <dig> components trrap or p <dig>  were able to bring down pp <dig>  confirming that pp <dig> is a previously unrecognized component of this complex.
putative interacting proteins or complexes identified here will need to be confirmed by co-immunoprecipitation experiments or other methods, and then ultimately all complexes, including the trrap complex, will need to be studied to define the role of pp <dig> binding to that protein or group of proteins.
a comparison of the proteins eluted and identified from rat and human nuclei reveals that most of the proteins are the same , but several differences are apparent.
first, some differences are not surprising given that the hela cell line expresses numerous proteins that differ from the rat liver cell proteome.
in addition, there are quite a number of proteins in the rat samples that appear in multiple bands excised from the gel, but this only occurs a few times in the hela bands.
this is likely due to the fact that preparation of the rat nuclei from liver tissue is more time consuming and thus we expect increased proteolysis of proteins and therefore many more hits of the same protein in bands of many masses.
comparing rat liver versus cells grown in culture also means that we started with much more protein from the rat preparation and this certainly contributed to the identification of more lower abundance proteins.
to date several approaches have been used to define the pp1-interactome of the cell, including total  elution from microcystin-sepharose followed by overlay analysis, silac mass spectrometry based pp <dig> binding partner investigation and most recently an antibody array approach for selected putative targets  <cit> .
any methodology has its limitations; our work certainly represents a subgroup of the total pp <dig> targeted protein/complexes of the nucleus for reasons discussed above and because our nuclear extraction procedure likely only released some fraction of the total pp <dig> complexes.
it is clear that many more pp <dig> binding partners exist than originally thought and it will be the combination of multiple approaches that will ultimately define the plethora of roles for pp <dig> in the nucleus and other cellular compartments.
CONCLUSIONS
we have developed a method to specifically elute proteins that reside in pp <dig> complexes or bind directly to pp <dig> after retention on the affinity matrix microcystin-sepharose.
this approach should be valuable to isolate new pp <dig> binding proteins and to link pp <dig> function to as yet unrecognized pp <dig> regulated cellular processes across a broad range of tissues, cell types and organisms.
this technique also lends further credibility to the notion that it could be possible to develop peptides as tools for the specific disruption of protein protein interactions with potential usage for therapeutic purposes.

 the phosphorylation of proteins is one of the most prevalent covalent modifications known, affecting essentially every aspect of cellular function. the protein kinases and phosphatases responsible are highly conserved across species and, with few exceptions, the kinases belong to one large gene family while the phosphatase complement is more complex and can be divided into three broad groups based on protein sequence, catalytic signature and substrate preference. the action of protein phosphatases is tightly controlled with cellular targeting being an important means of regulation. most phospho-serine and threonine dephosphorylation can be attributed to the ppm family and the more diverse ppp family, which includes pp pp2a, pp2b, and pp through to pp pp is thought to not exist as a free catalytic subunit in the cell, but to reside in complexes with a large array of targeting or regulatory subunits that define its function. numerous pp docking proteins have been identified, but they most likely represent only a small fraction of the total number in the cell. the microcystins are a group of cyclic peptides that bind with remarkable specificity and affinity to the type one, 2a and several recently identified protein phosphatases of the ppp family. microcystin covalently couples to a conserved cysteine residue of ppp family members through its methyl-dehydroalanine residue. nishiwaki et al first used microcystin-sepharose to purify pp2a. we exploited a different synthetic approach whereby the carbon-carbon double bond of methyl-dehydroalanine in microcystin couples the latter to aminoethanethiol, which is then linked to a sepharose bead. this generates a high affinity binding matrix for the microcystin-sensitive protein phosphatases that does not covalently couple the phosphatase. microcystin-sepharose has proved to be a powerful tool to purify these protein phosphatases and their associated regulatory subunits from a variety of tissues and cell types. with only a few characterized exceptions, pp interacting proteins bind pp through their primary docking sequence called the rvxf/w motif. their molecular interaction with pp has been visualized via pp1-peptide and pp regulatory subunit structures. it has also emerged that additional or secondary interaction sites often play a role in binding pp and likely contribute to pp isoform specificity recognition, substrate docking and modulation of pp activity. based on a compilation of demonstrated rvxf/w interaction motifs, the panning of a random peptide library, and mutagenesis and modeling studies we noted preferences for particular amino acids within and adjacent to the rvxf/w motif. this led us to speculate that a pp interaction motif peptide, based on this comparison, could be a unique means to specifically disrupt pp1-targetting or regulatory subunit interactions. slight variation in the rvxf/w-motif combined with the now recognized additional, secondary pp interaction sites provide sufficient interaction specificity which may allow the development of drugs or peptide mimetics to abolish specific pp binding protein interactions in vivo. the idea of targeting protein-protein interaction domains with drugs has historically not been favored by the pharmaceutical industry yet, due to improved understanding of the underlying molecular mechanisms, it is now a concept that is growing in popularity. this idea has been explored with pka anchoring proteins where optimal ri and rii subunit binding peptides were derived from parent peptides and used to target pka in vivo and displace it from its normal anchoring site. in addition to in vivo targeting, disrupting pp1-regulatory subunit interactions with a peptide would be an effective means to aid in identifying proteins in pp complexes and thus uncover new cellular processes regulated by this protein phosphatase.  we initiated our pp peptide displacement study selecting rvxf/w containing peptides from the pp targeting subunits nipp and zap. they were synthesized and tested for their ability to displace pp binding proteins from complexes retained on the microcystin matrix. in brief, we isolated rat liver nuclei, extracted proteins and incubated extracts with microcystin-sepharose to bind the microcystin-sensitive protein phosphatases. after extensive column washing, we incubated the matrix with the nipp or zap peptides to selectively displace proteins retained on the matrix through pp this implies that pp itself and other microcystin-sensitive phosphatase complexes should be retained on the matrix and can subsequently be eluted with the chaotrophic agent sodium thiocyanate, which will also displace other bound proteins including some that may be retained by binding non-specifically to the sepharose bead. blotting for pp and the pp2a regulatory subunit pr show this to be true with no retention of these phosphatase subunits on a control matrix coupled with tris alone. the nipp peptide readily displaced pp regulatory subunits, yet we chose to continue working with the zap peptide because it is small, readily soluble and an excellent match to the optimal pp binding peptide discovered through panning a random peptide library. indeed, the zap peptide rvrw motif and the additional c-terminal amino acids adl and n-terminal basic amino acids were most frequently obtained in the random peptide library screen. we thus predict that the zap sequence gkkrvrwadle is the most suitable, high affinity peptide that will compete and displace most other proteins docked to pp through variations of the rvxf/w-motif. we acknowledge that even with an'optimal binding peptide' there are certainly other pp interacting proteins on the matrix that may not be displaced because of completely novel pp interaction sites and/or additional interaction sites outside of the rvxf/w-motif that could maintain the interaction with pp even if the rvxf/w site is displaced. thus, the pp binding partners uncovered here still most likely only represent a sub-population of the total nuclear pp binding partners. rvrw peptide eluted samples were blotted for the known nuclear pp binding proteins zap, p and nipp. this revealed that these proteins were readily displaced from the matrix by the peptide with essentially no further regulatory subunit being eluted with nascn. a direct comparison of elution conditions with the gkkrvrwadle peptide showed that displacement was much more effective in a higher ionic strength buffer. having established that the gkkrvrwadle peptide is an excellent tool to displace pp regulatory subunits, we tried several peptide concentrations to optimize displacement from the matrix. this demonstrated that mm peptide was sufficient to displace zap and p but to mm peptide was needed to remove all of the more abundant and perhaps higher affinity binding nipp protein. at the mm peptide concentration we noted that displacement was in fact not as effective for zap or p in subsequent experiments we eluted by first incubating with mm, then mm peptide to ensure all pp complex proteins were displaced. to ensure the specificity of the method, we included independent negative controls. nuclear phosphatases bound to the microcystin matrix were equally divided into parts and eluted with either the gkkrvrwadle peptide, the gkkrvrwadle peptide where the key pp interacting amino acids were replaced by alanine, or a scrambled zap peptide. these fractions were blotted for zap, p and nipp confirming the specificity of the displacement technique. to extend this method to pp complexes outside the nucleus, we isolated glycogen particles from skeletal muscle where the well characterized pp regulatory subunit gm resides and functions to target pp to glycogen. as shown in figure 1g, the peptide gkkrvrwadle effectively displaced gm from the microcystin-matrix. in addition, prior to the rvrw displacement, we performed an incubation with the gkkraraadle peptide and, as predicted, due to the absence of the key v and w residues in the docking motif, this peptide did not displace the gm protein from the matrix which supports the idea that the peptide elution is specific for pp complexes. all subsequent experiments included the gkkraraadle peptide elution and buffer wash step prior to gkkrvrwadle peptide displacement. having developed this methodology, we scaled it up to specifically release pp interacting proteins from the matrix with the aim of identifying novel, less abundant nuclear pp regulatory subunits and/or proteins that reside in pp complexes. we predict many more unrecognized pp regulatory subunits exist whose identification may have been masked in previous studies when all microcystin-sensitive phosphatases and non-specifically bound proteins were released from the matrix using nascn alone. furthermore, peptides may not be detected during mass spectrometry in the presence of more abundant peptides. using an extract prepared from isolated rat liver nuclei we could show that gkkrvrwadle peptide displacement gave a banding pattern on sds-page that was clearly distinct from the subsequent nascn elution. individual gkkrvrwadle eluted bands were excised from the gel and proteins identified by mass spectrometry. many previously characterized and new potential pp binding proteins were identified. to validate our findings in different organisms, we then switched our attention to the human hela cell line. here the gkkraraadle, gkkrvrwadle and nascn column elutions again show distinct protein patterns. mass spectrometry identification of individual bands from the gkkrvrwadle displacement is shown in additional file and file for many of the identified proteins we were able to obtain antibodies and blotting of the individual elution steps confirmed that the gkkraraadle peptide did not elute any of the known or putative new pp interactors, supporting the idea that all of the gkkrvrwadle released proteins reside in pp complexes and that many are direct pp binding proteins. of note is the abundant presence of putative pp docking sites within the sequences of identified proteins. although we predict only pp binding/complex proteins should elute with docking site peptide, mass spectrometry reveals that some pp catalytic and pp2a components did release with gkkrvrwadle elution, while blotting showed that the vast majority of the total pool of these subunits were retained on the matrix. this is not surprising given that some complexes likely contain both pp and pp2a and other proteins may contain more than one pp dock site. the lack of displacement with the gkkraraadle peptide, and subsequent elution with gkkrvrwadle provide compelling evidence that pp directly docks or is in these protein complexes. to explore this further we performed immunoprecipitations with antibodies we obtained for many of these proteins and in every instance tested we could demonstrate the interaction with pp. these data again support the idea that we specifically eluted pp complex proteins from the matrix. one reason for using the rvrw peptide elution was to specifically displace proteins and not release any polypeptides that associate non-specifically. it is known that some proteins bind non-specifically to sepharose beads and thus we sought to determine if the proteins discovered here were retained on sepharose alone and eluted with the gkkrvrwadle peptide. therefore, as an additional control, we passed a hela nuclear extract over sepharose beads alone, washed as usual, then eluted with the gkkraraadle and gkkrvrwadle peptides and nascn, as described for the microcystin-matrix. these samples were then western blotted for a selection of interactors that were previously retained on the microcystin-sepharose and eluted with the gkkrvrwadle peptide. when the entire gkkraraadle, gkkrvrwadle and nascn fractions were individually concentrated and run on a single lane and blotted versus 1/ of the input we did weakly detect several of these proteins in the nascn elution, but only noted signals for filamin a and psf in the gkkrvrwadle peptide elution. given that this is in comparison to 1/ of the input, this represents a very minor background displacement and functions to remind us that, as in any exploratory work, additional experiments are needed to confirm true interactors. interestingly, psf has previously been defined as a pp docking protein. it is notable from the mass spectrometry data that we identified many nuclear processes or complexes not previously linked to pp for instance, we found numerous tip complex proteins. immunoprecipitation of the tip components trrap or p were able to bring down pp confirming that pp is a previously unrecognized component of this complex. putative interacting proteins or complexes identified here will need to be confirmed by co-immunoprecipitation experiments or other methods, and then ultimately all complexes, including the trrap complex, will need to be studied to define the role of pp binding to that protein or group of proteins. a comparison of the proteins eluted and identified from rat and human nuclei reveals that most of the proteins are the same, but several differences are apparent. first, some differences are not surprising given that the hela cell line expresses numerous proteins that differ from the rat liver cell proteome. in addition, there are quite a number of proteins in the rat samples that appear in multiple bands excised from the gel, but this only occurs a few times in the hela bands. this is likely due to the fact that preparation of the rat nuclei from liver tissue is more time consuming and thus we expect increased proteolysis of proteins and therefore many more hits of the same protein in bands of many masses. comparing rat liver versus cells grown in culture also means that we started with much more protein from the rat preparation and this certainly contributed to the identification of more lower abundance proteins. to date several approaches have been used to define the pp1-interactome of the cell, including total elution from microcystin-sepharose followed by overlay analysis, silac mass spectrometry based pp binding partner investigation and most recently an antibody array approach for selected putative targets. any methodology has its limitations; our work certainly represents a subgroup of the total pp targeted protein/complexes of the nucleus for reasons discussed above and because our nuclear extraction procedure likely only released some fraction of the total pp complexes. it is clear that many more pp binding partners exist than originally thought and it will be the combination of multiple approaches that will ultimately define the plethora of roles for pp in the nucleus and other cellular compartments.  we have developed a method to specifically elute proteins that reside in pp complexes or bind directly to pp after retention on the affinity matrix microcystin-sepharose. this approach should be valuable to isolate new pp binding proteins and to link pp function to as yet unrecognized pp regulated cellular processes across a broad range of tissues, cell types and organisms. this technique also lends further credibility to the notion that it could be possible to develop peptides as tools for the specific disruption of protein protein interactions with potential usage for therapeutic purposes. 
pp is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the rvxf/w motif. we demonstrate that a peptide based on the rvxf/w motif can effectively displace pp bound proteins from pp retained on the phosphatase affinity matrix microcystin-sepharose. this modification of the microcystin-sepharose technique offers an effective means of purifying novel pp regulatory subunits and associated proteins and provides a simple method to uncover a link between pp and additional cellular processes. protein phosphatase one is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. subsequent co-immunoprecipitation experiments confirmed that each identified binding protein was either a direct pp interactor or was in a complex that contains pp our results have linked pp to numerous new nuclear functions and proteins, including ki- rif- topoisomerase iiα, several nuclear helicases, nup and the trrap complex.
 protein phosphatase one is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. pp is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the rvxf/w motif.  we demonstrate that a peptide based on the rvxf/w motif can effectively displace pp bound proteins from pp retained on the phosphatase affinity matrix microcystin-sepharose. subsequent co-immunoprecipitation experiments confirmed that each identified binding protein was either a direct pp interactor or was in a complex that contains pp our results have linked pp to numerous new nuclear functions and proteins, including ki- rif- topoisomerase iiα, several nuclear helicases, nup and the trrap complex.  this modification of the microcystin-sepharose technique offers an effective means of purifying novel pp regulatory subunits and associated proteins and provides a simple method to uncover a link between pp and additional cellular processes. 