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Specific Increase in p85α Expression in Response to Dexamethasone Is Associated with Inhibition of Insulin-like Growth Factor-I Stimulated Phosphatidylinositol 3-Kinase Activity in Cultured Muscle Cells*

  • Francesco Giorgino
    Footnotes
    Affiliations
    Research Division, Joslin Diabetes Center, and Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02215 and the

    Istituto di Clinica Medica, Endocrinologia e Malattie Metaboliche, University of Bari School of Medicine, Bari, Italy 70124
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  • Michael T. Pedrini
    Footnotes
    Affiliations
    Research Division, Joslin Diabetes Center, and Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02215 and the
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  • Lucrezia Matera
    Affiliations
    Istituto di Clinica Medica, Endocrinologia e Malattie Metaboliche, University of Bari School of Medicine, Bari, Italy 70124
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  • Robert J. Smith
    Correspondence
    To whom reprint requests should be addressed
    Affiliations
    Research Division, Joslin Diabetes Center, and Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02215 and the
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  • Author Footnotes
    * This work was supported in part by the Juvenile Diabetes Foundation and National Institutes of Health Grants DK43038, DK48503, DK50411, Trauma Center Grant GM36428, and Diabetes and Endocrinology Research Center Grant DK36836 (to R. J. S.) and a grant from the Societa' Italiana di Diabetologia (Fondo per la Ricerca Eli Lilly, 1994-1996) (to F. G.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    Supported by postdoctoral fellowships from the Juvenile Diabetes Foundation International and the Mary K. Iacocca Foundation.
    Supported by a postdoctoral fellowship from the Max Kade Foundation.
    1 The abbreviations used are:PIphosphatidylinositolIGF-Iinsulin-like growth factor-IPDGFplatelet-derived growth factorIRS-1insulin receptor substrate-1SHsrc homologyp85regulatory subunit of PI 3-kinase of 85 kDap110catalytic subunit of PI 3-kinase of 110 kDaMEMminimal essential mediumPYphosphotyrosine.
Open AccessPublished:March 14, 1997DOI:https://doi.org/10.1074/jbc.272.11.7455
      The stimulation of phosphatidylinositol (PI) 3-kinase by insulin-like growth factor I (IGF-I) in L6 cultured skeletal muscle cells is inhibited by the glucocorticoid dexamethasone. The objective of this study was to investigate the mechanism of dexamethasone action by determining its effects on the expression of the p85α and p85β regulatory subunit isoforms of PI 3-kinase, their coupling with the p110 catalytic subunit, and their association with insulin receptor substrate 1 (IRS-1) in response to IGF-I stimulation. Dexamethasone induced a 300% increase in p85α protein content in the L6 cultured myoblast cell line, whereas it increased p110 content by only 38% and had no effect on p85β. The increase in p85α protein was associated with a coordinate increase in p85α mRNA. Stimulation with IGF-I induced the association of p85α and p85β with IRS-1, and this was accompanied by increased amounts of the p110 catalytic subunit and markedly increased PI 3-kinase activity in IRS-1 immunoprecipitates. In cells treated with dexamethasone, greater amounts of p85α and lower amounts of p85β, respectively, were found in IRS-1 immunoprecipitates, such that the α/β ratio was markedly higher than in control cells. In spite of the increase in both total and IRS-1-associated p85α following dexamethasone treatment, IRS-1-associated p110 catalytic subunit and PI 3-kinase activity were decreased by approximately 50%. Thus, dexamethasone induces a specific increase in expression of the p85α regulatory subunit that is not associated with a coordinate increase in the p110 catalytic subunit of PI 3-kinase. As a consequence, in dexamethasone-treated cells, p85α that is not coupled with p110 competes with both p85α·p110 and p85β·p110 complexes for association with IRS-1, leading to increased p85α but decreased p85β, p110, and PI 3-kinase activity in IRS-1 immunoprecipitates.

      INTRODUCTION

      Growth factor activation of transmembrane tyrosine kinase receptors results in rapid recruitment of phosphatidylinositol (PI)

      The abbreviations used are:

      PI
      phosphatidylinositol
      IGF-I
      insulin-like growth factor-I
      PDGF
      platelet-derived growth factor
      IRS-1
      insulin receptor substrate-1
      SH
      src homology
      p85
      regulatory subunit of PI 3-kinase of 85 kDa
      p110
      catalytic subunit of PI 3-kinase of 110 kDa
      MEM
      minimal essential medium
      PY
      phosphotyrosine.
      3-kinase activity to tyrosine-phosphorylated proteins. In intact cells, PI 3-kinase catalyzes the phosphorylation of PI 4,5-bisphosphate (PI-4,5-P2) at the 3′-position of the inositol ring, thus leading to elevation in intracellular PI 3,4,5-trisphosphate (PI-3,4,5-P3) (reviewed in
      • Cantley L.C.
      • Auger K.R.
      • Carpenter C.L.
      • Duckworth B.C.
      • Graziani A.
      • Kapeller R.
      • Soltoff S.
      ). Unlike the products of PI kinases in the classical PI cycle (PI-4-P and PI-4,5-P2), 3-phosphorylated phosphoinositides are not cleaved by phospholipase C-γ (
      • Serunian L.A.
      • Haber M.T.
      • Fukui T.
      • Kim J.W.
      • Rhee S.G.
      • Lowenstein J.M.
      • Cantley L.C.
      ,
      • Lips D.L.
      • Majerus P.W.
      • Gorga F.R.
      • Young A.T.
      • Benjamin T.L.
      ), and it has been suggested that they may serve as intracellular second messengers for yet unidentified in vivo targets (
      • Cantley L.C.
      • Auger K.R.
      • Carpenter C.L.
      • Duckworth B.C.
      • Graziani A.
      • Kapeller R.
      • Soltoff S.
      ,
      • Toker A.
      • Meyer M.
      • Reddy K.K.
      • Falck J.R.
      • Aneja R.
      • Aneja S.
      • Parra A.
      • Burns D.J.
      • Ballas L.M.
      • Cantley L.C.
      ). Extensive experimental evidence has established a key role for PI 3-kinase in the signal transduction mechanisms of a number of peptide growth factors, including epidermal growth factor, platelet-derived growth factor (PDGF), insulin, and insulin-like growth factor-I (IGF-I). PI 3-kinase has thus been implicated in the regulation of multiple general and specialized cellular processes, including membrane ruffling (
      • Kotani K.
      • Yonezawa K.
      • Hara K.
      • Ueda H.
      • Kitamura Y.
      • Sakaue H.
      • Ando A.
      • Chavanieu A.
      • Calas B.
      • Grigorescu F.
      ), receptor endocytosis (
      • Joly M.
      • Kazlauskas A.
      • Fay F.S.
      • Corvera S.
      ), mitogenesis (
      • Fantl W.J.
      • Escobedo J.A.
      • Martin G.A.
      • Turck C.W.
      • del Rosario M.
      • McCormick F.
      • Williams L.T.
      ,
      • Valius M.
      • Kazlauskas A.
      ), cell differentiation (
      • Tomiyama K.
      • Nakata H.
      • Sasa H.
      • Arimura S.
      • Nishio E.
      • Watanabe Y.
      ), and insulin stimulation of glucose transport (
      • Gould G.W.
      • Jess T.J.
      • Andrews G.C.
      • Herbst J.J.
      • Plevin R.J.
      • Gibbs E.M.
      ) and glycogen synthesis (
      • Shepherd P.R.
      • Nave B.T.
      • Siddle K.
      ,
      • Yamamoto-Honda R.
      • Tobe K.
      • Kaburagi Y.
      • Ueki K.
      • Asai S.
      • Yachi M.
      • Shirouzu M.
      • Yodoi J.
      • Akanuma Y.
      • Yokoyama S.
      • Yazaki Y.
      • Kadowaki T.
      ).
      Mammalian PI 3-kinase is a heterodimer composed of an 85-kDa (p85) regulatory subunit and a 110-kDa (p110) catalytic subunit (
      • Carpenter C.L.
      • Duckworth B.C.
      • Auger K.R.
      • Cohen B.
      • Schaffhausen B.S.
      • Cantley L.C.
      ,
      • Fry M.J.
      • Panayotou G.
      • Dhand R.
      • Ruiz-Larrea F.
      • Gout I.
      • Nguyen O.
      • Courtneidge S.A.
      • Waterfield M.D.
      ). Two distinct and closely related 85-kDa protein isoforms, p85α and p85β, have been cloned and shown to be the products of separate genes (
      • Escobedo J.A.
      • Navankasattusas S.
      • Kavanaugh W.M.
      • Milfay D.
      • Fried V.A.
      • Williams L.T.
      ). Both of these p85 proteins have the capacity to form stable high affinity complexes with the p110 component of PI 3-kinase (
      • Gout I.
      • Dhand R.
      • Panayotou G.
      • Fry M.J.
      • Hiles I.
      • Otsu M.
      • Waterfield M.D.
      ,
      • Pawson T.
      • Gish G.D.
      ). The two p85 isoforms have a multidomain structure, containing two SH2 (src homology 2) domains, one SH3 domain, and a region with significant sequence similarity to a GTPase-activating protein domain of the product of the breakpoint cluster region gene (
      • Dhand R.
      • Hara K.
      • Hiles I.
      • Bax B.
      • Gout I.
      • Panayotou G.
      • Fry M.J.
      • Yonezawa K.
      • Kasuga M.
      • Waterfield M.D.
      ). The presence of several functional domains suggests that the p85 proteins may have multiple interactive and regulatory roles. At present, functional differences between the p85α and p85β protein isoforms have not been established.
      Two forms of p110 also have been cloned, one from bovine brain designated p110 (
      • Hiles I.D.
      • Otsu M.
      • Volinia S.
      • Fry M.J.
      • Gout I.
      • Dhand R.
      • Panayotou G.
      • Ruiz-Larrea F.
      • Thompson A.
      • Totty N.F.
      • Hsuan J.J.
      • Courtneidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ), and a second human variant designated p110β (
      • Hu P.
      • Mondino A.
      • Skolnik E.Y.
      • Schlessinger J.
      ). Expression studies have demonstrated that both p110 proteins have intrinsic PI 3-kinase activity and can associate with the p85 component in intact cells (
      • Hiles I.D.
      • Otsu M.
      • Volinia S.
      • Fry M.J.
      • Gout I.
      • Dhand R.
      • Panayotou G.
      • Ruiz-Larrea F.
      • Thompson A.
      • Totty N.F.
      • Hsuan J.J.
      • Courtneidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ,
      • Hu P.
      • Mondino A.
      • Skolnik E.Y.
      • Schlessinger J.
      ). The domains in p85 and p110 required for subunit interaction have been identified and mapped to an amino acid sequence between the two SH2 domains of p85 and an NH2-terminal amino acid sequence of p110, respectively (
      • Hiles I.D.
      • Otsu M.
      • Volinia S.
      • Fry M.J.
      • Gout I.
      • Dhand R.
      • Panayotou G.
      • Ruiz-Larrea F.
      • Thompson A.
      • Totty N.F.
      • Hsuan J.J.
      • Courtneidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ,
      • Holt K.H.
      • Olson A.L.
      • Moye-Rowley W.S.
      • Pessin J.E.
      ). These studies have established a structural model of the PI 3-kinase complex, in which the p110 subunit contains catalytic activity and is tightly associated with the p85 subunit, which acts as an adaptor and/or regulatory subunit. Integrity of the p85·p110 complex appears to be necessary for p110 catalytic activity (
      • Klippel A.
      • Escobedo J.A.
      • Hirano M.
      • Williams L.T.
      ). Thus, overexpression of the p85 subunit or a portion of the p85 protein, such as an intact p85 SH2 domain, through transfection or microinjection of cells results in inhibition of PI 3-kinase activation and cell signaling (
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Lammers R.
      • Ullrich A.
      • Schlessinger J.
      ,
      • Jhun B.H.
      • Rose D.W.
      • Seely B.L.
      • Rameh L.
      • Cantley L.C.
      • Saltiel A.R.
      • Olefsky J.M.
      ). The physiological occurrence of selective up-regulation of p85 expression as a mechanism of inhibition of PI 3-kinase activity has not been investigated.
      The best characterized mode of PI 3-kinase activation in response to peptide growth factors involves changes induced in the p85 protein upon binding to certain phosphorylated tyrosine residues, which are then transmitted to the associated p110 catalytic subunit and cause its activation. In the case of the epidermal growth factor and PDGF receptors, the p85 protein binds directly to phosphorylated tyrosines in the receptor molecule through its SH2 domains (reviewed in
      • Cantley L.C.
      • Auger K.R.
      • Carpenter C.L.
      • Duckworth B.C.
      • Graziani A.
      • Kapeller R.
      • Soltoff S.
      ). In the case of the receptors for insulin and IGF-I, only a limited fraction of the total cell PI 3-kinase associates directly with the receptor, while most of PI 3-kinase interacts with specific tyrosine-phosphorylation sites of the receptor substrates IRS-1 and IRS-2 (
      • Yamamoto K.
      • Lapetina E.G.
      • Moxham C.P.
      ). Binding of p85α to tyrosine-phosphorylated IRS-1 or phosphorylated IRS-1-related YMXM peptide sequences results in increased catalytic activity of the PI 3-kinase complex (
      • Myers Jr., M.G.
      • Backer J.M.
      • Sun X.-J.
      • Shoelson S.E.
      • Hu P.
      • Schlessinger J.
      • Yoakim M.
      • Schaffhausen B.
      • White M.F.
      ,
      • Backer J.M.
      • Myers Jr., M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.-J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      • White M.F.
      ). Tyr-608 and Tyr-939 of IRS-1 appear to be the predominant sites for interaction with the amino-terminal SH2 domain of p85α (
      • Sun X.-J.
      • Crimmins D.L.
      • Myers Jr., M.G.
      • Miralpeix M.
      • White M.F.
      ). Although these studies have provided important insight into the mechanism of PI 3-kinase activation in response to insulin and IGF-I, limited information is available on the relative abundance of p85α and p85β within the IRS-1·PI 3-kinase complex and the coupling of IRS-1-associated p85 isoforms with catalytically active p110 subunits.
      In a previous report (
      • Giorgino F.
      • Smith R.J.
      ), we described the inhibition of IGF-I activated PI 3-kinase activity by the glucocorticoid dexamethasone in the L6 skeletal muscle cell line. The objective of this study was to investigate the effects of dexamethasone on the expression of p85α and p85β isoforms of PI 3-kinase in L6 cells, their coupling with the p110 catalytic subunit, and their association with IRS-1 in response to IGF-I stimulation. We show that L6 myoblasts express both p85α and p85β and that the expression of p85α, but not that of p85β, is specifically increased by dexamethasone. The increase in the cellular pool of p85α correlates with an increased amount of this protein associated with IRS-1 after IGF-I stimulation. However, despite the increase in IRS-1-associated p85α, both p110 and PI 3-kinase activity associated with IRS-1 are diminished. These data support the concept that a substantial fraction of p85α is “free” (i.e. not coupled with p110) in dexamethasone-treated cells. We propose that the selective increase in p85α expression may represent a novel physiological mechanism leading to inhibition of PI 3-kinase activity by glucocorticoids.

      DISCUSSION

      This study demonstrates that both the cellular amounts of the various subunits constituting the PI 3-kinase enzyme complex and PI 3-kinase subunit association with tyrosine-phosphorylated IRS-1 are differentially regulated by the glucocorticoid dexamethasone in undifferentiated L6 skeletal muscle cells. Dexamethasone markedly increased p85α in L6 myoblasts, but did not alter the levels of p85β and induced only a modest increase in cellular p110 content. Under these conditions, a greater amount of p85α and reduced amounts of both p85β and p110 were recruited to the IGF-I receptor substrate IRS-1 upon hormone stimulation. In addition, the activity of PI 3-kinase measured in IRS-1 immune complexes was significantly decreased by dexamethasone, likely reflecting the reduced amounts of IRS-1-associated p110 catalytic subunit.
      Glucocorticoids have been reported to increase the amount of p85 protein in rat skeletal muscle (
      • Saad M.J.A.
      • Folli F.
      • Kahn J.A.
      • Kahn C.R.
      ) and in F442A adipocytes (
      • Saad M.J.A.
      • Folli F.
      • Araki E.
      • Hashimoto N.
      • Csermely P.
      • Kahn C.R.
      ). In these previous studies, the p85 isoform pattern was not determined and, therefore, it is not known whether the effects of dexamethasone were isoform-specific. In L6 myoblasts, the increase in p85α protein content was associated with an increase in p85α mRNA and no change in p85β mRNA, suggesting that dexamethasone may act by specifically increasing expression of the p85α gene. The p85α and p85β isoforms possess 62% overall identity at the amino acid level and 58% nucleotide identity and, thus, are thought to be encoded by two distinct but related genes (
      • Otsu M.
      • Hiles I.
      • Gout I.
      • Fry M.J.
      • Ruis-Larrea F.
      • Panayotou G.
      • Thompson A.
      • Dhand R.
      • Hsuan J.
      • Totty N.
      • Smith A.D.
      • Morgan S.J.
      • Courtneidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ). Information on p85 gene structure is very limited at present and, in future studies, it will be important to identify the gene regulatory elements that dictate the tissue distribution of the two p85 isoforms as well as the glucocorticoid responsiveness limited to p85α. Our data would indicate that glucocorticoid response elements may be identified exclusively in the p85α gene (and not in the p85β gene).
      The selective regulation of the α isoform of p85 by dexamethasone supports the concept that p85α and p85β may have distinct biological roles. Although the related p85α and p85β regulatory subunits both have been shown to form stable complexes with the catalytic p110 component of PI 3-kinase (
      • Gout I.
      • Dhand R.
      • Panayotou G.
      • Fry M.J.
      • Hiles I.
      • Otsu M.
      • Waterfield M.D.
      ,
      • Pawson T.
      • Gish G.D.
      ), functional differences of p85α as compared to p85β previously have been reported. Studies conducted in T-lymphocytes have demonstrated that the two p85 isoforms have a different phosphorylation pattern upon T-cell activation (
      • Baltensperger K.
      • Kozma L.M.
      • Jaspers S.R.
      • Czech M.P.
      ). Following activation of the CD3 antigen complex in T-cells, rapid serine phosphorylation of p85α was observed, whereas phosphorylation of p85β was unchanged. In addition, the catalytic subunit p110 was shown to undergo rapid threonine phosphorylation when associated with p85β but not with p85α. It has recently been reported that much larger stimulation of PI 3-kinase is found in p85α compared to p85β immunoprecipitates upon insulin stimulation of CHO-T cells, even though both p85α and p85β associate with IRS-1(45). This has led to the suggestion that insulin causes recruitment of both p85α and p85β regulatory subunits to IRS-1 signaling complexes, but the activity of IRS-1-associated PI 3-kinase is stimulated only in the p85α·p110 complex, with little or no stimulation of the p85β·p110·PI 3-kinase complex. The results in the current study demonstrating specific augmentation of p85α protein content by dexamethasone indicate for the first time that the control of p85α and p85β expression represents an additional level of differential regulation of the two p85 isoforms.
      IGF-I stimulation of L6 myoblasts induced a severalfold increase in the association of both p85α and p85β isoforms with IRS-1 immune complexes. Both p85 isoforms have reportedly been shown to associate with IRS-1 signaling complexes upon insulin stimulation in COS-1 cells transiently transfected with the insulin receptor and in CHO-T cells stably overexpressing the insulin receptor (
      • Reif K.
      • Gout I.
      • Waterfield M.D.
      • Cantrell D.A.
      ). In the latter study, the α/β ratio in IRS-1 immune complexes generally reflected the α/β ratio in the total cell lysate, indicating no preferential recruitment of a given p85 isoform to IRS-1 (
      • Reif K.
      • Gout I.
      • Waterfield M.D.
      • Cantrell D.A.
      ). By contrast, our results indicate that p85β may preferentially associate with IRS-1 in signaling complexes, since the association of p85β with IRS-1 immune complexes was greater quantitatively than that of p85α in response to IGF-I stimulation (Fig. 7, Fig. 8). It is possible that association of p85α and p85β with IRS-1 signaling complexes may be modulated in a cell context specific manner and differ in cell lines expressing high levels of insulin receptors in which hyperphosphorylation of IRS-1 may occur. Interestingly, the amount of p85α in anti-phosphotyrosine immunoprecipitates from PDGF-stimulated L6 myoblasts was significantly higher than the amount of p85β and thus the α/β ratio was much greater than in IRS-1 immune complexes.
      F. Giorgino and R. J. Smith, unpublished results.
      This suggests that association of p85α and p85β with tyrosine-phosphorylated proteins in L6 skeletal muscle cells may differ depending on the specific protein target (i.e. IRS-1 versus the PDGF receptor).
      Ample experimental evidence has established the concept of IRS-1 acting as a “docking protein” capable of simultaneously binding multiple protein components through specific amino acid motifs containing phosphorylated tyrosine residues. GST fusions of the NH2-terminal SH2 domain of p85α were found to bind strongly to Tyr608-Met-Pro-Met and Tyr939-Met-Asn-Met and, to a lesser extent, to Tyr461-Ile-Cys-Met and Tyr987-Met-Tyr-Met in IRS-1 (
      • Sun X.-J.
      • Crimmins D.L.
      • Myers Jr., M.G.
      • Miralpeix M.
      • White M.F.
      ). At present, it is not known whether the same sites serve also for the two SH2 domains present in p85β. Although both p85α and p85β were shown to bind simultaneously to a single IRS-1 molecule in COS-1 cells transiently transfected with the insulin receptor, this was not the case in CHO-T cells stably expressing the insulin receptor (
      • Reif K.
      • Gout I.
      • Waterfield M.D.
      • Cantrell D.A.
      ). In the PDGF receptor, Tyr751 is a common binding site for both Nck and p85α, indicating that SH2 domains of different signaling molecules can compete for binding to the same phosphorylated tyrosine motif (
      • Nishimura R.
      • Li W.
      • Kashishian A.
      • Mondino A.
      • Zhou M.
      • Cooper J.
      • Schlessinger J.
      ). In L6 myoblasts treated with dexamethasone, the increase in IRS-1-bound p85α was associated with a coordinate decrease in IRS-1-associated p85β, which could potentially be explained by p85α and p85β competing for the same binding site(s) on tyrosine-phosphorylated IRS-1.
      Dexamethasone induced a cellular excess of p85α and a greater number of IRS-1·p85α complexes in L6 myoblasts. That the pool of IRS-1-associated p85α was largely composed of regulatory subunit lacking the p110 catalytic subunit is indicated by the detection of reduced amounts of IRS-1-associated p110 (demonstrated by both p110 immunoblotting and metabolic labeling experiments) and a corresponding decrease in PI 3-kinase activity. Although there was an increase in p110 protein in total cell lysates following treatment of L6 myoblasts with dexamethasone, this was much less pronounced than the increase in p85α protein (38 versus 300%, respectively). In interpreting these results, it is important to note that two p110 proteins (p110α and p110β) have been identified (
      • Hiles I.D.
      • Otsu M.
      • Volinia S.
      • Fry M.J.
      • Gout I.
      • Dhand R.
      • Panayotou G.
      • Ruiz-Larrea F.
      • Thompson A.
      • Totty N.F.
      • Hsuan J.J.
      • Courtneidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ,
      • Hu P.
      • Mondino A.
      • Skolnik E.Y.
      • Schlessinger J.
      ). We have used a monoclonal antibody raised against bovine p110 that recognizes only the p110α isoform. Therefore, it is not known whether or not dexamethasone affects cellular levels of p110β and what fraction of IRS-1-associated p85α in dexamethasone-treated cells was coupled with p110β. However, the decrease in IRS-1-associated p110α and IRS-1-associated PI 3-kinase activity in response to dexamethasone were coordinate. In addition, a single 35S-labeled protein of 110 kDa, possibly representing both p110α and p110β, was detectable in IRS-1 immunoprecipitates upon IGF-I stimulation. This band was less intense in L6 myoblasts treated with dexamethasone, confirming that the total amount of IRS-1-associated catalytic p110 subunit was decreased.
      A p85 subunit lacking a p110 subunit may have the capacity to bind to the specific tyrosine-phosphorylated motif in target proteins without localizing catalytic activity in the protein-protein signaling complex. Generation of non-coupled (i.e. monomeric or free) p85α can be achieved experimentally by transfection and overexpression of the p85α cDNA in mammalian cells. When this experiment was performed in 293 cells overexpressing the PDGF receptor, overexpression of p85α was shown to completely abrogate activation of PDGF receptor-associated PI 3-kinase activity (
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Lammers R.
      • Ullrich A.
      • Schlessinger J.
      ). In addition, microinjection of the p85α NH2-terminal SH2 domain into rat 1 fibroblasts overexpressing the insulin receptor was shown to inhibit insulin- and IGF-I-induced DNA synthesis by competing with endogenous PI 3-kinase for binding to IRS-1 (
      • Jhun B.H.
      • Rose D.W.
      • Seely B.L.
      • Rameh L.
      • Cantley L.C.
      • Saltiel A.R.
      • Olefsky J.M.
      ). These studies support the concept that cellular overexpression of the p85α regulatory subunit of the PI 3-kinase complex can lead to inhibition of PI 3-kinase activity and impairment of cell signaling through activation of this enzyme. Accordingly, the excess of free p85α induced by dexamethasone in L6 myoblasts may compete with both p85α·p110 and p85β·p110 complexes for binding to IRS-1. If there are functional specificities for IRS-1-bound p85α and p85β, respectively, this may have distinct effects by disrupting specific signaling responses not only through IRS-1·p85α·p110 complexes, but also through IRS-1·p85β·p110 complexes.
      There is evidence that free p85α binds to tyrosine-phosphorylated proteins with greater avidity than the intact p85·p110 complex (
      • Otsu M.
      • Hiles I.
      • Gout I.
      • Fry M.J.
      • Ruis-Larrea F.
      • Panayotou G.
      • Thompson A.
      • Dhand R.
      • Hsuan J.
      • Totty N.
      • Smith A.D.
      • Morgan S.J.
      • Courtneidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ,
      • Jhun B.H.
      • Rose D.W.
      • Seely B.L.
      • Rameh L.
      • Cantley L.C.
      • Saltiel A.R.
      • Olefsky J.M.
      ). Thus, preferential complex formation between IRS-1 and p85α may have occurred in L6 myoblasts treated with dexamethasone. It recently has been suggested that in situ concentrations of PI 3,4,5-P3 synthesized locally by the catalytic subunit of PI 3-kinase may bind to the SH2 domain of p85 and dissociate PI 3-kinase from the tyrosine phosphoprotein (
      • Rameh L.E.
      • Chen C-S.
      • Cantley L.C.
      ). The lack of PI 3,4,5-P3 production by the catalytically inactive p85α monomer induced by dexamethasone could provide a mechanism conferring greater affinity to this protein for binding to tyrosine-phosphorylated IRS-1 compared to p85α·p110 and p85β·p110 complexes. Such a mechanism could also lead to displacement of IRS-1-bound p85β·p110 by free p85α.
      Activation of PI 3-kinase by receptor and nonreceptor protein tyrosine kinases has been implicated in a broad spectrum of cellular responses, including mitogenesis (
      • Cantley L.C.
      • Auger K.R.
      • Carpenter C.L.
      • Duckworth B.C.
      • Graziani A.
      • Kapeller R.
      • Soltoff S.
      ,
      • Fantl W.J.
      • Escobedo J.A.
      • Martin G.A.
      • Turck C.W.
      • del Rosario M.
      • McCormick F.
      • Williams L.T.
      ,
      • Valius M.
      • Kazlauskas A.
      ), chemotaxis (
      • Wennstrom S.
      • Siegbahn A.
      • Koutaro Y.
      • Arvidsson A.-K.
      • Heldin C.-H.
      • Mori S.
      • Claesson-Welsh L.
      ,
      • Kundra V.
      • Escobedo J.A.
      • Kazlauskas A.
      • Kim H.K.
      • Rhee S.G.
      • Williams L.T.
      • Zetter B.R.
      ), membrane ruffling (
      • Kotani K.
      • Yonezawa K.
      • Hara K.
      • Ueda H.
      • Kitamura Y.
      • Sakaue H.
      • Ando A.
      • Chavanieu A.
      • Calas B.
      • Grigorescu F.
      ), activation of p70 S6 kinase (
      • Chung J.
      • Grammer T.C.
      • Lemon K.P.
      • Kazlauskas A.
      • Blenis J.
      ), insulin-dependent GLUT-4 translocation (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ), glycogen synthesis (
      • Shepherd P.R.
      • Nave B.T.
      • Siddle K.
      ,
      • Yamamoto-Honda R.
      • Tobe K.
      • Kaburagi Y.
      • Ueki K.
      • Asai S.
      • Yachi M.
      • Shirouzu M.
      • Yodoi J.
      • Akanuma Y.
      • Yokoyama S.
      • Yazaki Y.
      • Kadowaki T.
      ), activation of integrins in platelets (
      • Kovacsovics T.J.
      • Bachelot C.
      • Toker A.
      • Vlahos C.J.
      • Duckworth B.
      • Cantley L.C.
      • Hartwig J.H.
      ), histamine release (
      • Yano H.
      • Nakanishi S.
      • Kimura K.
      • Hanai N.
      • Saitoh Y.
      • Fukui Y.
      • Nonomura Y.
      • Matsuda Y.
      ), receptor down-regulation (
      • Joly M.
      • Kazlauskas A.
      • Fay F.S.
      • Corvera S.
      ), and inhibition of apoptosis (
      • Yao R.
      • Cooper G.M.
      ). All of these biological responses require a functionally active PI 3-kinase enzyme complex that reflects the coordinate expression of both p85 and p110 PI 3-kinase subunits in the cell. The generation of excess p85α regulatory subunit relative to the other components of the PI 3-kinase complex along with inhibition of IRS-1-associated PI 3-kinase activity in response to dexamethasone demonstrated in this study suggests a novel mechanism of PI 3-kinase regulation. This mechanism may not be limited to muscle cells and may occur in other cell types under physiological circumstances and/or in disease states characterized by high levels of endogenous cortisol or exogenously administered glucocorticoids.

      Acknowledgments

      We acknowledge Karen TenDyke for excellent technical support.

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