Matsuzaki Hidenori, Yamamoto Toshiyoshi, Kikkawa Ushio
Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan.
Biochemistry. 2004 Apr 13;43(14):4284-93. doi: 10.1021/bi0498712.
Protein kinase B (PKB) alpha, having the pleckstrin homology (PH) and catalytic domains in its amino- and carboxyl-terminal regions, respectively, is activated in the signaling pathway of growth factors as a downstream target of phosphatidylinositol 3-kinase and becomes an active form in heat-shocked cells in a manner independent of the lipid kinase. Therefore, the activation mechanisms of PKBalpha were compared in platelet-derived growth factor (PDGF)-stimulated and heat-shocked cells by monitoring the protein kinase activity and phosphorylation of the mutant molecules expressed in COS-7 cells. In heat-shocked cells, PKBalpha was activated to a certain level without phosphorylation on Thr-308 in the activation loop and on Thr-450 and Ser-473 in the carboxyl-terminal end region, which is critical for growth-factor-induced activation of PKBalpha. Metabolic labeling with (32)P-orthophosphate in the transfected cells revealed that there is no major phosphorylation site other than the three residues in PKBalpha. PKBalpha activated by heat shock was more stable than the enzyme stimulated by PDGF in the cells, and PKBalpha recovered from heat-shocked cells was resistant to the protein phosphatase treatment, whereas the enzyme obtained from the growth-factor-stimulated cells was inactivated by dephosphorylation. Heat shock also enhanced the association of the PH-domain fragment to the full-length PKBalpha in the transfected cells. On the other hand, the PH-domain fragment of PKBalpha, which moves from the cytosol to the plasma membrane upon PDGF stimulation by the interaction with the phosphatidylinositol 3-kinase products, did not translocate but stayed in the cytosol in heat-shocked NIH 3T3 cells. Furthermore, PKBalpha was associated with the nuclear region in heat-shocked cells, which is not observed in growth-factor-stimulated cells. These results indicate that heat shock induces the conformational change of PKBalpha that accompanies the protein complex formation and perinuculear/nuclear localization of the enzyme, to generate an active form by a mechanism distinct from that in the growth-factor-signaling pathway.
蛋白激酶B（PKB）α在其氨基末端和羧基末端区域分别具有普列克底物蛋白同源（PH）结构域和催化结构域，作为磷脂酰肌醇3激酶的下游靶点在生长因子信号通路中被激活，并以一种独立于脂质激酶的方式在热休克细胞中转变为活性形式。因此，通过监测在COS-7细胞中表达的突变分子的蛋白激酶活性和磷酸化情况，比较了血小板衍生生长因子（PDGF）刺激的细胞和热休克细胞中PKBα的激活机制。在热休克细胞中，PKBα在激活环中的苏氨酸-308以及羧基末端区域的苏氨酸-450和丝氨酸-473未发生磷酸化的情况下被激活到一定水平，而这些位点对于生长因子诱导的PKBα激活至关重要。用（32）P-正磷酸盐对转染细胞进行代谢标记显示，除了PKBα中的这三个残基外没有其他主要磷酸化位点。热休克激活的PKBα在细胞中比PDGF刺激的酶更稳定，从热休克细胞中回收的PKBα对蛋白磷酸酶处理具有抗性，而从生长因子刺激的细胞中获得的酶则通过去磷酸化而失活。热休克还增强了转染细胞中PH结构域片段与全长PKBα的结合。另一方面，PKBα的PH结构域片段在PDGF刺激下通过与磷脂酰肌醇3激酶产物相互作用从胞质溶胶转移到质膜，但在热休克的NIH 3T3细胞中不发生转位而是留在胞质溶胶中。此外，PKBα在热休克细胞中与核区域相关联，而在生长因子刺激的细胞中未观察到这种情况。这些结果表明，热休克诱导PKBα的构象变化，伴随着该酶的蛋白复合物形成和核周/核定位，通过一种不同于生长因子信号通路的机制产生活性形式。
