Activation of phosphatidylinositide 3-OH kinase (PI 3-kinase) is implicated in mediating a variety of growth factor-induced responses, among which are the inactivation of glycogen synthase kinase-3 (GSK-3) and the activation of the serine/threonine protein kinase B (PKB). GSK-3 inactivation occurs through phosphorylation of Ser-9, and several kinases, such as protein kinase C, mitogen-activated protein kinase-activated protein kinase-1 (p90 Rsk ), p70 S6kinase , and also PKB have been shown to phosphorylate this site in vitro. In the light of the many candidates to mediate insulin-induced GSK-3 inactivation we have investigated the role of PKB by constructing a PKB mutant that exhibits dominant-negative function (inhibition of growth factor-induced activation of PKB at expression levels similar to wild-type PKB), as currently no such mutant has been reported. We observed that the PKB mutant (PKB-CAAX) acts as an efficient inhibitor of PKB activation and also of insulin-induced GSK-3 regulation. Furthermore, it is shown that PKB and GSK-3 co-immunoprecipitate, indicating a direct interaction between GSK-3 and PKB. An additional functional consequence of this interaction is implicated by the observation that the oncogenic form of PKB, gagPKB induces a cellular relocalization of GSK-3 from the cytosolic to the membrane fraction. Our results demonstrate that PKB activation is both necessary and sufficient for insulin-induced GSK-3 inactivation and establish a linear pathway from insulin receptor to GSK-3. Regulation of GSK-3 by PKB is likely through direct interaction, as both proteins co-immunoprecipitate. This interaction also resulted in a translocation of GSK-3 to the membrane in cells expressing transforming gagPKB.Activation of membrane-bound receptors of both the serpentine and tyrosine kinase classes often results in the activation of the lipid kinase PI 1 3-kinase, of which several different isoforms have been described (1). Activation of PI 3-kinase results in the formation of 3Ј-phosphorylated phosphatidylinositol (PI-3P) lipids (i.e. PI 3,4-P 2 and PI 3,4,5-P 3 ). These lipids were long suspected to perform a second messenger function. However, until recently the nature of the PI-3P binding proteins and their role in cellular signaling remained unsolved.

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The small GTPase Rap1, which is activated by a large variety of stimuli, functions in the control of integrinmediated cell adhesion. Here we show that in human megakaryocytes and several other commonly used hematopoietic cell lines such as K562, Jurkat, and THP-1, stress induced by gentle tumbling of the samples resulted in rapid and strong activation of Rap1. This turbulence-induced activation could not be blocked by inhibitors previously shown to affect Rap1 activation in human platelets, such as the intracellular calcium chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid) and various protein kinase C inhibitors. Also inhibition of actin cytoskeleton dynamics did not influence this activation of Rap1, suggesting that this activation is mediated by cell surface receptors. Human platelets, however, were refractory to turbulence-induced activation of Rap1. To determine the consequences of Rap1 activation we measured adhesion of megakaryocytes to fibrinogen, which is mediated by the integrin ␣ IIb ␤ 3 , in the presence of inhibitors of Rap1 signaling. Introduction of both Rap1GAP and RalGDS-RBD in the megakaryoblastic cell line DAMI strongly reduced basal adhesion to immobilized fibrinogen. This inhibition was partially rescued by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate but not by ␣-thrombin. From these results we conclude that in megakaryocytes turbulence induces Rap1 activation that controls ␣ IIb ␤ 3 -mediated cell adhesion.The small GTPase Rap1 is a molecular switch that cycles between an inactive GDP and active GTP-bound conformation. The protein can be activated by a plethora of stimuli (1-6), indicating that Rap1 activation is a common event in signaling. In general this activation is mediated by second messengers such as cAMP, calcium ions, and diacylglycerol; and a number of guanine nucleotide exchange factors have been identified to mediate this activation (7). In human platelets, Rap1 is abundantly expressed and is rapidly activated by a large variety of agonists including ␣-thrombin, thromboxane A 2 , epinephrine, platelet-activating factor, ADP, and the phorbol ester TPA 1 (1). At least two different signaling pathways are involved in this activation, one mediated by G q , phospholipase C, and calcium and one mediated by protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) (1, 8 -10). In platelets, Rap1 activation is rapidly inhibited by agents that increase the intracellular cAMP concentration, like for instance prostaglandin I 2 (1). Activation of Rap1 is prior to platelet activation, suggesting a key role of Rap1 in this process. After platelet activation Rap1 relocalizes to the actin cytoskeleton (8,11,12). In the control of Rap1 in platelets, RapGAP, which can interact with the active ␣ subunit of the heterotrimeric G-protein G z , plays a role as well (13).In a variety of cell lines, Rap1 was found to be involved in the regulation of integrin-mediated cell adhesion. Rap1 controls T-cell receptor-, CD31-, and CD98-induced activation of ␣ L ␤ 2 (14 ...

Ral is a ubiquitously expressed Ras-like small GTPase. Several guanine nucleotide exchange factors for Ral have been identified, including members of the RalGDS family, which exhibit a Ras binding domain and are regulated by binding to RasGTP. Here we describe a novel type of RalGEF, RalGEF2. This guanine nucleotide exchange factor has a characteristic Cdc25-like catalytic domain at the N terminus and a pleckstrin homology (PH) domain at the C terminus. RalGEF2 is able to activate Ral both in vivo and in vitro. Deletion of the PH domain results in an increased cytoplasmic localization of the protein and a corresponding reduction in activity in vivo, suggesting that the PH domain functions as a membrane anchor necessary for optimal activity in vivo.