Peptides were separated on cellulose thin-layer plates by electrophoresis from left to right (anode on the right), followed by thin-layer chromatography from bottom to top

Peptides were separated on cellulose thin-layer plates by electrophoresis from left to right (anode on the right), followed by thin-layer chromatography from bottom to top. PKB from COS cells showed differences between insulin- and forskolin-stimulated cells that pointed to distinct activation mechanisms of PKB depending on whether insulin or cAMP was used. We looked at events downstream of PKB and found that PKA activation of PKB led to the phosphorylation and inhibition U-93631 of glycogen synthase kinase-3 (GSK-3) activity, a known in vivo substrate of PKB. Overexpression of a dominant unfavorable PKB led to the loss of inhibition of GSK-3 in both insulin- and forskolin-treated cells, demonstrating that PKB was responsible for this inhibition in both cases. Finally, we show by confocal microscopy that forskolin, similar to insulin, was able to induce translocation of PKB to the plasma membrane. This process was inhibited by high concentrations of wortmannin (300 nM), suggesting that forskolin-induced PKB movement may require phospholipids, which are probably U-93631 not generated by class I or class III PI3-kinase. However, high concentrations of wortmannin did not abolish PKB activation, which demonstrates that translocation per se is not important for PKA-induced PKB activation. Protein kinase B (PKB) (also called Akt and RAC [related to A and C protein kinase]) is usually a 60-kDa serine/threonine kinase which was cloned by virtue of its homology to PKA and PKC and is the cellular homologue of the product of the v-oncogene (7, 14, 33, 51). Two other isoforms of Rac1 PKB, termed PKB and PKB, have been identified and are overexpressed in ovarian, pancreatic, and breast malignancy cells (12, 13). Structurally, PKB contains a pleckstrin homology (PH) domain name amino terminal to the catalytic domain name, which is thought to mediate protein-lipid (26) and/or protein-protein interactions (20). The kinase is usually activated rapidly in response to stimulation of tyrosine kinase receptors such as those for platelet-derived growth factor (PDGF), insulin, basic fibroblast growth factor, and epidermal growth factor (11, 25, 35). Growth factor receptor stimulation of PKB has been shown to be dependent on phosphatidylinositol 3-kinase (PI3-kinase) activity for the following reasons: (i) it is sensitive to pharmacological inhibitors of PI3-kinase (35), (ii) PDGF mutant receptors which cannot interact with PI3-kinase fail to activate PKB (25), and (iii) constitutively active forms of PI3-kinase are able to stimulate PKB (11). A model has been proposed to explain activation of PKB in response to insulin and growth factors (2). First, stimulation of cells is usually thought to lead to an increase in the levels of phosphatidylinositol-3,4,5-triphosphate (PtdIns-3,4,5-P3) and PtdIns-3,4-P2 via PI3-kinase. Although it was initially reported that phospholipids could directly activate PKB by interacting with its PH domain name (26), more recently it has been shown that this interaction most likely fulfills this and/or additional functions. The first such function may be to localize PKB to the plasma membrane. Indeed, translocation of PKB has been shown to occur in response to interleukin 2 (1), peroxyvanadate (59), insulin-like growth factor I (IGF-1) (5), and insulin (27). In addition, the binding of phospholipids to the PH domain name of PKB might be necessary for alteration from the conformation of PKB and because of its phosphorylation by activating kinases. One particular PKB kinase which phosphorylates PKB on threonine 308 has been found out (4). This 63-kDa U-93631 monomeric enzyme was called 3-phosphoinositide-dependent proteins kinase-1, because it needs PtdIns-3,4,ptdIns-3 or 5-P3,4-P2 to be able to phosphorylate PKB (3, 52). Another PKB kinase, lately defined as an integrin-linked kinase (21), phosphorylates PKB on serine 473, the next residue important for PKB activity. To day, three different in vivo substrates of PKB have already been identified. The 1st one to become found out was glycogen synthase kinase-3 (GSK-3), which can be thought to donate to the phosphorylation of glycogen synthase, therefore resulting in its inactivation (18). Second, the center isoform.