CDK5-dependent Phosphorylation of the Rho Family GTPase TC10α Regulates Insulin-stimulated GLUT4 Translocation

Abstract

Insulin stimulation results in the activation of cyclin-dependent kinase-5 (CDK5) in lipid raft domains via a Fyn-dependent phosphorylation on tyrosine residue 15. In turn, activated CDK5 phosphorylates the Rho family GTP-binding protein TC10α on threonine 197 that is sensitive to the CDK5 inhibitor olomoucine and blocked by small interfering RNA-mediated knockdown of CDK5. The phosphorylation deficient mutant T197A-TC10α was not phosphorylated and excluded from the lipid raft domain, whereas the phosphorylation mimetic mutant (T197D-TC10α) was lipid raft localized. Insulin resulted in the GTP loading of T197D-TC10α but not T197A-TC10α and in parallel, T197D-TC10α but not T197A-TC10α depolymerized cortical actin and inhibited insulin-stimulated GLUT4 translocation. These data demonstrate that CDK5-dependent phosphorylation maintains TC10α in lipid raft compartments thereby disrupting cortical actin, whereas subsequent dephosphorylation of TC10α through inactivation of CDK5 allows for the re-assembly of F-actin. Because cortical actin reorganization is required for insulin-stimulated GLUT4 translocation, these data are consistent with a CDK5-dependent TC10α cycling between lipid raft and non-lipid raft compartments. Insulin stimulation results in the activation of cyclin-dependent kinase-5 (CDK5) in lipid raft domains via a Fyn-dependent phosphorylation on tyrosine residue 15. In turn, activated CDK5 phosphorylates the Rho family GTP-binding protein TC10α on threonine 197 that is sensitive to the CDK5 inhibitor olomoucine and blocked by small interfering RNA-mediated knockdown of CDK5. The phosphorylation deficient mutant T197A-TC10α was not phosphorylated and excluded from the lipid raft domain, whereas the phosphorylation mimetic mutant (T197D-TC10α) was lipid raft localized. Insulin resulted in the GTP loading of T197D-TC10α but not T197A-TC10α and in parallel, T197D-TC10α but not T197A-TC10α depolymerized cortical actin and inhibited insulin-stimulated GLUT4 translocation. These data demonstrate that CDK5-dependent phosphorylation maintains TC10α in lipid raft compartments thereby disrupting cortical actin, whereas subsequent dephosphorylation of TC10α through inactivation of CDK5 allows for the re-assembly of F-actin. Because cortical actin reorganization is required for insulin-stimulated GLUT4 translocation, these data are consistent with a CDK5-dependent TC10α cycling between lipid raft and non-lipid raft compartments. Similar to fibroblasts, pre-differentiated adipocytes are elongated and relatively flat cells that contain well defined actin stress fibers. In contrast, following differentiation into mature adipocytes the cells become highly spherical with a relatively thick cortical actin lining the inner surface of the plasma membrane. This cortical actin structure in adipocytes is composed of patches of punctate F-actin that emanates from organized caveolae-rosettes containing the caveolin protein refered to as Cav-actin (1Kanzaki M. Pessin J.E. J. Biol. Chem. 2002; 277: 25867-25869Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). This cortical organization of F-actin is dependent on caveolae-rosette organization as disruption of caveolae results in the dissolution of cortical actin, whereas depolymerization of cortical actin has no effect on caveolae-rosette organization (2Watson R.T. Shigematsu S. Chiang S.H. Mora S. Kanzaki M. Macara I.G. Saltiel A.R. Pessin J.E. J. Cell Biol. 2001; 154: 829-840Crossref PubMed Scopus (148) Google Scholar, 3Scherer P.E. Lisanti M.P. Baldini G. Sargiacomo M. Mastick C.C. Lodish H.F. J. Cell Biol. 1994; 127: 1233-1243Crossref PubMed Scopus (358) Google Scholar). Although insulin induces the rapid breakdown of stress fibers and the appearance of lamellipodia in pre-adipocytes, in differentiated 3T3L1 adipocytes and primary rat adipocytes insulin primarily results in dynamic actin rearrangements in both the cortical and perinuclear regions (4Omata W. Shibata H. Li L. Takata K. Kojima I. Biochem. J. 2000; 346: 321-328Crossref PubMed Google Scholar, 5Kanzaki M. Pessin J.E. J. Biol. Chem. 2001; 276: 42436-42444Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Caveolin-enriched domains in adipocytes have also been reported to associate with a variety of structural and functional proteins. For example, the structural protein flotillin is lipid raft localized and interacts with CAP, Cbl, and Fyn (6Liu J. Deyoung S.M. Zhang M. Dold L.H. Saltiel A.R. J. Biol. Chem. 2005; 280: 16125-16134Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). In particular, the unusual Rho family member GTP-binding protein TC10 appears also to be lipid raft localized in adipocytes and has been implicated in the regulation of insulin-stimulated GLUT4 translocation through the assembly of membrane docking regulating proteins (7Saltiel A.R. Pessin J.E. Traffic. 2003; 4: 711-716Crossref PubMed Scopus (135) Google Scholar). In parallel, TC10 appears to differentially regulate two distinct compartmentalized actin populations. Overexpression of TC10 was found to disrupt cortical actin when lipid raft localized through its endogenous carboxyl-terminal domain that specifies both farnesylation and dual acylation but not when mis-targeted through mutations that directed TC10 to non-lipid raft plasma membrane domains (8Hou J.C. Pessin J.E. Mol. Biol. Cell. 2003; 14: 3578-3591Crossref PubMed Google Scholar). Moreover, expression of a constitutive active (GTP-bound) TC10 mutant (TC10/Q75L) disrupted both Cav-actin and cortical actin structures, but induces massive actin polymerization in perinuclear regions (9Kanzaki M. Watson R.T. Hou J.C. Stamnes M. Saltiel A.R. Pessin J.E. Mol. Biol. Cell. 2002; 13: 2334-2346Crossref PubMed Scopus (79) Google Scholar). Recent studies using enhanced yellow fluorescent protein-tagged β-actin and real-time imaging also demonstrated insulin-regulated actin rearrangement in both cortical and perinuclear regions that was prevented by the Rho-specific toxin Clostridium difficile toxin B (5Kanzaki M. Pessin J.E. J. Biol. Chem. 2001; 276: 42436-42444Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Thus, several lines of evidence exist for both a direct action of TC10 in mediating insulin-stimulated GLUT4 translocation and indirect actions through modification of actin organization. In this regard, there are three TC10 isoforms, TC10α, TC10β, and long but only the constitutive over expression of TC10α inhibits insulin-stimulated GLUT4 translocation (10Chiang S.H. Hou J.C. Hwang J. Pessin J.E. Saltiel A.R. J. Biol. Chem. 2002; 277: 13067-13073Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). These isoforms differ at their amino terminus and previous studies suggested that this domain is responsible for alterations of actin structure and the inhibitory effect on insulin-stimulated GLUT4 translocation (8Hou J.C. Pessin J.E. Mol. Biol. Cell. 2003; 14: 3578-3591Crossref PubMed Google Scholar). However, in addition to the differences in the amino-terminal extension, we have observed that there are other differences in the carboxyl-terminal regions, in particular the presence of a CDK5 consensus phosphorylation site in TC10α but not in TC10β. We have therefore examined the CDK5-dependent phosphorylation of TC10α and demonstrated that this event is necessary for the regulation of TC10α function, localization, and reorganization of cortical F-actin in adipocytes. Reagents—The HA 2The abbreviations used are:HAhemagglutininCDKcyclin-dependent kinaseMAPKmitogen-activated protein kinaseRNAiRNA interferenceGSTglutathione S-transferasePBDp21-binding domaineGFPenchanced green fluorescent proteinsiRNAsmall interfering RNAWTwild typeTBSTTris-buffered saline Tween-20 polyclonal antibody was obtained from Sigma. CDK5, p35, and caveolin-1 antibodies were from Santa Cruz Biotechnology. Phospho-Thr MAPK/CDK substrate monoclonal antibody was from Cell Signaling. TC10 rabbit polyclonal antibody was prepared and affinity purified as previously described (1Kanzaki M. Pessin J.E. J. Biol. Chem. 2002; 277: 25867-25869Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). F-actin antibody was from Abcam. Omnia™ Ser/Thr recombinant kit was from BioSource. ProteoExtract Subcellular Proteome Extraction Kit was from Calbiochem. EZ-Detect Cdc42 Activation Kit was from Pierce. Caveole/Rafts Isolation Kit was from Sigma. CDK5 and Fyn RNAi were obtained from Invitrogen and Santa Cruz. ECL Plus Western Blotting Detection System was obtained from Amersham Biosciences. The horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies were obtained from Pierce. Cell culture media and reagents were from Invitrogen. All of other chemicals used in this study were purchased from Sigma. hemagglutinin cyclin-dependent kinase mitogen-activated protein kinase RNA interference glutathione S-transferase p21-binding domain enchanced green fluorescent protein small interfering RNA wild type Tris-buffered saline Tween-20 Cell Culture—3T3L1 preadipocytes were cultured in Dulbecco's modified Eagle's medium containing 25 mm glucose, 10! calf serum at 37 °C with 8! CO2. Confluent cultures were induced to differentiate into adipocytes as previously described (11Yamada E. Okada S. Saito T. Ohshima K. Sato M. Tsuchiya T. Uehara Y. Shimizu H. Mori M. J. Cell Biol. 2005; 168: 921-928Crossref PubMed Scopus (77) Google Scholar). Immunoprecipitation and Immunoblotting—Scraped frozen cells were rocked for 30 min at 4 °C with Nonidet P-40 lysis buffer (25 mm Hepes, pH 7.4, 10! glycerol, 50 mm sodium fluoride, 10 mm sodium pyrophosphate, 137 mm sodium chloride, 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, 1 mg/ml pepstatin, 5 mg/ml leupeptin). Insoluble material was separated from the soluble extract by centrifugation for 30 min at 4 °C, and the total protein amount in the supernatant was determined by the BCA method. After the addition of 4.5 μg of antibody to the whole cell lysates, samples (typically 2–3 mg of lysates) were incubated for 2 h at 4 °C. Then 50 μl of protein was and samples were rocked for 1 h at 4 °C. After the samples were three with the Nonidet P-40 lysis The samples were in buffer mm pH glycerol, mm and at °C for 5 were separated by and to The samples were with monoclonal or polyclonal antibody as in the and Subcellular F-actin we by using the ProteoExtract Subcellular Proteome Extraction After cells were in Extraction cells were by centrifugation 10 4 The was in Extraction and rocked for 30 min at 4 °C and for 10 min at at 4 °C. The was in Extraction and rocked for 10 min at 4 °C. Then was for 10 min at at 4 °C. The was in Extraction and this was used as a protein to F-actin rearrangement samples were on and to membrane and F-actin and were by Western TC10 activation kit was were with buffer and into After for 5 min at 4 °C, the was for min at at 4 °C. μg of domain was to the and μg of The was well and rocked for min at 4 °C. Then samples were with buffer three After the TC10α to was samples were on and to a membrane and with TC10 of 3T3L1 adipocytes were by and with a total of 1 mg of S. Mori M. Pessin J.E. S. and Scholar). The cells were to to culture for and the adipocytes were serum for 4 h to in the or presence of insulin at 37 °C for the in In CDK5 and TC10α Omnia™ Ser/Thr Kit was used to CDK5 CDK5 was from mg of 3T3L1 adipocytes in the or presence of insulin CDK5 or recombinant was with or TC10α in kinase buffer containing and and the kinase was at 30 °C for The samples were to the and incubated at for The samples were with and incubated with MAPK/CDK substrate monoclonal antibody at for The samples were with and incubated with horseradish peroxidase-conjugated antibody at for Then samples were with and Tris-buffered saline and incubated with for min and the was with The was at with a of buffer Isolation containing was for After cells were and to the was rocked for 30 min at 4 °C. The was with and the was to Then the was of 5 of with and The of was 2 for 1 of were at for 4 h at 4 °C. After from the to of the were described in caveolin-1 were found in from the and for the data the are for is the μg of with μg of was to 3T3L1 adipocytes. The cells were to to culture for and the adipocytes were serum for 2 h in the or presence of insulin for min at 37 °C. adipocytes were in saline and for 10 min in saline containing and The samples were on with were using a of GLUT4 of GLUT4 translocation was determined as described (11Yamada E. Okada S. Saito T. Ohshima K. Sato M. Tsuchiya T. Uehara Y. Shimizu H. Mori M. J. Cell Biol. 2005; 168: 921-928Crossref PubMed Scopus (77) Google Scholar, S. Mori M. Pessin J.E. S. and Scholar). 3T3L1 adipocytes were with 4 μg of μg of other as in or the cells were to 4 °C and incubated with a antibody by horseradish peroxidase-conjugated anti-mouse IgG The cell horseradish was determined by with the (11Yamada E. Okada S. Saito T. Ohshima K. Sato M. Tsuchiya T. Uehara Y. Shimizu H. Mori M. J. Cell Biol. 2005; 168: 921-928Crossref PubMed Scopus (77) Google Scholar, S. Mori M. Pessin J.E. S. and Scholar). are as were for using the of and The of was at The 2 was used for of CDK5 and in 3T3L1 is a kinase that the presence of its and for functional S. Saito T. 2003; PubMed Scopus Google Scholar). Although CDK5 is in has been that CDK5 is only in to the expression of and L.H. T. E. PubMed Google Scholar, J. T. 1994; PubMed Scopus Google Scholar, L.H. I. T. E. 1994; PubMed Scopus Google Scholar, J. M. H. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). However, studies have observed the expression of and in cells M. PubMed Scopus Google Scholar, L. J. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the activation and of CDK5 in other we examined CDK5 and are in differentiated 3T3L1 adipocytes. in of CDK5 and by demonstrated the presence of both of these proteins in differentiated adipocytes. Although the expression of CDK5 there was a in the expression of in differentiated adipocytes with preadipocytes In CDK5 is tyrosine phosphorylated by the tyrosine kinase Fyn Y. Y. Ohshima T. T. M. T. Y. S. Y. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Insulin stimulation in 3T3L1 adipocytes resulted in CDK5 tyrosine phosphorylation as with a antibody for However, this phosphorylation was and to the of phosphorylation by Because insulin has been reported to Fyn M. J. PubMed Scopus Google we Fyn expression by interference that resulted in in Fyn protein these both the and insulin-stimulated phosphorylation of CDK5 was effect on CDK5 protein the of CDK5 on insulin-stimulated GLUT4 translocation, we two we CDK5 and in 3T3L1 adipocytes to CDK5 S. Saito T. Ohshima Y. S. J. PubMed Scopus Google Scholar). in insulin-stimulated GLUT4 translocation was with adipocytes. In a we CDK5 expression by interference that resulted in in CDK5 protein these there was a of insulin-stimulated GLUT4 translocation for we examined several CDK5 and resulted in insulin-stimulated GLUT4 translocation, whereas of with a CDK5 also resulted in of insulin-stimulated GLUT4 translocation not In kinase using CDK5 from and insulin-stimulated cells that insulin stimulation results in in CDK5 kinase these data demonstrate that insulin stimulation results in the kinase activation and tyrosine phosphorylation of CDK5 in a Fyn-dependent TC10α by TC10α has a CDK5 phosphorylation site at we 3T3L1 differentiated adipocytes with a for a HA wild type TC10α or the for threonine mutant that is to CDK5-dependent phosphorylation In the was phosphorylated as by HA by with a MAPK/CDK substrate monoclonal Insulin stimulation for 5 min resulted in TC10α phosphorylation that to of the phosphorylation following min of insulin the other the T197A-TC10α mutant protein was a substrate with these the total amount of TC10α protein relatively with a CDK5-dependent of TC10α, of the cells with a inhibitor both the and insulin-stimulated phosphorylation with no in the amount of TC10α protein expression that olomoucine of TC10α phosphorylation was to a of CDK5, we CDK5 expression using a Although there was no in TC10α the knockdown of CDK5 inhibited insulin-stimulated TC10α phosphorylation both a as a substrate for CDK5 and the TC10α both of phosphorylation by recombinant two and TC10α were also phosphorylated by CDK5 from 3T3L1 adipocytes from insulin-stimulated 3T3L1 adipocytes phosphorylation of both two These data demonstrated that insulin not only the kinase of CDK5 but that TC10α is a direct substrate of CDK5 CDK5 to TC10α is to be lipid raft we the of CDK5 by its plasma membrane with lipid raft caveolin-1 J. Cell 2003; PubMed Scopus Google Scholar). the was by from 3T3L1 adipocytes as previously described P.E. 2003; PubMed Scopus Google Scholar). CDK5 with caveolin-1 in adipocytes as well as cells T197D-TC10α that TC10 phosphorylation Because CDK5 is lipid raft we examined the of CDK5-dependent phosphorylation of TC10α to regulate TC10α plasma membrane in was with whereas the amount of T197A-TC10α with caveolin-1 was In contrast, expression of mutant to phosphorylation (T197D-TC10α) amount of with The amount of T197A-TC10α that with caveolin-1 was not to differences in protein expression as the of TC10α was These data are consistent with phosphorylation a event the of TC10α between non-lipid raft and lipid TC10α and studies have demonstrated that insulin the activation of TC10α by GTP through the of GTP for M. J. PubMed Scopus Google Scholar). the of TC10α phosphorylation on GTP we of the that only the GTP activated of the Rho family of small GTP-binding proteins G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in insulin stimulation resulted in in of of a in GTP whereas the with T197A-TC10α was both in the and insulin-stimulated In contrast, T197D-TC10α a to the domain that was not following insulin Thus, these data that phosphorylation the of TC10α to become activated through TC10α F-actin studies have observed that the lipid raft of TC10α results in a disruption of cortical actin organization with a of insulin-stimulated GLUT4 translocation (1Kanzaki M. Pessin J.E. J. Biol. Chem. 2002; 277: 25867-25869Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, R.T. Shigematsu S. Chiang S.H. Mora S. Kanzaki M. Macara I.G. Saltiel A.R. Pessin J.E. J. Cell Biol. 2001; 154: 829-840Crossref PubMed Scopus (148) Google Scholar, 5Kanzaki M. Pessin J.E. J. Biol. Chem. 2001; 276: 42436-42444Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, J. Deyoung S.M. Zhang M. Dold L.H. Saltiel A.R. J. Biol. Chem. 2005; 280: 16125-16134Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, A.R. Pessin J.E. Traffic. 2003; 4: 711-716Crossref PubMed Scopus (135) Google Scholar, J.C. Pessin J.E. Mol. Biol. Cell. 2003; 14: 3578-3591Crossref PubMed Google Scholar, M. Watson R.T. Hou J.C. Stamnes M. Saltiel A.R. Pessin J.E. Mol. Biol. Cell. 2002; 13: 2334-2346Crossref PubMed Scopus (79) Google Scholar, S.H. Hou J.C. Hwang J. Pessin J.E. Saltiel A.R. J. Biol. Chem. 2002; 277: 13067-13073Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, M. J. PubMed Scopus Google Scholar). the data in are consistent with phosphorylation in both GTP loading and lipid raft localization, and were and for actin of and T197D-TC10α resulted in a disruption of F-actin in adipocytes with cells with the In contrast, adipocytes with the CDK5 phosphorylation mutant no in F-actin Moreover, the total amount of F-actin was not by insulin stimulation and the observed with the TC10α phosphorylation in the Thus, these data are consistent with the disruption of cortical F-actin to the of TC10α to the lipid raft domains that are in by the phosphorylation TC10α and GLUT4 the of TC10α to insulin-stimulated GLUT4 translocation with cortical F-actin we determined the effect of the TC10α were with a and or the T197D-TC10α or T197A-TC10α Overexpression of and T197D-TC10α no effect on the of GLUT4 but inhibited insulin-stimulated GLUT4 translocation to the plasma whereas T197A-TC10α was inhibitory These data are in using a that the amount of GLUT4 at the cell surface (11Yamada E. Okada S. Saito T. Ohshima K. Sato M. Tsuchiya T. Uehara Y. Shimizu H. Mori M. J. Cell Biol. 2005; 168: 921-928Crossref PubMed Scopus (77) Google Scholar, S. Mori M. Pessin J.E. S. and Scholar). Thus, these data are consistent with phosphorylation of TC10α as for the depolymerization of F-actin and of GLUT4 translocation. the TC10α of insulin-stimulated GLUT4 translocation was CDK5 expression was by RNAi previously observed the knockdown of CDK5 resulted in enhanced insulin-stimulated GLUT4 translocation of no effect on the of GLUT4 translocation but inhibited insulin-stimulated GLUT4 translocation However, CDK5 knockdown prevented the inhibitory of and resulted in a of insulin-stimulated GLUT4 translocation the other of T197A-TC10α in these resulted in a small of insulin-stimulated GLUT4 translocation that was by knockdown of CDK5 of T197D-TC10α no effect on the of GLUT4 translocation but inhibited insulin-stimulated GLUT4 translocation these the CDK5 was to insulin-stimulated GLUT4 translocation These data are consistent with CDK5-dependent phosphorylation of responsible for the inhibitory of TC10α on insulin-stimulated GLUT4 translocation. studies have observed that of a active mutant and a mutant inhibited insulin-stimulated GLUT4 translocation M. J. PubMed Scopus Google Scholar, S.H. Kanzaki M. Watson R.T. Macara I.G. Pessin J.E. Saltiel A.R. 2001; PubMed Scopus Google Scholar). Moreover, other Rho family GTP-binding TC10α a carboxyl-terminal domain that TC10α to plasma membrane lipid raft (8Hou J.C. Pessin J.E. Mol. Biol. Cell. 2003; 14: 3578-3591Crossref PubMed Google Scholar). the inhibitory effect of TC10α was reported to on lipid raft whereas of TC10α to non-lipid raft domains of the plasma membrane were effect (8Hou J.C. Pessin J.E. Mol. Biol. Cell. 2003; 14: 3578-3591Crossref PubMed Google Scholar). Thus, the that the inhibitory action of TC10α is of domain activation but that a protein with the structural domain of TC10α is TC10α regulation appears to in the insulin regulation of GLUT4 translocation as insulin stimulation results in GTP loading and subsequent of the required for GLUT4 docking S.H. Kanzaki M. Watson R.T. Macara I.G. Pessin J.E. Saltiel A.R. 2001; PubMed Scopus Google Scholar). In TC10α appears to have on cortical F-actin organization and (1Kanzaki M. Pessin J.E. J. Biol. Chem. 2002; 277: 25867-25869Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, R.T. Shigematsu S. Chiang S.H. Mora S. Kanzaki M. Macara I.G. Saltiel A.R. Pessin J.E. J. Cell Biol. 2001; 154: 829-840Crossref PubMed Scopus (148) Google Scholar, 5Kanzaki M. Pessin J.E. J. Biol. Chem. 2001; 276: 42436-42444Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, J. Deyoung S.M. Zhang M. Dold L.H. Saltiel A.R. J. Biol. Chem. 2005; 280: 16125-16134Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, A.R. Pessin J.E. Traffic. 2003; 4: 711-716Crossref PubMed Scopus (135) Google Scholar, J.C. Pessin J.E. Mol. Biol. Cell. 2003; 14: 3578-3591Crossref PubMed Google Scholar, M. Watson R.T. Hou J.C. Stamnes M. Saltiel A.R. Pessin J.E. Mol. Biol. Cell. 2002; 13: 2334-2346Crossref PubMed Scopus (79) Google Scholar, S.H. Hou J.C. Hwang J. Pessin J.E. Saltiel A.R. J. Biol. Chem. 2002; 277: 13067-13073Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, M. J. PubMed Scopus Google Scholar, S.H. Kanzaki M. Watson R.T. Macara I.G. Pessin J.E. Saltiel A.R. 2001; PubMed Scopus Google Scholar). Thus, the of insulin TC10α mediating actin as previous studies have also demonstrated that of wild or TC10α but not TC10β, inhibited insulin-stimulated GLUT4 translocation, and disrupted cortical F-actin (10Chiang S.H. Hou J.C. Hwang J. Pessin J.E. Saltiel A.R. J. Biol. Chem. 2002; 277: 13067-13073Abstract Full Text Full Text PDF PubMed Scopus (50) Google is that differences were of the amino the presence of a CDK5 phosphorylation site in TC10α that is not in TC10β. Thus, we that CDK5-dependent phosphorylation of TC10α for the of TC10α TC10β. In this regard, we have the presence of the CDK5 in differentiated 3T3L1 adipocytes as well as the presence of the CDK5 p35, is for CDK5 kinase (11Yamada E. Okada S. Saito T. Ohshima K. Sato M. Tsuchiya T. Uehara Y. Shimizu H. Mori M. J. Cell Biol. 2005; 168: 921-928Crossref PubMed Scopus (77) Google Scholar, 2003; PubMed Scopus Google Scholar). the tyrosine kinase was reported to CDK5 on that resulted in CDK5 kinase M. S. M. L.H. 2000; Full Text Full Text PDF Scopus Google we have also observed that the family kinase Fyn also phosphorylates CDK5 at in 3T3L1 to that found cells M. PubMed Scopus Google Scholar). In we have observed a CDK5 phosphorylation at that also a rapid but phosphorylation in adipocytes. The that CDK5 a relatively of phosphorylation was consistent with a relatively of CDK5 kinase The insulin-stimulated in phosphorylation with in CDK5 substrate kinase Thus, data that CDK5 is activated in the as well as activated following insulin stimulation and that Fyn is the tyrosine kinase that CDK5 In addition to its in CDK5 protein kinase has also been reported to associate with the isoforms of the Rho family that are of actin polymerization M. W. L.H. PubMed Scopus Google Scholar). TC10α is a member of the Rho family that is lipid raft localized and we have observed that CDK5 to to that observed for TC10α also appears to be a CDK5 substrate as of the consensus phosphorylation site and with the CDK5 inhibitor olomoucine or CDK5 RNAi TC10α phosphorylation on evidence that TC10α is a CDK5 substrate and expression of a mutant as well as phosphorylation mimetic mutant demonstrated that phosphorylation at this site not only lipid raft and but also F-actin organization and insulin-stimulated GLUT4 translocation. these we the following for the of GLUT4 translocation by In the TC10α is phosphorylated and lipid raft the of cortical F-actin. Insulin stimulation TC10α phosphorylation thereby its from non-lipid raft to lipid raft plasma membrane Because TC10α has actin depolymerization this results in a in the amount of cortical F-actin. However, subsequent dephosphorylation results in non-lipid raft thereby cortical F-actin organization. dynamic actin is necessary for GLUT4 translocation (5Kanzaki M. Pessin J.E. J. Biol. Chem. 2001; 276: 42436-42444Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, M. J. PubMed Scopus Google this of the GLUT4 to the plasma membrane localized docking This also for the effect of of TC10α to GLUT4 translocation and cortical CDK5 is of TC10α results in of TC10α in lipid raft domains the to actin depolymerization and the dynamic between actin depolymerization and In data that CDK5 in the and insulin-stimulated phosphorylation of a site in the TC10α This phosphorylation event the of TC10α to lipid raft or non-lipid raft domains and thereby the cortical actin polymerization and depolymerization of This regulation of cortical F-actin thereby allows or of GLUT4 to their plasma membrane docking event for membrane and in adipocytes.