Ingeborg Hers

Protein kinase B (Akt) plays a central role in cellular regulation, although many of the physiologically relevant substrates for the kinase remain to be identified. In this study, we have isolated a protein from primary epididymal adipocytes with an apparent molecular weight of 125,000. This protein exhibited immunoreactivity, in an insulin-dependent manner, with a phosphospecific antibody raised against the protein kinase B substrate consensus sequence RXRXX(pS/pT) as well as a phosphospecific antibody that recognizes serine 21/9 of GSK-3α/β. MALDI-TOF mass spectrometry revealed the protein to be ATP-citrate lyase, suggesting that the two phosphospecific antibodies recognize phosphoserine 454, a previously reported insulin- and isoproterenol-stimulated ATP-citrate lyase phosphorylation site. Indeed, both insulin and isoproterenol stimulated the phosphorylation of this protein on the site recognized by the phosphospecific antibodies in a wortmannin-sensitive and -insensitive manner, respectively. In addition, transient expression of a constitutively active protein kinase B in primary adipocytes mimicked the effect of insulin on ATP-citrate lyase phosphorylation. Furthermore, ATP-citrate lyase was phosphorylated in vitro by recombinant protein kinase B on the same site. Taken together, these results demonstrate that serine 454 of ATP-citrate lyase is a novel and majorin vivo substrate for protein kinase B. Protein kinase B (Akt) plays a central role in cellular regulation, although many of the physiologically relevant substrates for the kinase remain to be identified. In this study, we have isolated a protein from primary epididymal adipocytes with an apparent molecular weight of 125,000. This protein exhibited immunoreactivity, in an insulin-dependent manner, with a phosphospecific antibody raised against the protein kinase B substrate consensus sequence RXRXX(pS/pT) as well as a phosphospecific antibody that recognizes serine 21/9 of GSK-3α/β. MALDI-TOF mass spectrometry revealed the protein to be ATP-citrate lyase, suggesting that the two phosphospecific antibodies recognize phosphoserine 454, a previously reported insulin- and isoproterenol-stimulated ATP-citrate lyase phosphorylation site. Indeed, both insulin and isoproterenol stimulated the phosphorylation of this protein on the site recognized by the phosphospecific antibodies in a wortmannin-sensitive and -insensitive manner, respectively. In addition, transient expression of a constitutively active protein kinase B in primary adipocytes mimicked the effect of insulin on ATP-citrate lyase phosphorylation. Furthermore, ATP-citrate lyase was phosphorylated in vitro by recombinant protein kinase B on the same site. Taken together, these results demonstrate that serine 454 of ATP-citrate lyase is a novel and majorin vivo substrate for protein kinase B. protein kinase B (or Akt) cAMP-dependent protein kinase phosphoinositide 3-OH-kinase ATP-citrate lyase matrix-assisted laser-desorption time of flight glycogen synthase kinase-3 hemagglutinin mitogen-activate protein kinase The protein kinase B (PKB,1 also known as Akt) family comprises three isoforms (PKBα, -β, and -γ) that are highly conserved serine/threonine kinases originally identified through their homology to the transforming retroviral oncogene v-Akt, cAMP- dependent protein kinase (PKA) and protein kinase C (1Staal S.P. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5034-5037Crossref PubMed Scopus (646) Google Scholar, 2Jones 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 (443) Google Scholar, 3Coffer P.J. Woodgett J.R. Eur. J. Biochem. 1991; 201: 475-481Crossref PubMed Scopus (389) Google Scholar). PKB was subsequently found to be activated by a variety of stimuli such as insulin, growth factors, G-protein-coupled receptor agonists, and integrin binding and to function downstream of phosphoinositide 3-OH-kinase (PI3K) (reviewed in Refs. 4Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Crossref PubMed Scopus (969) Google Scholar and5Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1399) Google Scholar). PKB is involved in numerous cellular responses to insulin and growth factors. This includes suppression of apoptosis, at least in part through its ability to phosphorylate the pro-apoptotic protein BAD (6Datta S.R. Dudek H. Tao X. Masters S., Fu, H.A. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4946) Google Scholar, 7delPeso L. GonzalezGarcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1987) Google Scholar), and the regulation of gene expression at the levels of both transcription (via transcription factor phosphorylation (8Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P., Hu, L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5434) Google Scholar, 9Guo S. Rena G. Cichy S., He, X. Cohen P. Unterman T. J. Biol. Chem. 1999; 274: 17184-17192Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 10Du K.Y. Montminy M. J. Biol. Chem. 1998; 273: 32377-32379Abstract Full Text Full Text PDF PubMed Scopus (823) Google Scholar)) and translation (via the regulation of eI4E-binding proteins (11Takata M. Ogawa W. Kitamura T. Hino Y. Kuroda S. Kotani K. Klip A. Gingras A.C. Sonenberg N. Kasuga M. J. Biol. Chem. 1999; 274: 20611-20618Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar)). PKB is also involved in the stimulation of glucose uptake by insulin in muscle and adipose tissues. Transgenic mice that lack PKBβ, the isoform of PKB that predominates in adipose tissue, have markedly impaired glucose tolerance and are insulin-resistant and diabetic (12Cho H., Mu, J. Kim J.K. Thorvaldsen J.L. Chu Q. Crenshaw E.B. Kaestner K.H. Bartolomei M.S. Shulman G.I. Birnbaum M.J. Science. 2001; 292: 1728-1731Crossref PubMed Scopus (1547) Google Scholar). PKB has been implicated in mediating insulin-stimulated glucose uptake, at least in part through stimulating the translocation of the insulin-responsive glucose transporter, GLUT4, to the plasma membrane (13Cong L.N. Chen H., Li, Y. Zhou L. McGibbon M.A. Taylor S.I. Quon M.J. Mol. Endocrinol. 1997; 11: 1881-1890Crossref PubMed Google Scholar, 14Wang Q. Somwar R. Bilan P.J. Liu Z. Jin J. Woodgett J.R. Klip A. Mol. Cell. Biol. 1999; 19: 4008-4018Crossref PubMed Scopus (502) Google Scholar, 15Foran P.G.P. Fletcher L.M. Oatey P.B. Mohammed N. Dolly J.O. Tavareá J.M. J. Biol. Chem. 1999; 274: 28087-28095Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). PKB also plays an important role in the stimulation of glycogen synthase by insulin via the phosphorylation and inactivation of glycogen synthase kinase-3 (GSK-3α and GSK-3β (16Cross D.A.E. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4376) Google Scholar)). In many cases, for example GLUT4 translocation, the substrates of PKB that subsequently mediate the response have not been discovered. To identify potentially novel PKB targets in adipocytes, we have utilized a proteomic approach using a commercially available antibody raised against the minimal PKB consensus phosphorylation site found on almost all of its known substrates, RXRXX(pS/pT) (17Alessi D.R. Caudwell F.B. Andjelkovic M. Hemmings B.A. Cohen P. FEBS Lett. 1996; 399: 333-338Crossref PubMed Scopus (550) Google Scholar,18Obata T. Yaffe M.B. Leparc G.G. Piro E.T. Maegawa H. Kashiwagi A. Kikkawa R. Cantley L.C. J. Biol. Chem. 2000; 275: 36108-36115Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). In the process of our study, we identified ATP-citrate lyase (ACL) as a novel PKB substrate in primary rat adipocytes. Except where otherwise stated, all chemicals were from BDH (Lutterworth, Leicester, UK), and biochemicals were from Sigma Chemical Co. (Poole, Dorset, UK). Adenosine 5′-[32P]trisphosphate was from ICN Radiochemicals (Basingstoke, UK). Plasmids containing wild-type PKB (HA-PKB), constitutively active myristoylated PKB (Myr-PKB), and constitutively active p110 subunit of PI3K (p110.CAAX) were kindly provided by Dr. D. Alessi (University of Dundee), Dr. B. Hemmings (Friedrich Miescher Institute, Basel), and Dr. J. Downward (Cancer Research UK, London), respectively. All the PKB sequences included an N-terminal HA tag and were subcloned into the vector pcDNA3 (Invitrogen) for expression in rat adipocytes. Anti-ACL antisera, raised in either rabbit or chicken, were provided by Prof. R. M. Denton (University of Bristol). Male Wistar rats (150–200 g) were fed ad libitumon a stock laboratory diet (B&K Universal Ltd., Hull, UK). Adipocytes were isolated from the epididymal fat pads of Wistar rats as described previously (19Diggle T.A. Moule S.K. Avison M.B. Flynn A. Foulstone E.J. Proud C.G. Denton R.M. Biochem. J. 1996; 316: 447-453Crossref PubMed Scopus (83) Google Scholar). Cells were subsequently washed in Krebs-bicarbonate-HEPES buffer, pH 7.4 (130 mmNaCl, 4.7 mm KCl, 1.5 mm MgSO4, 1.2 mm CaCl2, 2.5 mmNaH2PO4, 15.5 mmNaHCO3, 10 mm HEPES, and 11 mmglucose) without bovine serum albumin. The cells were pre-treated with 100 nm wortmannin or 10 μm U0126 (Promega, Southampton, UK) for 30 min as indicated in the figure legends and subsequently incubated with 83 nm insulin or 10 nm isoproterenol for 10 min as required. The reaction was terminated by extracting the cells 1:1 (packed cell volume/volume) in ice-cold Nonidet P-40 extraction buffer (50 mm Tris, pH 7.5, containing 1 mm EDTA, 120 mm NaCl, 50 mm NaF, 40 mm β-glycerophosphate, 1 mm benzamidine, 1% Nonidet P-40, 1 μmmicrocystin, 7.2 mm mercaptoethanol, 5 mmorthovanadate, and 1 μg/ml each of pepstatin, leupeptin, and antipain). Alternatively, the cells were extracted (1:1, v/v) in ice-cold TES extraction buffer (20 mm Tris, pH 7.5, 5 mm EDTA, 250 mm sucrose, 2 mmNa3VO4, 10 mm NaF, 1 mmNa4P2O7, 100 mmphenylmethylsulfonyl fluoride, and 1 μg/ml each of pepstatin, antipain, and leupeptin) for subsequent protein sub-fractionation studies. Cell extracts were centrifuged at 10,000 × gfor 10 min at 4 °C, and the infranatant was taken for subsequent analysis. Cells were prepared as described previously (19Diggle T.A. Moule S.K. Avison M.B. Flynn A. Foulstone E.J. Proud C.G. Denton R.M. Biochem. J. 1996; 316: 447-453Crossref PubMed Scopus (83) Google Scholar) and washed in intracellular Krebs buffer (4 mm NaCl ,125 mm KCl, 1 mm EGTA, 1 mm MgCl2, 2.5 mmNaH2PO4, 15.5 mmNaHCO3, 10 mm HEPES, and 11 mmglucose). A 0.4-cm electrode gap Gene Pulser cuvette (Bio-Rad, Richmond, CA) was used to electroporate 500 μl of cells (30% cytocrit) in the presence of 5 μg of plasmid was by at and a of using a Gene Pulser cells were to a Universal Ltd., UK) and incubated for 30 min at the with 4 of pH 7.4 1% bovine serum 2 mm nm 100 μg/ml and mm The cells were incubated for at and subsequently washed into Krebs-bicarbonate-HEPES buffer without bovine serum to the Cell extracts prepared as described were by at 10,000 for 10 min and using a to fat and an previously in buffer A (50 mm Tris, pH 1 mm EDTA, 1 mm EGTA, mm β-glycerophosphate, 1 mm and 1 μg/ml each of pepstatin, antipain, and A of buffer A containing NaCl was 2.5 at a of 50 and were with the that the PKB substrate were and to the previously in buffer A. were against a of buffer A containing 1 NaCl 2.5 at a of 100 and were of protein was by and its apparent was by The was from the and using The were by MALDI-TOF using a MALDI-TOF mass with a The used was from 10 in a 1:1 of UK) and The was a of with an of and an extraction time of were using as MALDI-TOF was against the using the W. Chem. 2000; PubMed Scopus Google Scholar). was by 2.5 μg of protein and 5 μl of rabbit with μl of cell for 2 at 4 The were washed three in extraction buffer and in PKB buffer (20 mm HEPES, pH 7.5, mm β-glycerophosphate, 1 mm and subsequently used for in vitro phosphorylation. Alternatively, cell were by at × for 30 min at 4 °C, and the was incubated with 10 μl of for 2 at 4 The were by at × for 30 min at 4 and in Protein were by on to to a using the and antibodies were to the The was used at a In all cases, were with the of a or antibodies as at a of by were incubated for 30 min with of recombinant PKB from Dr. of UK) in the presence of 50 μm and 5 to μl with PKB were 30 min by the of buffer, to by was by or with the isolated primary adipocytes from rat epididymal fat pads were incubated in the or presence of insulin and were to by with a phosphospecific antibody raised against the consensus sequence found in almost all known PKB substrates in insulin stimulated the phosphorylation of many of and as in of the cells in the presence of the PI3K wortmannin the insulin-stimulated phosphorylation of many of these suggesting that are by or downstream of the of phosphorylation using the antibody was to the of insulin stimulation of primary rat adipocytes with Denton R.M. Biochem. J. PubMed Scopus Google Scholar, Denton R.M. Biochem. J. PubMed Scopus Google Scholar). This that this antibody recognizes the of the insulin-stimulated in rat fat incubated primary adipocytes in the presence of wortmannin or U0126 by in the or presence of insulin for 10 the cell were to by and the were by using the antibody was an protein from the at in The of the protein with the antibody was and insulin in a wortmannin-sensitive A three proteins of apparent and were also wortmannin-sensitive and in 4 and 5 mm NaCl, Taken together, these that the phosphorylation of the and proteins is in a each of these proteins insulin-stimulated PKB The protein was to To this we the from the for by on a using protein The were by with the and this revealed that the of the protein not to the in the C. K. J. and J. M. of the revealed protein to be to be from the with and by MALDI-TOF mass The were to using and the of the protein was as ATP-citrate lyase and the with either of two antibodies to the of the has been previously reported to the phosphorylation of in a wortmannin-sensitive S.K. G.I. T.A. Foulstone E.J. Proud C.G. Denton R.M. Biochem. J. 1995; PubMed Scopus Google Scholar), although the kinase involved has not been identified. has also been reported to the phosphorylation of a serine on in rat adipocytes, and this site has been identified as serine 454, in the J.L. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.L. T.A. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. N. Biochem. J. PubMed Scopus Google Scholar). 454 of to the consensus sequence for phosphorylation by PKB where is a (17Alessi D.R. Caudwell F.B. Andjelkovic M. Hemmings B.A. Cohen P. FEBS Lett. 1996; 399: 333-338Crossref PubMed Scopus (550) Google Scholar, T. Yaffe M.B. Leparc G.G. Piro E.T. Maegawa H. Kashiwagi A. Kikkawa R. Cantley L.C. J. Biol. Chem. 2000; 275: 36108-36115Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar)). the in the is in the serine 454 sequence of a at and at and and with the that PKB phosphorylate serine 454, the at the is reported to be by and is well (17Alessi D.R. Caudwell F.B. Andjelkovic M. Hemmings B.A. Cohen P. FEBS Lett. 1996; 399: 333-338Crossref PubMed Scopus (550) Google Scholar, T. Yaffe M.B. Leparc G.G. Piro E.T. Maegawa H. Kashiwagi A. Kikkawa R. Cantley L.C. J. Biol. Chem. 2000; 275: 36108-36115Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). Furthermore, the M.B. Leparc G.G. J. T. S. Cantley L.C. 2001; 19: PubMed Scopus Google Scholar) serine 454 a of is to the PKB in BAD and are known in vivo PKB to the of serine 454 insulin the phosphorylation of serine and in the and to serine are known substrates for phosphorylation by glycogen synthase kinase-3 Woodgett J.R. Y. D. Biochem. J. PubMed Scopus Google Scholar, S. G. PubMed Scopus Google Scholar, S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, K. S. Woodgett J.R. Biochem. J. PubMed Scopus Google Scholar)). is via phosphorylation by PKB on serine and serine in P. S. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). The sequence serine 454 of the PKB phosphorylation on and GSK-3β we also found that a phosphospecific antibody raised against phosphoserine 21/9 of was with by and that this was stimulated by insulin in a wortmannin-sensitive To that the and antibodies were with the was with antibodies from primary and with phosphospecific in from primary adipocytes was with both the and and this was by insulin in a wortmannin-sensitive The isoproterenol is well known to serine 454 phosphorylation on to via the of J.L. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.L. T.A. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). in of the and antibodies was by isoproterenol to an to that with insulin in a Taken the demonstrate that insulin-stimulated serine 454 phosphorylation on be using either of the and and isoproterenol also stimulated the phosphorylation of and GSK-3β the effect of insulin on phosphorylation was almost by the effect of isoproterenol was be that the proteins of and found in cell and 4 and using the antibody were not or were found to be with the such these proteins potentially novel PKB substrates, and their are in our 454 of has been previously to be phosphorylated by an wortmannin-sensitive kinase and by in vitro J.L. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.L. T.A. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. N. Biochem. J. PubMed Scopus Google Scholar). results that PKB be the wortmannin-sensitive serine 454 kinase identified by and S. N. Biochem. J. PubMed Scopus Google Scholar). To PKB is of in recombinant active PKB was incubated with in the presence of in is vitro PKB into was was incubated in the presence of recombinant The phosphorylation of was recognized by the suggesting that was on serine 454 results demonstrate that is a substrate for phosphorylation by PKB in to active PKB the phosphorylation of in cells incubated in the of To this we primary rat adipocytes by This results in of the cells K. and R. M. rat adipocytes were with a constitutively active PKB to the plasma membrane by or a constitutively active PI3K subunit the cells were incubated in the or presence of insulin, and phosphorylation was in cell by with the or in both the constitutively active PKB and stimulated phosphorylation to the same as insulin as using both the and phosphospecific The results the phosphorylation of and were by the constitutively active and as well as insulin Taken together, these results that PKB is for the phosphorylation of in adipocytes. In this we to that ATP-citrate lyase is an in vivo substrate for protein kinase B and that this kinase is for the previously reported insulin-stimulated phosphorylation of serine This not as a novel in vivo PKB also the phosphorylation consensus sequence that be by PKB on an protein to that serine 454 on is a substrate for PKB includes the two phosphospecific antibodies with the PKB consensus sequence with insulin stimulation and in a wortmannin-sensitive have revealed that serine 454 is the known insulin-stimulated phosphorylation site on S. G. PubMed Scopus Google Scholar) and that in a sequence that with the used to the and on serine 454 via the of J.L. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.L. T.A. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar), is also recognized by the and antibodies in a PKB in vitro on a site that is recognized by the antibody and a constitutively active PKB or a constitutively active PI3K (p110.CAAX) that PKB in primary phosphorylation in the of is a that the of and from in an and was identified as a in PubMed Scopus Google Scholar, S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The by this is utilized by to is used for the of by phosphorylation have been identified on serine and serine and receptor an in serine 454 the via the of a in the phosphorylation of the and serine at least in part through the inactivation of although the of a also be involved S. J. Biol. Chem. Full Text PDF PubMed Google 2000; PubMed Scopus Google Scholar). many of from isoproterenol or insulin of cells in vitro phosphorylation by have apparent effect on Biochem. PubMed Scopus Google Scholar, 1995; PubMed Scopus Google Scholar, J. M. P.J. of Scholar). M. 2000; PubMed Scopus Google Scholar) reported that recombinant from was activated in vitro serine 454 was phosphorylated by the phosphorylation of and serine by was without that the to in the of from was to phosphorylation its and subsequent the lack such is to a serine 454 of by insulin and isoproterenol with their and on a phosphorylation also be important in of such as its with and S. Biochem. PubMed Scopus Google Scholar) have reported that the phosphorylation of and serine is by insulin not by isoproterenol in rat adipose these two on function through in the phosphorylation of serine and serine In the of serine 454 phosphorylation the phosphoserine for phosphorylation of and serine by 2000; PubMed Scopus Google Scholar, S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. PubMed Scopus Google Scholar). In the of insulin, serine 454 phosphorylation and serine as a of and the of a and serine S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, 2000; PubMed Scopus Google Scholar). serine 454 phosphorylation for and serine the insulin is and is and are to these that the effect of insulin on serine 454 and is by we that insulin receptor to the phosphorylation of insulin receptor substrates, of of plasma membrane phosphoinositide of via and and serine 454 phosphorylation. The by insulin are and are stimulated via in and kinase M. Denton R.M. 1997; PubMed Google Scholar). the by insulin is not has the insulin-stimulated kinase that been identified. The phosphospecific PKB substrate antibody that we used in this study, was raised to the PKB consensus sequence not to the in the for this is in Indeed, the that PKB in is To we are of that that PKB phosphorylate a protein substrate that the in the to the phosphorylation this serine of K.Y. Montminy M. J. Biol. Chem. 1998; 273: 32377-32379Abstract Full Text Full Text PDF PubMed Scopus (823) Google Scholar). serine of in the sequence serine 454 of is also a for phosphorylation by K.Y. Montminy M. J. Biol. Chem. 1998; 273: 32377-32379Abstract Full Text Full Text PDF PubMed Scopus (823) Google Scholar). All known sequences in protein substrates for PKB to to the M.A. Alessi D.R. J. Cell Sci. 2001; PubMed Google Hemmings B.A. Biochem. Sci. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The consensus PKB phosphorylation has been from a of two that (17Alessi D.R. Caudwell F.B. Andjelkovic M. Hemmings B.A. Cohen P. FEBS Lett. 1996; 399: 333-338Crossref PubMed Scopus (550) Google Scholar,18Obata T. Yaffe M.B. Leparc G.G. Piro E.T. Maegawa H. Kashiwagi A. Kikkawa R. Cantley L.C. J. Biol. Chem. 2000; 275: 36108-36115Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). The was reported to be the substrate for phosphorylation by recombinant PKB T. Yaffe M.B. Leparc G.G. Piro E.T. Maegawa H. Kashiwagi A. Kikkawa R. Cantley L.C. J. Biol. Chem. 2000; 275: 36108-36115Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). of the at to the phosphoserine the ability of this to as a In a with an at for an was phosphorylated with a of in of the at for a be the for serine 454 in is a at this be to although this to be is important to the PKB substrate consensus sequence has been from using not the for an protein substrate in this be homology for PKB substrates on the results the that also be important in a site to phosphorylation by Furthermore, Alessi and M.A. Alessi D.R. J. Cell Sci. 2001; PubMed Google Scholar) have the of a PKB site on substrates in a to the on that as a site. This a site for PKB phosphorylation an to be such a PKB or such a site in PKB substrate identified to In our that serine 454 on is a novel in vivo substrate for Furthermore, we demonstrate that the of a phosphospecific antibody to the PKB substrate consensus sequence is a proteomic approach for novel PKB are to Denton (University of for are also to Alessi (University of Dundee), Hemmings (Friedrich Miescher Institute, Basel), and Downward (Cancer Research UK, for the of recombinant and and to (University of for with MALDI-TOF mass

Abstract Integrin αIIbβ3 is a highly abundant heterodimeric platelet receptor that can transmit information bidirectionally across the plasma membrane, and plays a critical role in hemostasis and thrombosis. Upon platelet activation, inside-out signaling pathways increase the affinity of αIIbβ3 for fibrinogen and other ligands. Ligand binding and integrin clustering subsequently stimulate outside-in signaling, which initiates and amplifies a range of cellular events driving essential platelet processes such as spreading, thrombus consolidation, and clot retraction. Integrin αIIbβ3 has served as an excellent model for the study of integrin biology, and it has become clear that integrin outside-in signaling is highly complex and involves a vast array of enzymes, signaling adaptors, and cytoskeletal components. In this review, we provide a concise but comprehensive overview of αIIbβ3 outside-in signaling, focusing on the key players involved, and how they cooperate to orchestrate this critical aspect of platelet biology. We also discuss gaps in the current understanding of αIIbβ3 outside-in signaling and highlight avenues for future investigation.

BACKGROUND: Platelets are central to the process of hemostasis, rapidly aggregating at sites of blood vessel injury and acting as coagulation nidus sites. On interaction with the subendothelial matrix, platelets are transformed into balloonlike structures as part of the hemostatic response. It remains unclear, however, how and why platelets generate these structures. We set out to determine the physiological relevance and cellular and molecular mechanisms underlying platelet membrane ballooning. METHODS AND RESULTS: Using 4-dimensional live-cell imaging and electron microscopy, we show that human platelets adherent to collagen are transformed into phosphatidylserine-exposing balloonlike structures with expansive macro/microvesiculate contact surfaces, by a process that we termed procoagulant spreading. We reveal that ballooning is mechanistically and structurally distinct from membrane blebbing and involves disruption to the platelet microtubule cytoskeleton and inflation through fluid entry. Unlike blebbing, procoagulant ballooning is irreversible and a consequence of Na(+), Cl(-), and water entry. Furthermore, membrane ballooning correlated with microparticle generation. Inhibition of Na(+), Cl(-), or water entry impaired ballooning, procoagulant spreading, and microparticle generation, and it also diminished local thrombin generation. Human Scott syndrome platelets, which lack expression of Ano-6, also showed a marked reduction in membrane ballooning, consistent with a role for chloride entry in the process. Finally, the blockade of water entry by acetazolamide attenuated ballooning in vitro and markedly suppressed thrombus formation in vivo in a mouse model of thrombosis. CONCLUSIONS: Ballooning and procoagulant spreading of platelets are driven by fluid entry into the cells, and are important for the amplification of localized coagulation in thrombosis.

Activation of platelets by collagen is mediated by the complex glycoprotein VI (GPVI)/Fc receptor gamma (FcR gamma chain). In the current study, the role of 2 Src family kinases, Fyn and Lyn, in GPVI signaling has been examined using murine platelets deficient in one or both kinases. In the fyn(-/-) platelets, tyrosine phosphorylation of FcR gamma chain, phopholipase C (PLC) activity, aggregation, and secretion are reduced, though the time of onset of response is unchanged. In the lyn(-/-) platelets, there is a delay of up to 30 seconds in the onset of tyrosine phosphorylation and functional responses, followed by recovery of phosphorylation and potentiation of aggregation and alpha-granule secretion. Tyrosine phosphorylation and aggregation in response to stimulation by collagen-related peptide is further attenuated and delayed in fyn(-/-)lyn(-/-) double-mutant platelets, and potentiation is not seen. This study provides the first genetic evidence that Fyn and Lyn mediate FcR immune receptor tyrosine-based activation motif phosphorylation and PLC gamma 2 activation after the ligation of GPVI. Lyn plays an additional role in inhibiting platelet activation through an uncharacterized inhibitory pathway. (Blood. 2000;96:4246-4253)