Cluster Analysis of Insulin Action in Adipocytes Reveals a Key Role for Akt at the Plasma Membrane
2009; Elsevier BV; Volume: 285; Issue: 4 Linguagem: Inglês
10.1074/jbc.m109.060236
ISSN1083-351X
AutoresYvonne Ng, Georg Ramm, James G. Burchfield, Adelle C.F. Coster, Jacqueline Stöckli, David E. James,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoThe phosphatidylinositol 3-kinase/Akt pathway regulates many biological processes, including insulin-regulated GLUT4 insertion into the plasma membrane. However, Akt operates well below its capacity to facilitate maximal GLUT4 translocation. Thus, reconciling modest changes in Akt expression or activity as a cause of metabolic dysfunction is complex. To resolve this, we examined insulin regulation of components within the signaling cascade in a quantitative kinetic and dose-response study combined with hierarchical cluster analysis. This revealed a strong relationship between phosphorylation of Akt substrates and GLUT4 translocation but not whole cell Akt phosphorylation. In contrast, Akt activity at the plasma membrane strongly correlated with GLUT4 translocation and Akt substrate phosphorylation. Additionally, two of the phosphorylated sites in the Akt substrate AS160 clustered separately, with Thr(P)-642 grouped with other Akt substrates. Further experiments suggested that atypical protein kinase Cζ phosphorylates AS160 at Ser-588 and that these two sites are mutually exclusive. These data indicate the utility of hierarchical cluster analysis for identifying functionally related biological nodes and highlight the importance of subcellular partitioning of key signaling components for biological specificity. The phosphatidylinositol 3-kinase/Akt pathway regulates many biological processes, including insulin-regulated GLUT4 insertion into the plasma membrane. However, Akt operates well below its capacity to facilitate maximal GLUT4 translocation. Thus, reconciling modest changes in Akt expression or activity as a cause of metabolic dysfunction is complex. To resolve this, we examined insulin regulation of components within the signaling cascade in a quantitative kinetic and dose-response study combined with hierarchical cluster analysis. This revealed a strong relationship between phosphorylation of Akt substrates and GLUT4 translocation but not whole cell Akt phosphorylation. In contrast, Akt activity at the plasma membrane strongly correlated with GLUT4 translocation and Akt substrate phosphorylation. Additionally, two of the phosphorylated sites in the Akt substrate AS160 clustered separately, with Thr(P)-642 grouped with other Akt substrates. Further experiments suggested that atypical protein kinase Cζ phosphorylates AS160 at Ser-588 and that these two sites are mutually exclusive. These data indicate the utility of hierarchical cluster analysis for identifying functionally related biological nodes and highlight the importance of subcellular partitioning of key signaling components for biological specificity. IntroductionThe receptor tyrosine kinase family is both large and diverse controlling a broad spectrum of fundamental biological processes, including cell death, differentiation, and proliferation. Curiously, these diverse processes are controlled by a limited subset of canonical signaling modules typified by the phosphatidylinositol 3-kinase (PI 3The abbreviations used are: PIphosphatidylinositolPMplasma membraneIRSinsulin receptor substratePDGFplatelet-derived growth factorPDGFRPDGF receptorDMEMDulbecco's modified Eagle's mediumBSAbovine serum albuminPBSphosphate-buffered salineMES4-morpholineethanesulfonic acidPKCprotein kinase CMAPKmitogen-activated protein kinaseTCLtotal cell lysateLDMlow density microsomeTIRFtotal internal refection fluorescence. 3-kinase)/Akt and Ras/MAPK pathways. But how do relatively few signaling pathways control such a diversity of actions? To answer this question, it is essential to identify pathway components and to understand how they interact in different cells under a range of conditions. Considerable information about the components that include the PI 3-kinase/Akt pathway is known (1Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4598) Google Scholar). Activation of a receptor tyrosine kinase at the plasma membrane (PM) generates a binding site for the p85 regulatory subunit of PI 3-kinase allowing for production of phosphatidylinositol 3,4,5-trisphosphate at the PM. Phosphatidylinositol 3,4,5-trisphosphate serves as a docking site for proteins such as PDK1 (phosphoinositide-dependent kinase 1) and Akt that possess lipid-binding domains. This presumably concentrates Akt with its upstream regulatory kinases PDK1 and mammalian target of rapamycin-rictor complex (2Sarbassov D.D. Guertin D.A. Ali S.M. Sabatini D.M. Science. 2005; 307: 1098-1101Crossref PubMed Scopus (5160) Google Scholar, 3Hresko R.C. Mueckler M. J. Biol. Chem. 2005; 280: 40406-40416Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar) resulting in Akt phosphorylation at Thr-308 and Ser-473, respectively. Phosphorylation at these sites leads to a regulatory conformational change in Akt that facilitates its interaction with downstream substrates. Numerous Akt substrates possessing the Akt kinase consensus motif RXRXX(S/T) have been identified, and these have been implicated in a range of fundamental biological processes (4Manning B.D. Cantley L.C. Cell. 2007; 129: 1261-1274Abstract Full Text Full Text PDF PubMed Scopus (4620) Google Scholar).Evidence points to a major role for Akt in almost all of the metabolic actions of insulin (5Whiteman E.L. Cho H. Birnbaum M.J. Trends Endocrinol. Metab. 2002; 13: 444-451Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar). This includes the regulation of glucose transport that is mediated via the regulated translocation of the GLUT4 (glucose transporter 4) to the PM, glycogen synthesis, protein synthesis, lipolysis, and transcription. In each of these cases, Akt substrates that control these processes have been identified as follows: the RabGAP AS160/TBC1D4 (6Kane S. Sano H. Liu S.C. Asara J.M. Lane W.S. Garner C.C. Lienhard G.E. J. Biol. Chem. 2002; 277: 22115-22118Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar); GSK3 (glycogen synthase kinase 3) (7Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4327) Google Scholar); the RhebGAP TSC2 (tuberous sclerosis protein 2) (8Potter C.J. Pedraza L.G. Xu T. Nat. Cell Biol. 2002; 4: 658-665Crossref PubMed Scopus (771) Google Scholar); PDE3B (phosphodiesterase 3B) (9Kitamura T. Kitamura Y. Kuroda S. Hino Y. Ando M. Kotani K. Konishi H. Matsuzaki H. Kikkawa U. Ogawa W. Kasuga M. Mol. Cell. Biol. 1999; 19: 6286-6296Crossref PubMed Scopus (307) Google Scholar), and FoxO (10Biggs 3rd, W.H. Meisenhelder J. Hunter T. Cavenee W.K. Arden K.C. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 7421-7426Crossref PubMed Scopus (938) Google Scholar). Although many of these molecules are ubiquitous and many growth factors control the PI 3-kinase/Akt pathway, there are several facets of the insulin pathway that are unique as follows: insulin regulation of metabolism is confined to muscle, adipose tissue, and liver (5Whiteman E.L. Cho H. Birnbaum M.J. Trends Endocrinol. Metab. 2002; 13: 444-451Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar); the insulin pathway utilizes the insulin receptor substrate (IRS) scaffold protein family to orchestrate upstream activation of the Akt pathway (11White M.F. Mol. Cell. Biochem. 1998; 182: 3-11Crossref PubMed Scopus (623) Google Scholar); and metabolism is thought to be controlled by certain signaling isoforms within the PI 3-kinase/Akt pathway, including the p110α PI 3-kinase catalytic subunit (12Knight Z.A. Gonzalez B. Feldman M.E. Zunder E.R. Goldenberg D.D. Williams O. Loewith R. Stokoe D. Balla A. Toth B. Balla T. Weiss W.A. Williams R.L. Shokat K.M. Cell. 2006; 125: 733-747Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar, 13Foukas L.C. Claret M. Pearce W. Okkenhaug K. Meek S. Peskett E. Sancho S. Smith A.J. Withers D.J. Vanhaesebroeck B. Nature. 2006; 441: 366-370Crossref PubMed Scopus (384) Google Scholar) and the Akt2 isoform (14Cho H. Mu J. Kim J.K. Thorvaldsen J.L. Chu Q. Crenshaw 3rd, E.B. Kaestner K.H. Bartolomei M.S. Shulman G.I. Birnbaum M.J. Science. 2001; 292: 1728-1731Crossref PubMed Scopus (1536) Google Scholar, 15Jiang Z.Y. Zhou Q.L. Coleman K.A. Chouinard M. Boese Q. Czech M.P. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 7569-7574Crossref PubMed Scopus (303) Google Scholar, 16Katome T. Obata T. Matsushima R. Masuyama N. Cantley L.C. Gotoh Y. Kishi K. Shiota H. Ebina Y. J. Biol. Chem. 2003; 278: 28312-28323Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar).Impaired insulin action or insulin resistance plays a central role in a range of metabolic diseases, including type 2 diabetes. Here, it is generally considered that a defect in one or more of the upstream components of the PI 3-kinase/Akt pathway results in attenuation of signal transmission through Akt leading to global dysregulation of insulin action. For example, a modest reduction in tyrosine phosphorylation of IRS1 will lead to a modest reduction in PI 3-kinase activation in turn leading to a modest reduction in Akt activation and so on. This implies the existence of a robust coupling mechanism between each of the components that define the system. One prediction from this is that each of the components that include a functionally related node will exhibit behavioral kinship that should be measurable. However, we have recently observed features of the Akt node that do not support this hypothesis. Biological outputs such as GLUT4 translocation achieve a maximal stimulatory level at insulin concentrations that are sufficient to activate as little as 10–20% of the total cellular Akt pool (17Hoehn K.L. Hohnen-Behrens C. Cederberg A. Wu L.E. Turner N. Yuasa T. Ebina Y. James D.E. Cell Metab. 2008; 7: 421-433Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 18Whitehead J.P. Molero J.C. Clark S. Martin S. Meneilly G. James D.E. J. Biol. Chem. 2001; 276: 27816-27824Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Thus, this implies that Akt operates well below its maximal capacity. Under physiological conditions, most processes have evolved to operate over the steepest and most responsive part of their intrinsic range. However, this appears not to be the case for Akt. Intuitively, it is difficult to envisage how minute changes in Akt activity could accurately translate into large biological changes of the kind observed for GLUT4 translocation and so on. Consistent with this anomalous behavior, we recently observed that in a range of insulin resistance models where defects in Akt activity were in some cases evident, there was little parity between this and phosphorylation of other substrates such as the AS160 or GLUT4 translocation (17Hoehn K.L. Hohnen-Behrens C. Cederberg A. Wu L.E. Turner N. Yuasa T. Ebina Y. James D.E. Cell Metab. 2008; 7: 421-433Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). In this study, we set out to investigate the topology of a range of components within the insulin-signaling network to test the behavioral kinship hypothesis. We found that a simple dose-response analysis provided a powerful behavioral index of individual components. Hierarchical cluster analysis allowed us to sort components into related groups providing the first clue that Akt phosphorylation at either Thr-308 or Ser-473 in whole cells maintains a poor relationship with insulin-dependent phosphorylation of a range of Akt substrates, including AS160, GSK3, FoxO, and TSC2. In contrast, there was a strong relationship between Akt phosphorylation at the PM with GLUT4 translocation and AS160 phosphorylation at its major 14-3-3-binding site Thr-642. Interestingly, among the Akt substrates, AS160 is clustered closest to GLUT4 translocation. This is striking because AS160, but none of the other substrates, has been implicated as a major regulator of GLUT4 translocation. Hence, these studies support a model of behavioral kinship suggesting that similar analyses might be useful in mapping functional signaling nodes within complex networks. Our studies also show that subcellular localization of signaling components is likely one of the major determinants of specificity, and so in terms of mapping mechanistic disease loci this may represent a major target.DISCUSSIONHierarchical clustering of the canonical PI 3-kinase/Akt pathway in 3T3-L1 adipocytes using dose and temporal variables reveals several novel topological features of this network. First, Akt substrates, including TSC2, GSK3, AS160, and FoxO, belonged to a distinct cluster that could be segregated from non-Akt substrates such as S6 kinase and p42/p44 MAPK. Second, a disparity was observed between phosphorylation of a range of Akt substrates and Akt itself, and this was resolved by considering the spatial localization of active Akt, implicating an important role for Akt at the PM. Third, Akt phosphorylation at the PM was highly clustered with AS160 phosphorylation at Thr-642 as well as with GLUT4 translocation providing independent evidence for a functional link between these components. Finally, phosphorylation of AS160 at an alternate putative Akt site, Ser-588 did not cluster with Thr-642 phosphorylation nor with other Akt substrates suggesting that this is not an Akt substrate. Further functional studies revealed that Ser-588 in AS160 is a substrate of atypical PKCζ and that phosphorylation at the Ser-588 and Thr-642 sites is mutually exclusive suggesting a sequential mode of regulation.The starting point for these studies was that the ED50 for insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes was significantly less than that of insulin-stimulated Akt activation. This did not fit with the concept that the Akt pathway is necessary and sufficient for this particular biological action of insulin because if this were the case one would predict direct transmission between individual pathway components and thus a more predictable behavior of the pathway as a whole. This was borne out in analysis of a group of Akt substrates, which elicited behavior more analogous to GLUT4 translocation than to Akt phosphorylation itself. This discrepancy at the Akt node was resolved with the realization that Akt displays spatially restricted behavior in that Akt phosphorylation at the PM, but not in the whole cell lysate, was highly correlated with GLUT4 translocation. It is perhaps not surprising that a functional pool of Akt resides at the PM because this is where the enzyme is activated. As to whether this reflects preferential localization of a particular Akt isoform to the plasma membrane, as recently suggested by Gonzalez and McGraw (31Gonzalez E. McGraw T.E. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 7004-7009Crossref PubMed Scopus (140) Google Scholar), will require further study. The observation that Akt phosphorylation at the PM is highly clustered with the phosphorylation of several Akt substrates raises the possibility that phosphorylation of these substrates may well occur at the PM or another restricted compartment. This conclusion was supported by subcellular fractionation studies, which showed an enrichment of the phospho-Akt substrates in the PM fraction. Consistent with the PM as a central location of the Akt node, it has been shown that selective targeting of Akt to the PM (32Kohn A.D. Summers S.A. Birnbaum M.J. Roth R.A. J. Biol. Chem. 1996; 271: 31372-31378Abstract Full Text Full Text PDF PubMed Scopus (1086) Google Scholar, 33Ng Y. Ramm G. Lopez J.A. James D.E. Cell Metab. 2008; 7: 348-356Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) results in Akt activation concomitant with robust GLUT4 translocation and phosphorylation of AS160, GSK3, and S6 kinase.Cluster analysis of multisite phosphorylation of AS160 further exemplified the utility of this approach for dissecting novel features of signaling pathways. Although phosphorylation of AS160 at Thr-642 was highly clustered with GLUT4 translocation, this was not the case for another putative Akt site at Ser-588. Further analysis revealed that phosphorylation of AS160 at Ser-588 is probably regulated by atypical PKCζ and not by Akt directly, and phosphorylation at Ser-588 and Thr-642 is mutually exclusive. This raises the possibility that AS160 regulation is more complex than previously realized involving either a multisite phosphorylation cascade or two functionally distinct pools of AS160. Phosphorylation of Thr-642 on AS160 is important for 14-3-3 binding and GLUT4 trafficking (20Ramm G. Larance M. Guilhaus M. James D.E. J. Biol. Chem. 2006; 281: 29174-29180Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), and many agonists that regulate GLUT4 translocation induce phosphorylation at this site (28Stöckli J. Davey J.R. Hohnen-Behrens C. Xu A. James D.E. Ramm G. Mol. Endocrinol. 2008; 22: 2703-2715Crossref PubMed Scopus (52) Google Scholar). On the other hand, this was not the case for Ser-588, and the importance of Ser-588 phosphorylation is still not well understood (28Stöckli J. Davey J.R. Hohnen-Behrens C. Xu A. James D.E. Ramm G. Mol. Endocrinol. 2008; 22: 2703-2715Crossref PubMed Scopus (52) Google Scholar). It should also be noted that AS160 possesses several other regulated phosphorylation sites (34Geraghty K.M. Chen S. Harthill J.E. Ibrahim A.F. Toth R. Morrice N.A. Vandermoere F. Moorhead G.B. Hardie D.G. MacKintosh C. Biochem. J. 2007; 407: 231-241Crossref PubMed Scopus (139) Google Scholar) that require more detailed analysis.Despite the fact that the Akt substrates do cluster based on dose-response and time course variables, it was of interest that taking dose response alone into account, which tends to dominate this relationship, then among the four Akt substrates studied, the strongest link to GLUT4 translocation was with AS160. This is striking in view of the known role of AS160 in GLUT4 trafficking in 3T3-L1 adipocytes (21Larance M. Ramm G. Stöckli J. van Dam E.M. Winata S. Wasinger V. Simpson F. Graham M. Junutula J.R. Guilhaus M. James D.E. J. Biol. Chem. 2005; 280: 37803-37813Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 24Sano H. Kane S. Sano E. Mîinea C.P. Asara J.M. Lane W.S. Garner C.W. Lienhard G.E. J. Biol. Chem. 2003; 278: 14599-14602Abstract Full Text Full Text PDF PubMed Scopus (721) Google Scholar). In contrast, FoxO, TSC2, and GSK3 regulate transcription, protein synthesis, and glycogen synthesis, respectively (4Manning B.D. Cantley L.C. Cell. 2007; 129: 1261-1274Abstract Full Text Full Text PDF PubMed Scopus (4620) Google Scholar). Thus, it is of interest to discern other unique features of AS160 regulation that distinguish it from these other substrates. First, the specific activity of Thr(P)-642 AS160 in the PM was considerably higher than that of other substrates. Second, within the PM, Thr(P)-642 AS160 was confined to a detergent-insoluble subdomain as was a limited pool of active Akt. Finally, the stoichiometry of AS160 phosphorylation at Thr(P)-642 was very low with the majority of the protein being found in the LDM fraction. These observations highlight the fact that unlike other Akt substrates, such as FoxO, insulin regulation of AS160 is confined to a very discrete subpopulation of AS160 molecules. These data raise novel insights into the process as well as new questions concerning AS160 regulation. Previously, it was thought that AS160 functions by binding to intracellular GLUT4 vesicles in the absence of insulin and inhibiting a Rab required for GLUT4 trafficking with its Rab GTPase-activating protein (RabGAP) domain (21Larance M. Ramm G. Stöckli J. van Dam E.M. Winata S. Wasinger V. Simpson F. Graham M. Junutula J.R. Guilhaus M. James D.E. J. Biol. Chem. 2005; 280: 37803-37813Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Insulin-dependent phosphorylation of AS160 was suggested to inhibit its RabGAP activity, thus promoting Rab GTP loading and GLUT4 trafficking (21Larance M. Ramm G. Stöckli J. van Dam E.M. Winata S. Wasinger V. Simpson F. Graham M. Junutula J.R. Guilhaus M. James D.E. J. Biol. Chem. 2005; 280: 37803-37813Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 24Sano H. Kane S. Sano E. Mîinea C.P. Asara J.M. Lane W.S. Garner C.W. Lienhard G.E. J. Biol. Chem. 2003; 278: 14599-14602Abstract Full Text Full Text PDF PubMed Scopus (721) Google Scholar). In this model, however, there is no a priori role for AS160 at the PM. Based on these data, we feel it is necessary to consider an alternative model where the major functional pool of AS160 resides in a specific location within the PM. This localization may serve to bring AS160 into proximity of a discrete pool of active Akt and possibly other molecules such as the putative Rab substrate. In this model, phosphorylation of AS160 may not only affect the RabGAP activity of AS160 but also its ability to act as an effector for vesicle docking and fusion with the PM, an event that might ultimately be linked to its GTPase-activating protein activity to recycle the components that underpin this process. It will be of interest to determine how AS160 and Akt are targeted to the discrete PM subdomain and as to whether these proteins form a complex at this site. Recent studies have identified Akt-binding proteins that may serve such a function. These include APPL1 (35Saito T. Jones C.C. Huang S. Czech M.P. Pilch P.F. J. Biol. Chem. 2007; 282: 32280-32287Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) and ClipR-59 (27Ding J. Du K. Mol. Cell. Biol. 2009; 29: 1459-1471Crossref PubMed Scopus (28) Google Scholar), both of which have been implicated in insulin action and GLUT4 trafficking. It is, however, clear from this study that the regulation of AS160 by Akt and that of other Akt substrates is distinct, which is consistent with the possibility that different Akt-binding proteins selectively tether the kinase to unique substrates. For example, endosomal partitioning of Akt by APPL1 appears to control phosphorylation of GSK3 but not TSC2 (36Schenck A. Goto-Silva L. Collinet C. Rhinn M. Giner A. Habermann B. Brand M. Zerial M. Cell. 2008; 133: 486-497Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). In contrast, targeting of Akt to lipid rafts controls the phosphorylation of a different subset of substrates (26Adam R.M. Mukhopadhyay N.K. Kim J. Di Vizio D. Cinar B. Boucher K. Solomon K.R. Freeman M.R. Cancer Res. 2007; 67: 6238-6246Crossref PubMed Scopus (103) Google Scholar).These studies validate the prediction that functionally linked intermediates in complex signal transduction networks display highly related behavior. This behavior can be mapped based upon analysis of a relatively simple set of variables that include the dose-response relationship and the kinetics of the response. By subjecting such data to hierarchical cluster analysis, it is feasible to make novel predictions about the topology and composition of signaling pathways. This study supports the emerging concept that subcellular localization plays a central role in determining the sensitivity, specificity, and kinetics of signaling that may skew interpretation of studies that have overlooked this parameter. In this context, we found that the dose response of Akt activation at the PM but not in the whole cell lysate correlates with GLUT4 translocation. Further studies are required to identify alternate Akt substrates in the PM that could play an important role in this process. These findings bring us a step closer to understanding the physiology of insulin action and how it might go awry. IntroductionThe receptor tyrosine kinase family is both large and diverse controlling a broad spectrum of fundamental biological processes, including cell death, differentiation, and proliferation. Curiously, these diverse processes are controlled by a limited subset of canonical signaling modules typified by the phosphatidylinositol 3-kinase (PI 3The abbreviations used are: PIphosphatidylinositolPMplasma membraneIRSinsulin receptor substratePDGFplatelet-derived growth factorPDGFRPDGF receptorDMEMDulbecco's modified Eagle's mediumBSAbovine serum albuminPBSphosphate-buffered salineMES4-morpholineethanesulfonic acidPKCprotein kinase CMAPKmitogen-activated protein kinaseTCLtotal cell lysateLDMlow density microsomeTIRFtotal internal refection fluorescence. 3-kinase)/Akt and Ras/MAPK pathways. But how do relatively few signaling pathways control such a diversity of actions? To answer this question, it is essential to identify pathway components and to understand how they interact in different cells under a range of conditions. Considerable information about the components that include the PI 3-kinase/Akt pathway is known (1Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4598) Google Scholar). Activation of a receptor tyrosine kinase at the plasma membrane (PM) generates a binding site for the p85 regulatory subunit of PI 3-kinase allowing for production of phosphatidylinositol 3,4,5-trisphosphate at the PM. Phosphatidylinositol 3,4,5-trisphosphate serves as a docking site for proteins such as PDK1 (phosphoinositide-dependent kinase 1) and Akt that possess lipid-binding domains. This presumably concentrates Akt with its upstream regulatory kinases PDK1 and mammalian target of rapamycin-rictor complex (2Sarbassov D.D. Guertin D.A. Ali S.M. Sabatini D.M. Science. 2005; 307: 1098-1101Crossref PubMed Scopus (5160) Google Scholar, 3Hresko R.C. Mueckler M. J. Biol. Chem. 2005; 280: 40406-40416Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar) resulting in Akt phosphorylation at Thr-308 and Ser-473, respectively. Phosphorylation at these sites leads to a regulatory conformational change in Akt that facilitates its interaction with downstream substrates. Numerous Akt substrates possessing the Akt kinase consensus motif RXRXX(S/T) have been identified, and these have been implicated in a range of fundamental biological processes (4Manning B.D. Cantley L.C. Cell. 2007; 129: 1261-1274Abstract Full Text Full Text PDF PubMed Scopus (4620) Google Scholar).Evidence points to a major role for Akt in almost all of the metabolic actions of insulin (5Whiteman E.L. Cho H. Birnbaum M.J. Trends Endocrinol. Metab. 2002; 13: 444-451Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar). This includes the regulation of glucose transport that is mediated via the regulated translocation of the GLUT4 (glucose transporter 4) to the PM, glycogen synthesis, protein synthesis, lipolysis, and transcription. In each of these cases, Akt substrates that control these processes have been identified as follows: the RabGAP AS160/TBC1D4 (6Kane S. Sano H. Liu S.C. Asara J.M. Lane W.S. Garner C.C. Lienhard G.E. J. Biol. Chem. 2002; 277: 22115-22118Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar); GSK3 (glycogen synthase kinase 3) (7Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4327) Google Scholar); the RhebGAP TSC2 (tuberous sclerosis protein 2) (8Potter C.J. Pedraza L.G. Xu T. Nat. Cell Biol. 2002; 4: 658-665Crossref PubMed Scopus (771) Google Scholar); PDE3B (phosphodiesterase 3B) (9Kitamura T. Kitamura Y. Kuroda S. Hino Y. Ando M. Kotani K. Konishi H. Matsuzaki H. Kikkawa U. Ogawa W. Kasuga M. Mol. Cell. Biol. 1999; 19: 6286-6296Crossref PubMed Scopus (307) Google Scholar), and FoxO (10Biggs 3rd, W.H. Meisenhelder J. Hunter T. Cavenee W.K. Arden K.C. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 7421-7426Crossref PubMed Scopus (938) Google Scholar). Although many of these molecules are ubiquitous and many growth factors control the PI 3-kinase/Akt pathway, there are several facets of the insulin pathway that are unique as follows: insulin regulation of metabolism is confined to muscle, adipose tissue, and liver (5Whiteman E.L. Cho H. Birnbaum M.J. Trends Endocrinol. Metab. 2002; 13: 444-451Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar); the insulin pathway utilizes the insulin receptor substrate (IRS) scaffold protein family to orchestrate upstream activation of the Akt pathway (11White M.F. Mol. Cell. Biochem. 1998; 182: 3-11Crossref PubMed Scopus (623) Google Scholar); and metabolism is thought to be controlled by certain signaling isoforms within the PI 3-kinase/Akt pathway, including the p110α PI 3-kinase catalytic subunit (12Knight Z.A. Gonzalez B. Feldman M.E. Zunder E.R. Goldenberg D.D. Williams O. Loewith R. Stokoe D. Balla A. Toth B. Balla T. Weiss W.A. Williams R.L. Shokat K.M. Cell. 2006; 125: 733-747Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar, 13Foukas L.C. Claret M. Pearce W. Okkenhaug K. Meek S. Peskett E. Sancho S. Smith A.J. Withers D.J. Vanhaesebroeck B. Nature. 2006; 441: 366-370Crossref PubMed Scopus (384) Google Scholar) and the Akt2 isoform (14Cho H. Mu J. Kim J.K. Thorvaldsen J.L. Chu Q. Crenshaw 3rd, E.B. Kaestner K.H. Bartolomei M.S. Shulman G.I. Birnbaum M.J. Science. 2001; 292: 1728-1731Crossref PubMed Scopus (1536) Google Scholar, 15Jiang Z.Y. Zhou Q.L. Coleman K.A. Chouinard M. Boese Q. Czech M.P. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 7569-7574Crossref PubMed Scopus (303) Google Scholar, 16Katome T. Obata T. Matsushima R. Masuyama N. Cantley L.C. Gotoh Y. Kishi K. Shiota H. Ebina Y. J. Biol. Chem. 2003; 278: 28312-28323Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar).Impaired insulin action or insulin resistance plays a central role in a range of metabolic diseases, including type 2 diabetes. Here, it is generally considered that a defect in one or more of the upstream components of the PI 3-kinase/Akt pathway results in attenuation of signal transmission through Akt leading to global dysregulation of insulin action. For example, a modest reduction in tyrosine phosphorylation of IRS1 will lead to a modest reduction in PI 3-kinase activation in turn leading to a modest reduction in Akt activation and so on. This implies the existence of a robust coupling mechanism between each of the components that define the system. One prediction from this is that each of the components that include a functionally related node will exhibit behavioral kinship that should be measurable. However, we have recently observed features of the Akt node that do not support this hypothesis. Biological outputs such as GLUT4 translocation achieve a maximal stimulatory level at insulin concentrations that are sufficient to activate as little as 10–20% of the total cellular Akt pool (17Hoehn K.L. Hohnen-Behrens C. Cederberg A. Wu L.E. Turner N. Yuasa T. Ebina Y. James D.E. Cell Metab. 2008; 7: 421-433Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 18Whitehead J.P. Molero J.C. Clark S. Martin S. Meneilly G. James D.E. J. Biol. Chem. 2001; 276: 27816-27824Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Thus, this implies that Akt operates well below its maximal capacity. Under physiological conditions, most processes have evolved to operate over the steepest and most responsive part of their intrinsic range. However, this appears not to be the case for Akt. Intuitively, it is difficult to envisage how minute changes in Akt activity could accurately translate into large biological changes of the kind observed for GLUT4 translocation and so on. Consistent with this anomalous behavior, we recently observed that in a range of insulin resistance models where defects in Akt activity were in some cases evident, there was little parity between this and phosphorylation of other substrates such as the AS160 or GLUT4 translocation (17Hoehn K.L. Hohnen-Behrens C. Cederberg A. Wu L.E. Turner N. Yuasa T. Ebina Y. James D.E. Cell Metab. 2008; 7: 421-433Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). In this study, we set out to investigate the topology of a range of components within the insulin-signaling network to test the behavioral kinship hypothesis. We found that a simple dose-response analysis provided a powerful behavioral index of individual components. Hierarchical cluster analysis allowed us to sort components into related groups providing the first clue that Akt phosphorylation at either Thr-308 or Ser-473 in whole cells maintains a poor relationship with insulin-dependent phosphorylation of a range of Akt substrates, including AS160, GSK3, FoxO, and TSC2. In contrast, there was a strong relationship between Akt phosphorylation at the PM with GLUT4 translocation and AS160 phosphorylation at its major 14-3-3-binding site Thr-642. Interestingly, among the Akt substrates, AS160 is clustered closest to GLUT4 translocation. This is striking because AS160, but none of the other substrates, has been implicated as a major regulator of GLUT4 translocation. Hence, these studies support a model of behavioral kinship suggesting that similar analyses might be useful in mapping functional signaling nodes within complex networks. Our studies also show that subcellular localization of signaling components is likely one of the major determinants of specificity, and so in terms of mapping mechanistic disease loci this may represent a major target.
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