Protein kinase B (PKB), also known as Akt or RAC-PK, is a serine/threonine kinase that can be activated by growth factors via phosphatidylinositol 3-kinase. In this article we show that PKC but not PKC␣ and PKC␦ can co-immunoprecipitate PKB from CHO cell lysates. Association of PKB with PKC was also found in COS-1 cells transiently expressing PKB and PKC, and moreover we found that this association is mediated by the AH domain of PKB. Stimulation of COS-1 cells with plateletderived growth factor (PDGF) resulted in a decrease in the PKB-PKC interaction. The use of kinase-inactive mutants of both kinases revealed that dissociation of the complex depends upon PKB activity. Analysis of the activities of the interacting kinases showed that PDGFinduced activation of PKC was not affected by co-expression of PKB. However, both PDGF-and p110-CAAXinduced activation of PKB were significantly abolished in cells co-expressing PKC. In contrast, co-expression of a kinase-dead PKC mutant showed an increased induction of PKB activity upon PDGF treatment. Downstream signaling of PKB, such as the inhibition of glycogen synthase kinase-3, was also reduced by coexpression of PKC. A clear inhibitory effect of PKC was found on the constitutively active double PKB mutant (T308D/S473D). In summary, our results demonstrate that PKB interacts with PKC in vivo and that PKC acts as a negative regulator of PKB.Protein kinase B (PKB), 1 also referred to as c-Akt or RAC-PK is a 60-kDa serine/threonine kinase which is the cellular homologue of the viral oncogene v-Akt (1-3). So far, three isoforms of PKB have been isolated: PKB␣, PKB␤, and PKB␥ (1, 2, 4, 5). Overexpression of PKB family members has been correlated with different cancers such as breast cancer and some pancreatic and ovarian cancers (2, 6, 7). Recently, PKB has been found to yield an anti-apoptotic signal, which is crucial for cell survival in both fibroblasts and neuronal cells (8, 9). Other reports have indicated a role for PKB in the regulation of glycogen synthesis by inhibition of glycogen synthase kinase-3 (GSK-3) (10, 11). In addition, glucose uptake and metabolism in 3T3-L1 adipocytes have been shown to be regulated by PKB by mediating the translocation of the glucose transporter GLUT4 to the plasma membrane (12, 13). Moreover, a role for PKB has been described in the regulation of protein synthesis through indirect activation of the p70 ribosomal S6 kinase (p70 S6K ) (14). PKB comprises a NH 2 -terminal Akt homology (AH) domain of 148 amino acids, a catalytic domain of 264 amino acids showing high homology with cyclic AMP-dependent protein kinase A (PKA) and protein kinase C (PKC) and a short COOHterminal tail of 68 amino acids. A pleckstrin homology (PH) domain of 106 amino acids is present within the AH domain. Treatment of cells with different growth factors, insulin, or phosphatase inhibitors results in rapid activation of PKB (10,14,15). Also heat shock, hyperosmolarity stress, and intracellular cAMP elevation were shown to activate PKB in vivo (16,17). Growth facto...

Plant cell PMs contain a family of multifunctional H+-ATPases (Sussman, 1994). These H+-ATPases provide the driving force for ion and solute uptake, control the cytoplasmic pH, lower the cell-wall pH to enable cell elongation, and control the activity of voltage-sensitive transporters by means of the electrical membrane potential. In view of these numerous functions, it is not surprising that the activity of the pumps is controlled in a number of ways (Palmgren, 1991). Tight control is exerted by the C terminus of the pump, which acts as an autoinhibitor; a series of phosphorylation sites in this domain are potential control

The fusicoccin-binding protein was solubilised from purified oat root plasma membranes. The solubilised protein retained full binding activity, provided that protease inhibitors were included. Sodium fluoride reduced the high-affinity [3H]fusicoccin binding to almost zero in a concentrationdependent way, with an optimum at approximately 20 mM sodium fluoride. The presence of magnesium (> 100 pM) was required for the inhibitory action of fluoride, whereas addition of low amounts of aluminium (25 pM It is concluded that the inhibitory effect of fluoride upon the binding of fusicoccin is indirect and mediated through activated GTP-binding proteins. A hypothesis on the mechanism of fusicoccin action is presented wherein the fusicoccin-binding protein is one component of a signal-transduction chain, two or more steps downstream of a heterotrimeric GTP-binding protein.The fungal toxin fusicoccin has evoked great interest amongst plant physiologists, ever since its discovery was reported (Ballio et al., 1964). The toxin affects all higher plant cells tested thus far and typical responses of these cells are hyperpolarization of the membrane potential, increased H' efflux and K+ influx followed by elongation growth of the cell. Initially, it was suggested that fusicoccin might interfere with the signal-transduction pathway of hormones like auxin and abscisic acid. Recent studies by Hager et al. (1991) have cast doubts on a similar mode of action of auxin and fusicoccin; indole-3-acetic acid did promote the incorporation of H+-ATPase molecules into the plasma membrane by vesicle fusion while fusicoccin had no measurable effect. Fusicoccin might act through interference with regulatory mechanisms of the H+ pump and reports about the presence of endogenous fusicoccin-like ligands in the plant (Aducci et al., 1980),

The role of diacylglycerol (DG) formation from phosphatidylcholine in mitogenic signal transduction is poorly understood. We have generated this lipid at the plasma membrane by treating Rat-1 fibroblasts with bacterial phosphatidylcholine-specific phospholipase C (PC-PLC). This treatment leads to activation of mitogen-activated protein kinase (MAPK). However, unlike platelet-derived growth factor (PDGF) or epidermal growth factor (EGF), PC-PLC fails to activate Ras and to induce DNA synthesis, and activates MAPK only transiently (< 45 min). Down-regulation of protein kinase C (PKC) -α, -Δ and -ε isotypes has little or no effect on MAPK activation by either PC-PLC or growth factors. However, Ro 31-8220, a highly selective inhibitor of all PKC isotypes, including atypical PKC-ζ but not Raf-1, blocks MAPK activation by PDGF and PC-PLC, but not that by EGF, suggesting that atypical PKC mediates the PDGF and PC-PLC signal. In line with this, PKC-ζ is activated by PC-PLC and PDGF, but not by EGF, as shown by a kinase assay in vitro, using biotinylated ε-peptide as a substrate. Furthermore, dominant-negative PKC-ζ inhibits, while (wild-type) PKC-ζ overexpression enhances MAPK activation by PDGF and PC-PLC. The results suggest that DG generated by PC-PLC can activate the MAPK pathway independent of Ras and phorbol-ester-sensitive PKC but, instead, via PKC-ζ.