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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-3␣/␤. 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 isoproterenolstimulated 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 major in vivo substrate for protein kinase B.

, 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.

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.

Insulin-stimulated glucose uptake involves the recruitment of the glucose transporter 4 isoform (GLUT4) from an intracellular location to the plasma membrane of fat and muscle cells. Although the activation of the PI3-kinase/protein kinase B (PKB) pathway is central to this effect of insulin, the key substrates for PKB that are involved require identification. Here we report that serine318 on the FYVE domain-containing PtdIns(3)P 5-kinase (PIKfyve) is a novel substrate for PKB, and show that phosphorylation stimulates the PtdIns(3)P 5-kinase activity of the enzyme. We also demonstrate that PIKfyve is phosphorylated on serine318 in intact cells in response to insulin, in a PI3-kinase-dependent manner, and that PIKfyve colocalises with a highly motile subpopulation of insulin-regulated aminopeptidase (IRAP)/GLUT4 vesicles. Finally, we demonstrate that overexpression of a PIKfyve[S318A] mutant in 3T3-L1 adipocytes enhances insulin-stimulated IRAP/GLUT4 vesicle translocation to the plasma membrane suggesting a role for PKB-dependent phosphorylation of PIKfyve in insulin-regulated IRAP/GLUT4 trafficking. The phosphorylation and activation of PIKfyve by PKB provides a novel signalling paradigm that may link plasma membrane-localised PtdIns(3,4,5)P3 signals via a protein kinase cascade to regulated PtdIns(3,5)P2 production, and thereby to the control of trafficking of other membrane cargos.

IntroductionVascular damage leads to the rapid recruitment of platelets to the site of injury. Platelets adhere to the newly exposed von Willebrand factor (VWF) and collagen in the extracellular matrix, leading to subsequent platelet activation and the secretion of dense and ␣ granule content into the extracellular media. Platelet granules contain a range of autocrine agonists that can activate or modulate the function of other platelets to form aggregates, including ADP, serotonin, and possibly Gas6 1,2 and tachykinins. 3 Another factor that is found in the ␣ granules in platelets is insulin-like growth factor-1 (IGF-1), 4,5 a growth factor involved in cell proliferation, differentiation, and migration. 6 The presence of IGF-1 at the site of vascular injury is essential in the wound-healing process, 7 and local delivery by platelets may therefore support the healing process.IGF-1 is a peptide hormone whose concentration in plasma increases in response to growth hormone, largely due to increased production by the liver. 8 The effect of IGF-1 is modulated by multiple IGF-binding proteins (IGFBPs), which bind IGF-1 and thereby serve as transporter proteins and storage pools. 6 The receptor for IGF-1 has a wide tissue expression and is closely related to the insulin receptor. The insulin receptor is involved in plasma glucose homeostasis and therefore signals mainly to metabolic responses, whereas the IGF receptor couples primarily to mitogenic responses. Both receptors are heterodimers composed of two ligand-binding ␣ subunits linked by disulphide bridges to two transmembrane ␤ subunits that possess ligand-stimulated tyrosine kinase activity. Ligand binding and subsequent activation leads to the phosphorylation of the major substrates Shc and the insulin receptor substrate (IRS) proteins IRS-1 and IRS-2. Tyrosine phosphorylation of IRS proteins on specific residues creates binding sites for Src homology (SH2)-containing proteins such as the tyrosine phosphatase SHP-2 and the regulatory p85 subunit of phosphoinositide-3 kinase (PI3K), thereby increasing their enzyme activity and/or localizing them to their substrates. Activation of PI3K leads to a rapid rise in PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 , which results in the recruitment of protein kinase B (PKB) to the plasma membrane and subsequent phosphorylation of PKB on Thr308 by PDK1 and Ser473 by second kinase, which is likely to be the mTOR/Rictor complex. 9 Subsequent activation of PKB is involved in numerous cellular responses to insulin and IGF-1. 10,11 Insulin resistance and altered blood IGF-1 levels have been implicated in the development of cardiovascular disease. 12,13 Insulin resistance is described as the reduced ability of insulin to lower glucose concentrations in the blood, and is closely associated with obesity and diabetes. 13 Increased free IGF-1 levels and decreased levels of IGFBP-1 are found in serum of obese subjects compared with lean subjects, whereas total IGF-1 levels are within the normal range. 14 Raised local levels of IGF-1 are also li...

Background: The constitutively active kinase GSK3β is a negative regulator of thrombin-stimulated platelet function.Results: Interfering with PKCα and Akt blocked thrombin-mediated GSK3α/β phosphorylation and reduced platelet function.Conclusion: PKCα and Akt phosphorylate GSK3α/β resulting in reduced GSK3α/β activity and increased thrombin-mediated platelet function.Significance: This study shows a novel mechanism by which GSK3α/β is regulated and contributes to platelet function.

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