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Regulation of Protein Kinase B in Rat Adipocytes by Insulin, Vanadate, and Peroxovanadate

1997; Elsevier BV; Volume: 272; Issue: 34 Linguagem: Inglês

10.1074/jbc.272.34.21520

1083-351X

Jonny Wijkander, Lena Stenson Holst, Tova Rahn, Svante Resjö, Isabelle Castan‐Laurell, Vincent C. Manganiello, Per Belfrage, Eva Degerman,

Nitric Oxide and Endothelin Effects

Protein kinase B (PKB) (also referred to as RAC/Akt kinase) has been shown to be controlled by various growth factors, including insulin, using cell lines and transfected cells. However, information is so far scarce regarding its regulation in primary insulin-responsive cells. We have therefore used isolated rat adipocytes to examine the mechanisms, including membrane translocation, whereby insulin and the insulin-mimicking agents vanadate and peroxovanadate control PKB. Stimulation of adipocytes with insulin, vanadate, or peroxovanadate caused decreased PKB mobility on sodium dodecyl sulfate-polyacrylamide gels, indicative of increased phosphorylation, which correlated with an increase in kinase activity detected with the peptide KKRNRTLTK. This peptide was found to detect activated PKB selectively in crude cytosol and partially purified cytosol fractions from insulin-stimulated adipocytes. The decrease in electrophoretic mobility and activation of PKB induced by insulin was reversed both in vitro by treatment of the enzyme with alkaline phosphatase and in the intact adipocyte upon removal of insulin or addition of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin. Significant translocation of PKB to membranes could not be demonstrated after insulin stimulation, but peroxovanadate, which appeared to activate PI 3-kinase to a higher extent than insulin, induced substantial translocation. The translocation was prevented by wortmannin, suggesting that PI 3-kinase and/or the 3-phosphorylated phosphoinositides generated by PI 3-kinase are indeed involved in the membrane targeting of PKB. Protein kinase B (PKB) (also referred to as RAC/Akt kinase) has been shown to be controlled by various growth factors, including insulin, using cell lines and transfected cells. However, information is so far scarce regarding its regulation in primary insulin-responsive cells. We have therefore used isolated rat adipocytes to examine the mechanisms, including membrane translocation, whereby insulin and the insulin-mimicking agents vanadate and peroxovanadate control PKB. Stimulation of adipocytes with insulin, vanadate, or peroxovanadate caused decreased PKB mobility on sodium dodecyl sulfate-polyacrylamide gels, indicative of increased phosphorylation, which correlated with an increase in kinase activity detected with the peptide KKRNRTLTK. This peptide was found to detect activated PKB selectively in crude cytosol and partially purified cytosol fractions from insulin-stimulated adipocytes. The decrease in electrophoretic mobility and activation of PKB induced by insulin was reversed both in vitro by treatment of the enzyme with alkaline phosphatase and in the intact adipocyte upon removal of insulin or addition of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin. Significant translocation of PKB to membranes could not be demonstrated after insulin stimulation, but peroxovanadate, which appeared to activate PI 3-kinase to a higher extent than insulin, induced substantial translocation. The translocation was prevented by wortmannin, suggesting that PI 3-kinase and/or the 3-phosphorylated phosphoinositides generated by PI 3-kinase are indeed involved in the membrane targeting of PKB. In recent years, the recognition of phosphatidylinositol 3-kinase (PI 3-kinase) 1The abbreviations used are: PI 3-kinase, phosphatidylinositol 3-kinase; PKB, protein kinase B; PH, pleckstrin homology; PMA, 4-β-phorbol 12-myristate 13-acetate; TES,N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; MAP kinase, mitogen-activated protein kinase.1The abbreviations used are: PI 3-kinase, phosphatidylinositol 3-kinase; PKB, protein kinase B; PH, pleckstrin homology; PMA, 4-β-phorbol 12-myristate 13-acetate; TES,N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; MAP kinase, mitogen-activated protein kinase. as an important link in insulin signal transduction has facilitated understanding of new signaling mechanisms. Activation of PI 3-kinase by insulin and growth factors leads to generation of 3-phosphorylated phosphoinositides such as phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (1Varticovski L. Harrison-Findik D. Keeler M.L. Susa M. Biochim. Biophys. Acta. 1994; 1226: 1-11Crossref PubMed Scopus (95) Google Scholar, 2Carpenter C.L. Cantley L.C. Curr. Opin. Cell Biol. 1996; 8: 153-158Crossref PubMed Scopus (575) Google Scholar), which are believed to act as second messengers. In a number of cultured cells the serine/threonine protein kinase B (PKB), also known as RAC or Akt, has been shown to be a target for PI 3-kinase-generated signals (3Franke T.F. Yang S.-I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1820) Google Scholar, 4Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (318) Google Scholar, 5Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar, 6Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4324) Google Scholar, 7Andjelkovic M. Jakubowicz T. Cron P. Ming X.-F. Han J.-W. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5699-5704Crossref PubMed Scopus (427) Google Scholar, 8Datta K. Bellacosa A. Chan T.O. Tsichlis P.N. J. Biol. Chem. 1996; 271: 30835-30839Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). PKB is expressed as three isoforms (9Jones P.F. Jakubowicz T. Pitossi F.J. Maurer F. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4171-4175Crossref PubMed Scopus (439) Google Scholar, 10Coffer P.J. Woodgett J.R. Eur. J. Biochem. 1991; 201: 475-481Crossref PubMed Scopus (387) Google Scholar, 11Bellacosa A. Testa J.R. Staal S.P. Tsichlis P.N. Science. 1991; 254: 274-277Crossref PubMed Scopus (786) Google Scholar, 12Jones P.F. Jakubowicz T. Hemmings B.A. Cell Regul. 1991; 2: 1001-1009Crossref PubMed Scopus (141) Google Scholar, 13Konishi H. Shinomura T. Kuroda S. Ono Y. Kikkawa U. Biochem. Biophys. Res. Commun. 1994; 205: 817-825Crossref PubMed Scopus (70) Google Scholar), all of which contain an amino-terminal pleckstrin homology (PH) domain (14Haslam R.J. Koide H.B. Hemmings B.A. Nature. 1993; 363: 309-310Crossref PubMed Scopus (386) Google Scholar), which may be involved in protein-protein or protein-lipid interactions (15Harlan J.E. Hajduk P.J. Yoon H.S. Fesik S.W. Nature. 1994; 371: 168-170Crossref PubMed Scopus (669) Google Scholar). A proposed mechanism for the PI 3-kinase-dependent activation of PKB involves translocation of PKB from the cytosol to membranes and subsequent phosphorylation by one or several membrane-associated kinase(s) (7Andjelkovic M. Jakubowicz T. Cron P. Ming X.-F. Han J.-W. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5699-5704Crossref PubMed Scopus (427) Google Scholar, 16Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). It has been demonstrated that PKB can bind to phosphatidylinositol 3,4-bisphosphate (17Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1293) Google Scholar, 18James S.R. Downes C.P. Gigg R. Grove S.J.A. Holmes A.B. Alessi D.R. Biochem. J. 1996; 315: 709-713Crossref PubMed Scopus (270) Google Scholar) and phosphatidylinositol 3,4,5-trisphosphate in vitro (18James S.R. Downes C.P. Gigg R. Grove S.J.A. Holmes A.B. Alessi D.R. Biochem. J. 1996; 315: 709-713Crossref PubMed Scopus (270) Google Scholar), most likely through its PH domain, suggesting that in vivo the formation of such lipids by activated PI 3-kinase could have a role in the translocation of PKB. It is a matter of controversy whether the 3-phosphorylated phosphoinositides not only bind but also to some extent activate PKB (3Franke T.F. Yang S.-I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1820) Google Scholar, 17Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1293) Google Scholar, 18James S.R. Downes C.P. Gigg R. Grove S.J.A. Holmes A.B. Alessi D.R. Biochem. J. 1996; 315: 709-713Crossref PubMed Scopus (270) Google Scholar, 19Matsuzaki H. Konishi H. Tanaka M. Ono Y. Takenawa T. Watanabe Y. Ozaki S. Kuroda S. Kikkawa U. FEBS Lett. 1996; 396: 305-308Crossref PubMed Scopus (20) Google Scholar).Because almost all of the studies concerning the proposed mechanism(s) for insulin regulation of PKB have been carried out using cell lines or transfected cells and some of the findings are conflicting, there is obviously need for such information also from insulin-responsive target tissues such as liver, muscle, or adipose tissue. Therefore, we have performed such studies in the isolated rat adipocyte. Major findings reported in this paper are that PKB in the intact adipocyte is rapidly and reversibly activated in response to physiological concentrations of insulin and upon stimulation with the insulin-mimicking agent peroxovanadate is translocated to membranes via a wortmannin-sensitive mechanism.DISCUSSIONThe major findings in this paper are that PKB is rapidly and reversibly activated via a wortmannin-sensitive mechanism by physiological concentrations of insulin in adipocytes, a major target cell for insulin, and that PKB is recruited to membranes via a wortmannin-sensitive mechanism in the intact cell.Kohn et al. (4Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (318) Google Scholar) observed that stimulation of adipocytes with insulin resulted in decreased electrophoretic mobility of PKB on SDS-polyacrylamide gels. Here we show that the decreased electrophoretic mobility of PKB is linked to activation of the kinase and that the activation is wortmannin-sensitive and also appears to involve phosphorylation. Furthermore, the activation is rapid (detectable within 1 min), reversible, and occurs in response to physiological concentrations of insulin (detectable with 100 pm). Therefore, the insulin-induced activation of PKB could well be important in metabolic actions of insulin. Insulin and insulin-like growth factor-1-mediated activation of PKB have been demonstrated using different cell lines and transfected cells, sometimes in the presence of as much as 100–1000 nminsulin (4Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (318) Google Scholar, 5Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar, 6Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4324) Google Scholar, 16Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 31Kohn 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 (1085) Google Scholar, 32Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar, 33Hurel S.J. Rochford J.J. Borthwick A.C. Wells A.M. Vandenheede J.R. Turnbull D.M. Yeaman S.J. Biochem. J. 1996; 320: 871-877Crossref PubMed Scopus (82) Google Scholar). At high concentration of insulin it cannot be excluded that the effects observed are mediated via the insulin-like growth factor-1 receptor and not by the insulin receptor (34Moses A.C. Tzusaki S. LeRoith D. Insulin-like Growth Factor: Molecular and Cellular Aspects. CRC Press, Boca Raton, FL1991: 245-270Google Scholar). Our results show that in primary rat adipocytes there is a physiological regulation of PKB by insulin.The K9 peptide (KKRNRTLTK) was originally designed as a substrate for p70 S6 kinase (35Leighton I.A. Dalby K.N. Caudwell F.B. Cohen P.T.W. Cohen P. FEBS Lett. 1995; 375: 289-293Crossref PubMed Scopus (109) Google Scholar). Our results show that K9 can be used to detect PKB activity selectively in cytosol fractions from stimulated adipocytes. The peptide Crosstide (GRPRTSSFAEG), which closely resembles the sequence containing the site phosphorylated by PKB in glycogen synthase kinase-3, has been used to determine PKB activity in immunoprecipitates as well as in partially purified fractions of PKB (6Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4324) Google Scholar). Both Crosstide and K9 have basic residues at positions n-3 andn-5 (where n is the site of phosphorylation). In the case of Crosstide the phosphate acceptor is a serine, and in K9 it is a threonine. In a set of experiments (results not shown) we compared kinase activity in adipocytes using three different peptide substrates; Crosstide, K9, and a K9 peptide with the most carboxyl-terminal threonine replaced by a serine (K9Thr → Ser). In contrast to the results in Fig. 2 B with K9 as substrate, Crosstide as well as K9Thr → Ser detected PMA-stimulated kinase activity in the cytosol fraction to the same extent as insulin-stimulated kinase activity. Because PMA stimulation does not result in activation of PKB in adipocytes (Fig. 2) or other cells (3Franke T.F. Yang S.-I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1820) Google Scholar, 4Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (318) Google Scholar, 5Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar) this indicates that kinase assay of crude adipocyte preparations with peptides containing a serine as phosphate acceptor (Crosstide and K9Thr → Ser) are less selective for PKB and will also detect other kinases such as p90 rsk. This conclusion is supported by a recent study where the substrate specificity of PKB, p90 rsk, and p70 S6 kinase were compared (36Alessi D.R. Caudwell F.B. Andjelkovic M. Hemmings B.A. Cohen P. FEBS Lett. 1996; 399: 333-338Crossref PubMed Scopus (550) Google Scholar).The importance of phosphorylation as a mechanism to activate PKB was demonstrated recently by Alessi et al. (32Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar). Site-directed mutagenesis studies of PKB revealed two activity-controlling phosphorylation sites. However, the protein kinase(s) and phosphatase(s) responsible for the phosphorylation of PKB in vivo have not yet been identified. In agreement with findings by others (4Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (318) Google Scholar, 5Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar, 6Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4324) Google Scholar), alkaline phosphatase treatment of PKB from insulin-stimulated adipocytes resulted in its deactivation as well as reversal of its electrophoretic mobility to that of PKB from control cells. A role for protein phosphatases in the regulation of PKB in intact cells is supported by the observation that treatment of Swiss 3T3 cells with okadaic acid induces activation and reduction in the electrophoretic mobility of PKB (7Andjelkovic M. Jakubowicz T. Cron P. Ming X.-F. Han J.-W. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5699-5704Crossref PubMed Scopus (427) Google Scholar). Our results demonstrated that the insulin-induced activation of PKB, as well as the corresponding decrease in electrophoretic mobility, was reversed upon removal of insulin or addition of wortmannin subsequent to insulin stimulation, indicating that the activation of PKB is reversed rapidly by protein phosphatase(s) in the intact cell.The finding that wortmannin blocked the peroxovanadate-induced translocation of PKB to membranes in intact cells is of considerable interest since it has been proposed that PKB can be targeted to membranes where it can be phosphorylated and activated by membrane-associated kinase(s) (7Andjelkovic M. Jakubowicz T. Cron P. Ming X.-F. Han J.-W. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5699-5704Crossref PubMed Scopus (427) Google Scholar, 16Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). However, membrane translocation of PKB has not been demonstrated in intact cells but is supported by observations that PKB binds to lipid vesicles containing phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (17Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1293) Google Scholar, 18James S.R. Downes C.P. Gigg R. Grove S.J.A. Holmes A.B. Alessi D.R. Biochem. J. 1996; 315: 709-713Crossref PubMed Scopus (270) Google Scholar) and that targeting of PKB to membranes by adding the src myristoylation sequence results in constitutive activation of PKB (5Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar, 8Datta K. Bellacosa A. Chan T.O. Tsichlis P.N. J. Biol. Chem. 1996; 271: 30835-30839Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 31Kohn 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 (1085) Google Scholar). In contrast to the results with peroxovanadate, we were unable to find significant amounts of PKB in membrane fractions from insulin-stimulated cells. We have not identified the factor responsible for the marked difference in translocation of PKB to membranes during stimulation of adipocytes with insulin or peroxovanadate. It is possible that higher concentrations of 3-phosphorylated phosphoinositides are generated in the presence of peroxovanadate, since severalfold greater amounts of PI 3-kinase were found in association with insulin receptor substrate-13after stimulation of adipocytes with peroxovanadate than after treatment with insulin. The amount of phosphatidylinositol 3,4,5-trisphosphate could also have been increased by inhibition of its degradation. Since vanadate has been shown to inhibit phosphatidylinositol 3,4,5-trisphosphate 5′-phosphatase in vitro (37Woscholski R. Waterfield M.D. Parker P.J. J. Biol. Chem. 1995; 270: 31001-31007Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), it is tempting to speculate that peroxovanadate could inhibit this phosphatase in the intact cell and thereby increase the level of phosphatidylinositol 3,4,5-trisphosphate.Little is known regarding the physiologically important downstream targets for PKB. Recently, it was demonstrated that the insulin-induced phosphorylation and inhibition of glycogen synthase kinase-3 in myotubes are mediated by PKB, indicating an important role for PKB in the regulation of glycogen synthesis (6Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4324) Google Scholar). In 3T3-L1 adipocytes the expression of constitutively active PKB resulted in increased glucose uptake, which was associated with increased translocation of GLUT4 to plasma membranes and increased expression of GLUT1 (31Kohn 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 (1085) Google Scholar). In these cells, increased glucose uptake in the presence of constitutively active PKB was associated with increased lipid synthesis. Another important effect of insulin is to counteract catecholamine-induced hydrolysis of stored adipose tissue triglycerides. A key enzyme in this action of insulin is phosphodiesterase 3B whose activation causes reduction of cellular cAMP (38Degerman E. Leroy M.J. Taira M. Belfrage P. Manganiello V. LeRoith D. Taylor S.I. Olefsky J.M. Diabetes Mellitus. Lippincott-Raven Publishers, Philadelphia1996: 197-204Google Scholar). We are currently investigating the role of PKB in the insulin-induced phosphorylation and activation of phosphodiesterase 3B. Preliminary results indicate that phosphodiesterase 3B could be a substrate for PKBin vitro. 4T. Rahn and J. Wijkander, unpublished data., 5During the review of our manuscript, Mouleet al. (39Moule S.K. Welsh G.I. Edgell N.J. Foulstone E.J. Proud C.G. Denton R.M. J. Biol. Chem. 1997; 272: 7713-7719Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar) and Cross et al. (40Cross D.A.E. Watt P.W. Shaw M. van der Kaay J. Downes C.P. Holder J.C. Cohen P. FEBS Lett. 1997; 406: 211-215Crossref PubMed Scopus (191) Google Scholar) reported on insulin-induced activation of PKB in rat primary adipocytes. In recent years, the recognition of phosphatidylinositol 3-kinase (PI 3-kinase) 1The abbreviations used are: PI 3-kinase, phosphatidylinositol 3-kinase; PKB, protein kinase B; PH, pleckstrin homology; PMA, 4-β-phorbol 12-myristate 13-acetate; TES,N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; MAP kinase, mitogen-activated protein kinase.1The abbreviations used are: PI 3-kinase, phosphatidylinositol 3-kinase; PKB, protein kinase B; PH, pleckstrin homology; PMA, 4-β-phorbol 12-myristate 13-acetate; TES,N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; MAP kinase, mitogen-activated protein kinase. as an important link in insulin signal transduction has facilitated understanding of new signaling mechanisms. Activation of PI 3-kinase by insulin and growth factors leads to generation of 3-phosphorylated phosphoinositides such as phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (1Varticovski L. Harrison-Findik D. Keeler M.L. Susa M. Biochim. Biophys. Acta. 1994; 1226: 1-11Crossref PubMed Scopus (95) Google Scholar, 2Carpenter C.L. Cantley L.C. Curr. Opin. Cell Biol. 1996; 8: 153-158Crossref PubMed Scopus (575) Google Scholar), which are believed to act as second messengers. In a number of cultured cells the serine/threonine protein kinase B (PKB), also known as RAC or Akt, has been shown to be a target for PI 3-kinase-generated signals (3Franke T.F. Yang S.-I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1820) Google Scholar, 4Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (318) Google Scholar, 5Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar, 6Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4324) Google Scholar, 7Andjelkovic M. Jakubowicz T. Cron P. Ming X.-F. Han J.-W. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5699-5704Crossref PubMed Scopus (427) Google Scholar, 8Datta K. Bellacosa A. Chan T.O. Tsichlis P.N. J. Biol. Chem. 1996; 271: 30835-30839Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). PKB is expressed as three isoforms (9Jones P.F. Jakubowicz T. Pitossi F.J. Maurer F. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4171-4175Crossref PubMed Scopus (439) Google Scholar, 10Coffer P.J. Woodgett J.R. Eur. J. Biochem. 1991; 201: 475-481Crossref PubMed Scopus (387) Google Scholar, 11Bellacosa A. Testa J.R. Staal S.P. Tsichlis P.N. Science. 1991; 254: 274-277Crossref PubMed Scopus (786) Google Scholar, 12Jones P.F. Jakubowicz T. Hemmings B.A. Cell Regul. 1991; 2: 1001-1009Crossref PubMed Scopus (141) Google Scholar, 13Konishi H. Shinomura T. Kuroda S. Ono Y. Kikkawa U. Biochem. Biophys. Res. Commun. 1994; 205: 817-825Crossref PubMed Scopus (70) Google Scholar), all of which contain an amino-terminal pleckstrin homology (PH) domain (14Haslam R.J. Koide H.B. Hemmings B.A. Nature. 1993; 363: 309-310Crossref PubMed Scopus (386) Google Scholar), which may be involved in protein-protein or protein-lipid interactions (15Harlan J.E. Hajduk P.J. Yoon H.S. Fesik S.W. Nature. 1994; 371: 168-170Crossref PubMed Scopus (669) Google Scholar). A proposed mechanism for the PI 3-kinase-dependent activation of PKB involves translocation of PKB from the cytosol to membranes and subsequent phosphorylation by one or several membrane-associated kinase(s) (7Andjelkovic M. Jakubowicz T. Cron P. Ming X.-F. Han J.-W. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5699-5704Crossref PubMed Scopus (427) Google Scholar, 16Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). It has been demonstrated that PKB can bind to phosphatidylinositol 3,4-bisphosphate (17Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1293) Google Scholar, 18James S.R. Downes C.P. Gigg R. Grove S.J.A. Holmes A.B. Alessi D.R. Biochem. J. 1996; 315: 709-713Crossref PubMed Scopus (270) Google Scholar) and phosphatidylinositol 3,4,5-trisphosphate in vitro (18James S.R. Downes C.P. Gigg R. Grove S.J.A. Holmes A.B. Alessi D.R. Biochem. J. 1996; 315: 709-713Crossref PubMed Scopus (270) Google Scholar), most likely through its PH domain, suggesting that in vivo the formation of such lipids by activated PI 3-kinase could have a role in the translocation of PKB. It is a matter of controversy whether the 3-phosphorylated phosphoinositides not only bind but also to some extent activate PKB (3Franke T.F. Yang S.-I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1820) Google Scholar, 17Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1293) Google Scholar, 18James S.R. Downes C.P. Gigg R. Grove S.J.A. Holmes A.B. Alessi D.R. Biochem. J. 1996; 315: 709-713Crossref PubMed Scopus (270) Google Scholar, 19Matsuzaki H. Konishi H. Tanaka M. Ono Y. Takenawa T. Watanabe Y. Ozaki S. Kuroda S. Kikkawa U. FEBS Lett. 1996; 396: 305-308Crossref PubMed Scopus (20) Google Scholar). Because almost all of the studies concerning the proposed mechanism(s) for insulin regulation of PKB have been carried out using cell lines or transfected cells and some of the findings are conflicting, there is obviously need for such information also from insulin-responsive target tissues such as liver, muscle, or adipose tissue. Therefore, we have performed such studies in the isolated rat adipocyte. Major findings reported in this paper are that PKB in the intact adipocyte is rapidly and reversibly activated in response to physiological concentrations of insulin and upon stimulation with the insulin-mimicking agent peroxovanadate is translocated to membranes via a wortmannin-sensitive mechanism. DISCUSSIONThe major findings in this paper are that PKB is rapidly and reversibly activated via a wortmannin-sensitive mechanism by physiological concentrations of insulin in adipocytes, a major target cell for insulin, and that PKB is recruited to membranes via a wortmannin-sensitive mechanism in the intact cell.Kohn et al. (4Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (318) Google Scholar) observed that stimulation of adipocytes with insulin resulted in decreased electrophoretic mobility of PKB on SDS-polyacrylamide gels. Here we show that the decreased electrophoretic mobility of PKB is linked to activation of the kinase and that the activation is wortmannin-sensitive and also appears to involve phosphorylation. Furthermore, the activation is rapid (detectable within 1 min), reversible, and occurs in response to physiological concentrations of insulin (detectable with 100 pm). Therefore, the insulin-induced activation of PKB could well be important in metabolic actions of insulin. Insulin and insulin-like growth factor-1-mediated activation of PKB have been demonstrated using different cell lines and transfected cells, sometimes in the presence of as much as 100–1000 nminsulin (4Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (318) Google Scholar, 5Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar, 6Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4324) Google Scholar, 16Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 31Kohn 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 (1085) Google Scholar, 32Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar, 33Hurel S.J. Rochford J.J. Borthwick A.C. Wells A.M. Vandenheede J.R. Turnbull D.M. Yeaman S.J. Biochem. J. 1996; 320: 871-877Crossref PubMed Scopus (82) Google Scholar). At high concentration of insulin it cannot be excluded that the effects observed are mediated via the insulin-like growth factor-1 receptor and not by the insulin receptor (34Moses A.C. Tzusaki S. LeRoith D. Insulin-like Growth Factor: Molecular and Cellular Aspects. CRC Press, Boca Raton, FL1991: 245-270Google Scholar). Our results show that in primary rat adipocytes there is a physiological regulation of PKB by insulin.The K9 peptide (KKRNRTLTK) was originally designed as a substrate for p70 S6 kinase (35Leighton I.A. Dalby K.N. Caudwell F.B. Cohen P.T.W. Cohen P. FEBS Lett. 1995; 375: 289-293Crossref PubMed Scopus (109) Google Scholar). Our results show that K9 can be used to detect PKB activity selectively in cytosol fractions from stimulated adipocytes. The peptide Crosstide (GRPRTSSFAEG), which closely resembles the sequence containing the site phosphorylated by PKB in glycogen synthase kinase-3, has been used to determine PKB activity in immunoprecipitates as well as in partially purified fractions of PKB (6Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4324) Google Scholar). Both Crosstide and K9 have basic residues at positions n-3 andn-5 (where n is the site of phosphorylation). In the case of Crosstide the phosphate acceptor is a serine, and in K9 it is a threonine. In a set of experiments (results not shown) we compared kinase activity in adipocytes using three different peptide substrates; Crosstide, K9, and a K9 peptide with the most carboxyl-terminal threonine replaced by a serine (K9Thr → Ser). In contrast to the results in Fig. 2 B with K9 as substrate, Crosstide as well as K9Thr → Ser detected PMA-stimulated kinase activity in the cytosol fraction to the same extent as insulin-stimulated kinase activity. Because PMA stimulation does not result in activation of PKB in adipocytes (Fig. 2) or other cells (3Franke T.F. Yang S.-I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1820) Google Scholar, 4Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (318) Google Scholar, 5Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar) this indicates that kinase assay of crude adipocyte preparations with peptides containing a serine as phosphate acceptor (Crosstide and K9Thr → Ser) are less selective for PKB and will also detect other kinases such as p90 rsk. This conclusion is supported by a recent study where the substrate specificity of PKB, p90 rsk, and p70 S6 kinase were compared (36Alessi D.R. Caudwell F.B. Andjelkovic M. Hemmings B.A. Cohen P. FEBS Lett. 1996; 399: 333-338Crossref PubMed Scopus (550) Google Scholar).The importance of phosphorylation as a mechanism to activate PKB was demonstrated recently by Alessi et al. (32Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar). Site-directed mutagenesis studies of PKB revealed two activity-controlling phosphorylation sites. However, the protein kinase(s) and phosphatase(s) responsible for the phosphorylation of PKB in vivo have not yet been identified. In agreement with findings by others (4Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (318) Google Scholar, 5Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar, 6Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4324) Google Scholar), alkaline phosphatase treatment of PKB from insulin-stimulated adipocytes resulted in its deactivation as well as reversal of its electrophoretic mobility to that of PKB from control cells. A role for protein phosphatases in the regulation of PKB in intact cells is supported by the observation that treatment of Swiss 3T3 cells with okadaic acid induces activation and reduction in the electrophoretic mobility of PKB (7Andjelkovic M. Jakubowicz T. Cron P. Ming X.-F. Han J.-W. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5699-5704Crossref PubMed Scopus (427) Google Scholar). Our results demonstrated that the insulin-induced activation of PKB, as well as the corresponding decrease in electrophoretic mobility, was reversed upon removal of insulin or addition of wortmannin subsequent to insulin stimulation, indicating that the activation of PKB is reversed rapidly by protein phosphatase(s) in the intact cell.The finding that wortmannin blocked the peroxovanadate-induced translocation of PKB to membranes in intact cells is of considerable interest since it has been proposed that PKB can be targeted to membranes where it can be phosphorylated and activated by membrane-associated kinase(s) (7Andjelkovic M. Jakubowicz T. Cron P. Ming X.-F. Han J.-W. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5699-5704Crossref PubMed Scopus (427) Google Scholar, 16Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). However, membrane translocation of PKB has not been demonstrated in intact cells but is supported by observations that PKB binds to lipid vesicles containing phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (17Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1293) Google Scholar, 18James S.R. Downes C.P. Gigg R. Grove S.J.A. Holmes A.B. Alessi D.R. Biochem. J. 1996; 315: 709-713Crossref PubMed Scopus (270) Google Scholar) and that targeting of PKB to membranes by adding the src myristoylation sequence results in constitutive activation of PKB (5Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar, 8Datta K. Bellacosa A. Chan T.O. Tsichlis P.N. J. Biol. Chem. 1996; 271: 30835-30839Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 31Kohn 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 (1085) Google Scholar). In contrast to the results with peroxovanadate, we were unable to find significant amounts of PKB in membrane fractions from insulin-stimulated cells. We have not identified the factor responsible for the marked difference in translocation of PKB to membranes during stimulation of adipocytes with insulin or peroxovanadate. It is possible that higher concentrations of 3-phosphorylated phosphoinositides are generated in the presence of peroxovanadate, since severalfold greater amounts of PI 3-kinase were found in association with insulin receptor substrate-13after stimulation of adipocytes with peroxovanadate than after treatment with insulin. The amount of phosphatidylinositol 3,4,5-trisphosphate could also have been increased by inhibition of its degradation. Since vanadate has been shown to inhibit phosphatidylinositol 3,4,5-trisphosphate 5′-phosphatase in vitro (37Woscholski R. Waterfield M.D. Parker P.J. J. Biol. Chem. 1995; 270: 31001-31007Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), it is tempting to speculate that peroxovanadate could inhibit this phosphatase in the intact cell and thereby increase the level of phosphatidylinositol 3,4,5-trisphosphate.Little is known regarding the physiologically important downstream targets for PKB. Recently, it was demonstrated that the insulin-induced phosphorylation and inhibition of glycogen synthase kinase-3 in myotubes are mediated by PKB, indicating an important role for PKB in the regulation of glycogen synthesis (6Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4324) Google Scholar). In 3T3-L1 adipocytes the expression of constitutively active PKB resulted in increased glucose uptake, which was associated with increased translocation of GLUT4 to plasma membranes and increased expression of GLUT1 (31Kohn 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 (1085) Google Scholar). In these cells, increased glucose uptake in the presence of constitutively active PKB was associated with increased lipid synthesis. Another important effect of insulin is to counteract catecholamine-induced hydrolysis of stored adipose tissue triglycerides. A key enzyme in this action of insulin is phosphodiesterase 3B whose activation causes reduction of cellular cAMP (38Degerman E. Leroy M.J. Taira M. Belfrage P. Manganiello V. LeRoith D. Taylor S.I. Olefsky J.M. Diabetes Mellitus. Lippincott-Raven Publishers, Philadelphia1996: 197-204Google Scholar). We are currently investigating the role of PKB in the insulin-induced phosphorylation and activation of phosphodiesterase 3B. Preliminary results indicate that phosphodiesterase 3B could be a substrate for PKBin vitro. 4T. Rahn and J. Wijkander, unpublished data., 5During the review of our manuscript, Mouleet al. (39Moule S.K. Welsh G.I. Edgell N.J. Foulstone E.J. Proud C.G. Denton R.M. J. Biol. Chem. 1997; 272: 7713-7719Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar) and Cross et al. (40Cross D.A.E. Watt P.W. Shaw M. van der Kaay J. Downes C.P. Holder J.C. Cohen P. FEBS Lett. 1997; 406: 211-215Crossref PubMed Scopus (191) Google Scholar) reported on insulin-induced activation of PKB in rat primary adipocytes. The major findings in this paper are that PKB is rapidly and reversibly activated via a wortmannin-sensitive mechanism by physiological concentrations of insulin in adipocytes, a major target cell for insulin, and that PKB is recruited to membranes via a wortmannin-sensitive mechanism in the intact cell. Kohn et al. (4Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (318) Google Scholar) observed that stimulation of adipocytes with insulin resulted in decreased electrophoretic mobility of PKB on SDS-polyacrylamide gels. Here we show that the decreased electrophoretic mobility of PKB is linked to activation of the kinase and that the activation is wortmannin-sensitive and also appears to involve phosphorylation. Furthermore, the activation is rapid (detectable within 1 min), reversible, and occurs in response to physiological concentrations of insulin (detectable with 100 pm). Therefore, the insulin-induced activation of PKB could well be important in metabolic actions of insulin. Insulin and insulin-like growth factor-1-mediated activation of PKB have been demonstrated using different cell lines and transfected cells, sometimes in the presence of as much as 100–1000 nminsulin (4Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (318) Google Scholar, 5Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar, 6Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4324) Google Scholar, 16Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 31Kohn 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 (1085) Google Scholar, 32Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar, 33Hurel S.J. Rochford J.J. Borthwick A.C. Wells A.M. Vandenheede J.R. Turnbull D.M. Yeaman S.J. Biochem. J. 1996; 320: 871-877Crossref PubMed Scopus (82) Google Scholar). At high concentration of insulin it cannot be excluded that the effects observed are mediated via the insulin-like growth factor-1 receptor and not by the insulin receptor (34Moses A.C. Tzusaki S. LeRoith D. Insulin-like Growth Factor: Molecular and Cellular Aspects. CRC Press, Boca Raton, FL1991: 245-270Google Scholar). Our results show that in primary rat adipocytes there is a physiological regulation of PKB by insulin. The K9 peptide (KKRNRTLTK) was originally designed as a substrate for p70 S6 kinase (35Leighton I.A. Dalby K.N. Caudwell F.B. Cohen P.T.W. Cohen P. FEBS Lett. 1995; 375: 289-293Crossref PubMed Scopus (109) Google Scholar). Our results show that K9 can be used to detect PKB activity selectively in cytosol fractions from stimulated adipocytes. The peptide Crosstide (GRPRTSSFAEG), which closely resembles the sequence containing the site phosphorylated by PKB in glycogen synthase kinase-3, has been used to determine PKB activity in immunoprecipitates as well as in partially purified fractions of PKB (6Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4324) Google Scholar). Both Crosstide and K9 have basic residues at positions n-3 andn-5 (where n is the site of phosphorylation). In the case of Crosstide the phosphate acceptor is a serine, and in K9 it is a threonine. In a set of experiments (results not shown) we compared kinase activity in adipocytes using three different peptide substrates; Crosstide, K9, and a K9 peptide with the most carboxyl-terminal threonine replaced by a serine (K9Thr → Ser). In contrast to the results in Fig. 2 B with K9 as substrate, Crosstide as well as K9Thr → Ser detected PMA-stimulated kinase activity in the cytosol fraction to the same extent as insulin-stimulated kinase activity. Because PMA stimulation does not result in activation of PKB in adipocytes (Fig. 2) or other cells (3Franke T.F. Yang S.-I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1820) Google Scholar, 4Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (318) Google Scholar, 5Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar) this indicates that kinase assay of crude adipocyte preparations with peptides containing a serine as phosphate acceptor (Crosstide and K9Thr → Ser) are less selective for PKB and will also detect other kinases such as p90 rsk. This conclusion is supported by a recent study where the substrate specificity of PKB, p90 rsk, and p70 S6 kinase were compared (36Alessi D.R. Caudwell F.B. Andjelkovic M. Hemmings B.A. Cohen P. FEBS Lett. 1996; 399: 333-338Crossref PubMed Scopus (550) Google Scholar). The importance of phosphorylation as a mechanism to activate PKB was demonstrated recently by Alessi et al. (32Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2495) Google Scholar). Site-directed mutagenesis studies of PKB revealed two activity-controlling phosphorylation sites. However, the protein kinase(s) and phosphatase(s) responsible for the phosphorylation of PKB in vivo have not yet been identified. In agreement with findings by others (4Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (318) Google Scholar, 5Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar, 6Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4324) Google Scholar), alkaline phosphatase treatment of PKB from insulin-stimulated adipocytes resulted in its deactivation as well as reversal of its electrophoretic mobility to that of PKB from control cells. A role for protein phosphatases in the regulation of PKB in intact cells is supported by the observation that treatment of Swiss 3T3 cells with okadaic acid induces activation and reduction in the electrophoretic mobility of PKB (7Andjelkovic M. Jakubowicz T. Cron P. Ming X.-F. Han J.-W. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5699-5704Crossref PubMed Scopus (427) Google Scholar). Our results demonstrated that the insulin-induced activation of PKB, as well as the corresponding decrease in electrophoretic mobility, was reversed upon removal of insulin or addition of wortmannin subsequent to insulin stimulation, indicating that the activation of PKB is reversed rapidly by protein phosphatase(s) in the intact cell. The finding that wortmannin blocked the peroxovanadate-induced translocation of PKB to membranes in intact cells is of considerable interest since it has been proposed that PKB can be targeted to membranes where it can be phosphorylated and activated by membrane-associated kinase(s) (7Andjelkovic M. Jakubowicz T. Cron P. Ming X.-F. Han J.-W. Hemmings B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5699-5704Crossref PubMed Scopus (427) Google Scholar, 16Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). However, membrane translocation of PKB has not been demonstrated in intact cells but is supported by observations that PKB binds to lipid vesicles containing phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (17Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1293) Google Scholar, 18James S.R. Downes C.P. Gigg R. Grove S.J.A. Holmes A.B. Alessi D.R. Biochem. J. 1996; 315: 709-713Crossref PubMed Scopus (270) Google Scholar) and that targeting of PKB to membranes by adding the src myristoylation sequence results in constitutive activation of PKB (5Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1871) Google Scholar, 8Datta K. Bellacosa A. Chan T.O. Tsichlis P.N. J. Biol. Chem. 1996; 271: 30835-30839Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 31Kohn 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 (1085) Google Scholar). In contrast to the results with peroxovanadate, we were unable to find significant amounts of PKB in membrane fractions from insulin-stimulated cells. We have not identified the factor responsible for the marked difference in translocation of PKB to membranes during stimulation of adipocytes with insulin or peroxovanadate. It is possible that higher concentrations of 3-phosphorylated phosphoinositides are generated in the presence of peroxovanadate, since severalfold greater amounts of PI 3-kinase were found in association with insulin receptor substrate-13after stimulation of adipocytes with peroxovanadate than after treatment with insulin. The amount of phosphatidylinositol 3,4,5-trisphosphate could also have been increased by inhibition of its degradation. Since vanadate has been shown to inhibit phosphatidylinositol 3,4,5-trisphosphate 5′-phosphatase in vitro (37Woscholski R. Waterfield M.D. Parker P.J. J. Biol. Chem. 1995; 270: 31001-31007Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), it is tempting to speculate that peroxovanadate could inhibit this phosphatase in the intact cell and thereby increase the level of phosphatidylinositol 3,4,5-trisphosphate. Little is known regarding the physiologically important downstream targets for PKB. Recently, it was demonstrated that the insulin-induced phosphorylation and inhibition of glycogen synthase kinase-3 in myotubes are mediated by PKB, indicating an important role for PKB in the regulation of glycogen synthesis (6Cross D.A.E. Alessi D.R. Cohen P. Andjelkovic M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4324) Google Scholar). In 3T3-L1 adipocytes the expression of constitutively active PKB resulted in increased glucose uptake, which was associated with increased translocation of GLUT4 to plasma membranes and increased expression of GLUT1 (31Kohn 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 (1085) Google Scholar). In these cells, increased glucose uptake in the presence of constitutively active PKB was associated with increased lipid synthesis. Another important effect of insulin is to counteract catecholamine-induced hydrolysis of stored adipose tissue triglycerides. A key enzyme in this action of insulin is phosphodiesterase 3B whose activation causes reduction of cellular cAMP (38Degerman E. Leroy M.J. Taira M. Belfrage P. Manganiello V. LeRoith D. Taylor S.I. Olefsky J.M. Diabetes Mellitus. Lippincott-Raven Publishers, Philadelphia1996: 197-204Google Scholar). We are currently investigating the role of PKB in the insulin-induced phosphorylation and activation of phosphodiesterase 3B. Preliminary results indicate that phosphodiesterase 3B could be a substrate for PKBin vitro. 4T. Rahn and J. Wijkander, unpublished data., 5During the review of our manuscript, Mouleet al. (39Moule S.K. Welsh G.I. Edgell N.J. Foulstone E.J. Proud C.G. Denton R.M. J. Biol. Chem. 1997; 272: 7713-7719Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar) and Cross et al. (40Cross D.A.E. Watt P.W. Shaw M. van der Kaay J. Downes C.P. Holder J.C. Cohen P. FEBS Lett. 1997; 406: 211-215Crossref PubMed Scopus (191) Google Scholar) reported on insulin-induced activation of PKB in rat primary adipocytes. We acknowledge gratefully the excellent technical assistance of Ann-Kristin Holmén Pålbrink and Maria Bogren.