Patent Application: US-7880808-A

Abstract:
the present invention provides binding motifs , binding sites , peptides , and 14 - 3 - 3 / mekk complexes . also provided are agents reactive with same , pharmaceutical compositions , molecules , and kits comprising same , and methods of producing same . the present invention further provides isolated nucleic acids and constructs comprising same , host cells comprising the constructs , and transgenic non - human animals . additionally , the present invention provides methods of : facilitating or inhibiting 14 - 3 - 3 / mekk interaction , regulating mekk activity , identifying agents that interact with mekk or 14 - 3 - 3 , detecting and monitoring the development of mekk - mediated conditions , and treating and / or preventing mekk - mediated conditions .

Description:
mekk2 and mekk3 play important roles in the activation of numerous mapk and nf - κb signaling pathways following cellular stress and activation by pro - inflammatory cytokines . for example , mekk3 is essential for inflammatory gene expression that is induced downstream of tnf receptor - 1 , il - 1 receptor , and toll - like receptor activation through the activation of jnk , p38 mapk , and nf - kb ( 26 , 28 ). accordingly , the mechanisms that control mekk2 and mekk3 activation , and their target specificities , are subjects of intense interest . specificity of signaling is coordinated through hierarchical phosphorylation cascades that are regulated , in part , through association between signaling proteins . the well - known phosphoprotein - binding molecule , 14 - 3 - 3 , plays many roles in controlling the specificity , signal strength , and spatial localization of numerous pathway components ( 29 ). 14 - 3 - 3 has been shown to interact with mekk3 , but the site of interaction , or the biological significance , has not yet been determined . in the present investigation , the inventor performed tryptic mapping , phosphoamino acid analysis , and immunoblot analysis with a phosphospecific antibody to identify t294 as a novel site of mekk3 phosphorylation . mutation of this residue to alanine abolished 14 - 3 - 3 interaction , as did incubation of mekk3 with a synthetic peptide containing phosphorylated , but not the unphosphorylated , t294 peptide . these novel and surprising results demonstrate that t294 is the primary mekk3 site of 14 - 3 - 3 interaction . as disclosed herein , the inventor determined that phosphorylation of t294 was dependent upon the catalytic activity of mekk3 , since mutation of the catalytic lysine at position 391 significantly diminished phosphorylation of t294 and dramatically reduced the interaction between mekk3 and 14 - 3 - 3 . this suggests that phosphorylation of t294 occurs by autophosphorylation . however , it is also possible that a downstream kinase , activated by mekk3 , phosphorylates t294 . phosphorylation of the activation loop s526 was also necessary for 14 - 3 - 3 binding , but it is unlikely that 14 - 3 - 3 directly interacts with this site . s526 phosphorylation was necessary for kinase activity , and mutation of this residue functionally mimicked kinase - dead mutation , k391m . the role that 14 - 3 - 3 association with mekk3 plays is of considerable interest . with raf - 1 , 14 - 3 - 3 association to s259 and s621 plays opposing roles in regulating raf - 1 activity ( 30 ). to probe the role of 14 - 3 - 3 association with mekk3 , the inventor examined the ability of mekk3 to induce the phosphorylation of the mapks , erk and p38 mapk , and the map ( 2 ) k , mkk4 , in cells expressing wildtype and t294a mekk3 . in these experiments , phosphorylation of erk , p38 mapk , and mkk4 appeared to be similar between wt and t294a mekk3 , indicating that t294a mekk3 retains catalytic activity in the absence of 14 - 3 - 3 binding . these results suggest that , unlike the activation loop s526a mutation of mekk3 , which abolishes kinase activity and results in a total loss of erk phosphorylation ( 9 , 10 ; and data not shown ), 14 - 3 - 3 binding to phospho - t294 might play a more subtle role in regulating downstream pathway activation . indeed , it has been suggested that 14 - 3 - 3 interaction with mekk3 could serve to protect s526 phosphorylation , thereby preserving kinase activity ( 10 ). additional work has also shown that dimerization of mekk2 and mekk3 is required for their full activation ( 31 ), and it is possible that 14 - 3 - 3 binding could play a role in this process . thus , endogenous mekk3 , which undergoes stimulus - dependent s526 autophosphorylation , could require 14 - 3 - 3 interaction for maximal activity . alternatively , 14 - 3 - 3 binding could play a role in mekk3 pathway specificity by coupling mekk3 to signal - dependent binding partners . correlation of t294 phosphorylation of endogenous mekk3 proteins with upstream signals will be possible using the phospho - t294 antibody that the inventor has generated in accordance with methods disclosed herein . the closely - related mekk2 protein kinase shares significant homology to mekk3 , but may have distinct signaling functions . mekk2 is highly homologous to mekk3 in the amino acid sequence surrounding the equivalent site of t283 , suggesting that mekk2 might associate with 14 - 3 - 3 at t283 ( fig7 ). notably , this sequence is not present in any other protein kinase with any degree of homology . therefore , the 14 - 3 - 3 - binding motif at t283m94 is likely unique to the mekk2 / 3 family . the present invention is described in the following examples , which are set forth to aid in the understanding of the invention , and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter . hek 293 cells were obtained from the american type culture collection , and maintained in dulbecco &# 39 ; s modified eagles medium ( dmem ) supplemented with 10 % fetal bovine serum and antibiotics at 37 ° c ., 5 % co 2 , and humidity . human mekk3 cdna was cloned from i . m . a . g . e . clone id # 7939519 by pcr , and inserted into pcmv10 - 3 × flag to introduce an n - terminal flag epitope . mutagenesis of pcmv10 - 3 × flag - mekk3 was performed using a quickchange kit ( strategene ), and mutations were sequence verified . hek 293 cells were plated onto 35 - mm - diameter dishes at 80 % confluency , and transfected with 100 to 500 ng of cdna using lipofectamine - 2000 ( gibco - brl ) following the manufacturer &# 39 ; s protocol . transfection medium was removed and replaced with complete dmem overnight . cells were lysed in 50 mm tris - hcl , ph 7 . 4 , 1 % triton x - 100 , 25 mm naf , 25 mm β - glycerophosphate , 5 mm edta , 0 . 05 % sds , 1 . 0 μg / ml microcystin lr , and protease inhibitors . anti - flag m2 antibody , conjugated to agarose beads ( sigma ), was added to lysates and incubated overnight at 4 ° c . beads were washed three times with lysis buffer , and proteins were eluted with 200 μl of lithium dodecyl sulfate ( lds ) sample buffer heated to 70 ° c . for 10 minutes . portions of the lysates prior to immunoprecipitation were also boiled with lds - containing sample buffer . lysates and immunoprecipitations were fractionated by sds polyacrylamide gel electrophoresis ( page ). proteins were transferred to a polyvinylidene difluoride ( pvdf ) membrane , blocked in 5 % skim milk for 30 min , and probed with the appropriate antibody overnight at 4 ° c . secondary decoration with horseradish - peroxidase - conjugated anti - rabbit or anti - mouse antibodies was performed at room temperature for 1 . 5 hours . proteins were visualized using ecl , according to the manufacturer &# 39 ; s protocol ( amersham ). for blotting using the licor odyssey infrared imager , membranes were probed with ir680 - anti - rabbit and ir800 - anti - mouse secondary antibodies . flag was stained with anti - flag m2 mouse monoclonal ( sigma ), and 14 - 3 - 3 isoforms were detected with anti - 14 - 3 - 3β ( h8 ; santa cruz biotechnology ). hek 293 cells were plated onto 35 - mm - diameter dishes at 80 % confluency , and transfected with 500 ng of pcmvi0 - flag - mekk3 using lipofectamine - 2000 ( gibco - brl ) according to the manufacturer &# 39 ; s protocol . after 24 hours , cells were placed in phosphate - free dmem medium with 1 mci / ml 32 p - labeled orthophosphate at 37 ° c . for 4 h . flag - mekk3 was immunoprecipitated from detergent - solubilized lysates , and fractionated on an 8 % gel . 32 p - labeled mekk3 was detected by autoradiography . 32 p - metabolically - labeled flag - mekk3 was isolated as described above , excised from the gel , and digested with 10 μg / ml tosylphenylalanylk chloromethyl ketone - treated trypsin ( promega ) in 50 mm ( nh 4 ) hco 3 , ph 7 . 8 , overnight at 37 ° c . gel fragments were pelleted by centrifugation , and the remaining supernatant was transferred to clean tubes and dried under vacuum . dried peptides were resuspended in 50 μl performic acid , incubated on ice for 2 hours , and dried under vacuum . peptides were washed with diminishing volumes of water , and resuspended in 5 μl of ph 1 . 9 electrophoresis buffer . electrophoresis was performed on 200 - μm microcrystalline cellulose plates ( kodak ) at 1000 v , 7 ° c ., for 30 min . the plates were chromatographed in the second dimension in chromatography buffer ( n - butanol / pyridine / acetic acid / water , 32 . 5 : 25 : 5 : 20 ). plates were dried , and phosphopeptides were visualized using an fx - imager ( biorad ) or film autoradiography . if cold synthetic phosphopeptides were also run , these were visualized with ninhydrin staining . phosphoamino acid analysis was performed by hydrolyzing 32 p - labelled mekk3 in 500 μl of 6 n hcl heated to 110 ° c . for 60 min . the hcl was removed under vacuum , and the phosphoamino acids were washed with diminishing volumes of water . separation was performed on cellulose plates using buffer consisting of 0 . 5 % pyridine and 5 % acetic acid at 1500 v , 7 ° c ., for 20 min in one direction , and then the plates were allowed to dry . once dry , phosphoamino acids were separated in a second dimension using ph 3 . 5 buffer at 1600v , 7 ° c ., for 13 min . 32 p - labeled phosphoamino acids were detected by autoradiography . in each of the samples , 1 μg of a mixture of phospho - l - serine , phospho - l - threonine , and phospho - l - tyrosine was also added ; this was visualized by ninhydrin staining . peptide 1 ( dgrrtfprir ) and phosphopeptide 1 ( dgrr ( pt ) fprir ) corresponding to the sequence surrounding t294 were synthesized by genscrip corp ( new jersey ). peptide 2 ( yndgrrtfprirr ) and phosphopeptide 2 ( yndgrr ( pt ) fprirr ) were generated by open biosystems ( huntsville , ala .). lysates containing flag - mekk3 were incubated with 200 μm of peptide or phosphopeptide where indicated . anti - flag m2 agarose was added and the mixtures were incubated overnight at 4 ° c . beads were washed three times and proteins were eluted and resolved by sds page . co - immunoprecipitated 14 - 3 - 3 was detected using a pan - specific anti - 14 - 3 - 3 antibody ( santa cruz ). discussed below are results obtained by the inventor in connection with the experiments of examples 1 - 7 : mekk3 is a phosphoprotein ( 9 - 12 ), but the extent and physiological significance of phosphorylation of mekk3 is not fully understood . in an initial survey , the inventor expressed wildtype ( wt ) mekk3 and mekk3 with various mutations in hek293 cells , and resolved detergent - solubilized lysates on a high - ratio acrylamide : bis - acrylamide gel . mekk3 resolved as numerous species over a 10 - kda range , with the majority of the wildtype protein migrating as a single band ( fig1 ). the mobility of mekk3 was dramatically increased with introduction of a kinase - dead mutation , k391m , suggesting that mekk3 autophosphorylates on a number of residues in cells . mekkk3 expressed in cells treated with staurosporine , a broadly - specific serine / threonine kinase inhibitor , also reduced the mekk3 band shift ( data not shown ), suggesting that staurosporine inhibits mekk3 or one or more kinases upstream of mekk3 . treatment with a pi3k inhibitor , wortmannin , had no effect on mobility shift , nor did the mek1 / 2 inhibitor , u0126 ( data not shown ). next , the three known sites of phosphorylation — s166 , s337 , and s526 — were individually mutated to alanine , and each allele was expressed in cells . the mobility shift of s166a was reduced compared with wildtype mekk3 ( fig1 ), confirming that this site is likely phosphorylated in cells , as previously suggested ( 11 ). the s337a mutation did not affect mobility shift on sds - page . finally , s526a reduced the mobility shift of mekk3 , comparable to k391m ( fig1 ). the loss of mobility shift of s526a was partially restored with the introduction of a phosphomimetic glutamic acid , s526d . the rescued mobility shift of s526d was completely abolished upon compounding this mutation with the kinase - dead lysine mutation , mekk3 s526d / k391m ( fig1 ). this suggested that s526d was permissive for autophosphorylation on additional , distinct sites . the inventor also introduced mutations at several other potential sites of phosphorylation , based on sequence similarity to known kinase phosphorylation motifs . these included s236 , s250 , and s357 ( table 1 ). none of these point mutations altered the mobility shift of mekk3 ( fig1 ). thus , the difference in mobility shift between kinase - dead mekk3 and wildtype mekk3 , as well as the observation that s526d induced a mobility shift to a greater degree than s526d / k391m , suggested that mekk3 autophosphorylates on one or more sites . the phosphoprotein binding molecule 14 - 3 - 3 is involved in regulating a diverse set of cellular proteins by altering activity , location , and stabilization ( 23 , 24 ). mekk3 has been shown to associate with 14 - 3 - 3 isoforms ( 22 ), but the mechanism and significance of this interaction is unknown . the inventor considered the possibility that mekk3 autophosphorylation might play a role in mediating mekk3 - 14 - 3 - 3 interaction , and found that mekk3 co - immunoprecipitated with one or more endogenous 14 - 3 - 3 isoforms ( fig2 a ). the family of 14 - 3 - 3 proteins was detected by immunoblotting with a pan - specific 14 - 3 - 3 antibody raised against a common sequence present in all family members . this interaction was specific for catalytically - active mekk3 , since the kinase - dead k391m mutant did not co - immunoprecipitate 14 - 3 - 3 ( fig2 a ). in addition , the intact s526 residue in the catalytic site , which is required for mekk3 activity ( 9 , 10 ), was also essential for 14 - 3 - 3 association , since mutation to alanine prevented 14 - 3 - 3 co - immunoprecipitation ( fig2 b ). this suggested that mekk3 activity was required for 14 - 3 - 3 association , possibly as a consequence of cis - or trans - autophosphorylation . the kinase inhibitor , staurosporine , also disrupted mekk3 - 14 - 3 - 3 interaction ( fig2 c ), again indicating that kinase activity — either mekk3 itself or an upstream kinase — was necessary for 14 - 3 - 3 association . in addition to s526 , two other sites of phosphorylation of mekk3 have been identified : s166 and s337 ( 11 ). another site , s250 , lies between s166 and s337 , and resembles a mode 1 14 - 3 - 3 binding site . the inventor speculated that these three sites might act co - operatively in binding with 14 - 3 - 3 , and that this might not be revealed when testing single point mutations . to test this possibility , the inventor created a triple mutation consisting of s166a / s250a / s337a . the mekk3 triple mutation , in which s166 , s250 , and s337 were changed to alanine , co - immunoprecipitated 14 - 3 - 3 in a manner similar to that of wildtype mekk3 ( fig3 ). this result conclusively demonstrated that phosphorylation of s166 , s250 , and s337 is not a requirement for regulating mekk3 - 14 - 3 - 3 binding . the inventor also tested other potential 14 - 3 - 3 binding sites , including s236 and s357 . these sites could be mode 1 14 - 3 - 3 binding sites based on sequence similarity to known 14 - 3 - 3 binding motifs ( table 1 ). however , mutation of s357 to alanine ( fig3 ) or s236 to alanine ( not shown ) did not prevent 14 - 3 - 3 co - immunoprecipitation . taken together , the inventor &# 39 ; s results indicate that 14 - 3 - 3 binding required phosphorylation of sites on mekk3 distinct from s166 , s236 , s250 , s337 , and s357 . the inventor &# 39 ; s observations of the mobility shift of mekk3 and mutants , and the profile of 14 - 3 - 3 co - immunoprecipitation , suggested that mekk3 could be phosphorylated on residues in addition to the confirmed sites of s166 , s337 , and s526 . the inventor also predicted that some of these additional sites could be a result of autophosphorylation , and might mediate mekk3 - 14 - 3 - 3 interaction . in order to identify the additional unknown site ( s ) of phosphorylation , the inventor expressed wt mekk3 and k391m mekk3 , and labeled cells with 32 p - orthophosphate ( fig4 a ). significant 32 p was incorporated into each of the expressed proteins , including k391m mekk3 , suggesting that both the active kinase and inactive kinase are targets of upstream kinases . next , the inventor performed phosphoamino acid analysis on the 32 p - labelled mekk3 ( fig4 b ). this revealed a mixture of phosphoserine and phosphothreonine , indicating that mekk3 was phosphorylated in cells on both amino acids . tyrosine phosphorylation was not detected . serine phosphorylation was expected ; however , threonine phosphorylation was surprising . the inventor examined the mekk3 sequence to identify potential sites of threonine phosphorylation . the threonine residue at 294 , within the sequence 291 . . . grrt 294 fpri . . . 298 ( of mekk3 isoform 2 ; t345 of the longer mekk3 isoform 1 ), was considered as a potential site of phosphorylation because of the arginine at − 2 , the phenylalanine at + 1 , and the proline at + 2 . this motif resembles the canonical phosphorylation motif for r / k - directed , basophilic kinases , and also resembles the mode 1 site for 14 - 3 - 3 protein binding . to test if this site was phosphorylated in cells , the inventor digested 32 p - labeled mekk3 with trypsin , and resolved the tryptic fragments by two - dimensional electrophoresis and cellulose chromatography . the inventor included in the sample the synthetic peptide [ phospho - t ] fpr , corresponding to the unique tryptic fragment generated from 291 . . . grrt 294 fpri . . . 298 . wildtype mekk3 generated numerous 32 p - labelled tryptic peptides ( fig4 c ). the synthetic peptide [ phospho - f ] fpr co - migrated exactly with spot a , revealing the identity of this tryptic spot as [ phospho - t ] fpr . the phosphorylation of this tryptic fragment , along with several other spots , was greatly diminished in tryptic maps generated from k391m mekk3 ( fig4 c ). the other spots surrounding spot a were likely partial digestion of the sequence flanking t294 , dgrr ( pt ) frirr . trypsin inefficiently cleaves between two arginine residues , and this would result in tryptic peptides of various lengths and charges . consistent with this , the inventor noted that the 2d - tryptic maps generated from t294a mekk3 showed that many of these additional spots were abolished , including spot a ( data not shown ). to further confirm that t294 was a site of phosphorylation , the inventor generated a phosphospecific antibody to phospho - t294 . serum from rabbits immunized with the synthetic peptide yndgrr ( pt ) fprirr , corresponding to phospho - 1294 , detected wt mekk3 , but not t294a mekk3 ( fig4 d ). in addition , k391m mekk3 was only weakly detected , consistent with the reduction in phosphorylation at t294 observed by tryptic mapping . together , these results demonstrate that t294 is a site of threonine phosphorylation of mekk3 . the inventor noted that the loss of threonine phosphorylation of k391m mekk3 coincided with loss of 14 - 3 - 3 association . in addition , the inventor observed that the t294 residue resided within a potential mode 1 14 - 3 - 3 binding site . accordingly , the inventor tested the interaction of 14 - 3 - 3 with mekk3 in which t294 was replaced with alanine . mutation of t294 to alanine completely abolished 14 - 3 - 3 co - immunoprecipitation ( fig5 a ), suggesting that this site plays an important role in stabilizing 14 - 3 - 3 — mekk3 interaction . however , it remained possible that phosphorylation of t294 was acting as an enhancer to facilitate 14 - 3 - 3 binding to a distinct site . to prove that t294 was the site of 14 - 3 - 3 binding , the inventor incubated wt mekk3 , prior to immunoprecipitation , with either phosphorylated or non - phosphorylated peptides corresponding to the sequence surrounding t294 , and observed whether these peptides could compete with mekk3 for 14 - 3 - 3 binding . the phosphorylated peptides abolished 14 - 3 - 3 co - immunoprecipitation , while the non - phosphorylated peptides had no effect on co - immunoprecipitation ( fig5 b ). together , these experiments established that t294 was the primary site of 14 - 3 - 3 protein interaction . the inventor expressed mekk3 in 293 cells , and examined the phosphorylation status of various mapk and map ( 2 ) ks . previously , others established that mekk3 is capable of activating components of the several mapk pathways in cell lines and in primary cells ( 3 , 6 , 10 , 25 - 27 ). in the inventor &# 39 ; s experiments , both wildtype and t294a mekk3 , but not kinase - dead k391m mekk3 , induced the phosphorylation of erk , mkk4 , and p38 mapk on activating sites within their catalytic domains ( fig6 ). these experiments demonstrate that t294a mekk3 is both active and competent for mapk pathway activation , and , unlike s621 of raf - 1 , phosphorylation of t294 is not essential for mekk3 activity . while the foregoing invention has been described in some detail for purposes of clarity and understanding , it will be appreciated by one skilled in the art , from a reading of the disclosure , that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims . 1 . deacon , k ., and blank , j . l . 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