A 3-phosphoinositide-dependent protein kinase-1 (PDK1) docking site is required for the phosphorylation of protein kinase Cζ (PKCζ) and PKC-related kinase 2 by PDK1
A 3-phosphoinositide-dependent protein kinase-1 (PDK1) docking site is required for the phosphorylation of protein kinase Cζ (PKCζ) and PKC-related kinase 2 by PDK1
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DOI：
10.1074/jbc.m000421200
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发表时间：
2000-07-07
影响因子：
4.8
通讯作者：
Alessi, DR
中科院分区：
文献类型：
--
作者：
Balendran, A;Biondi, RM;Alessi, DR
蛋白激酶 AGC 亚家族的成员，包括蛋白激酶 B、p70 S6 激酶和蛋白激酶 C (PKC) 同工型，通过两个残基的磷酸化来激活和/或稳定，一个残基位于激酶结构域的 T 环中，另一个位于激酶结构域 C 末端的疏水基序区域。非典型 PKC 同工型（例如 PKC zeta）和 PKC 相关激酶（例如 PRK2）也可通过其 T 环位点的磷酸化来激活，但其疏水基序中不具有可磷酸化的 Ser/Thr，而是含有酸性残基。 3-磷酸肌醇依赖性蛋白激酶 (PDK1) 通过磷酸化 T 环残基，在体外激活 AGC 激酶亚家族的许多成员，包括 PKC zeta 和 PRK2。在本研究中，我们证明 PKC zeta 和 PKC iota 以及 PRK1 和 PRK2 的疏水基序与 PDK1 的激酶结构域相互作用。全长 PKC zeta、全长 PRK2 或缺少 N 末端调节结构域的 PRK2 的疏水基序的保守残基的突变会消除或显着降低这些激酶与 PDK1 相互作用并在其 T 环位点磷酸化的能力。此外，细胞中 PRK2 疏水基序的过度表达会阻止 T 环磷酸化，从而抑制 PRK2 和 PKC zeta 的激活。这些发现表明 PRK2 和 PKC zeta 的疏水基序充当“对接位点”，使 PDK1 能够募集到这些底物上。这对于细胞中 PDK1 对其进行磷酸化至关重要。
Members of the AGC subfamily of protein kinases including protein kinase B, p70 S6 kinase, and protein kinase C (PKC) isoforms are activated and/or stabilized by phosphorylation of two residues, one that resides in the T-loop of the kinase domain and the other that is located C-terminal to the kinase domain in a region known as the hydrophobic motif. Atypical PKC isoforms, such as PKC zeta, and the PKC-related kinases, like PRK2, are also activated by phosphorylation of their T-loop site but, instead of possessing a phosphorylatable Ser/Thr in their hydrophobic motif, contain an acidic residue. The 3-phosphoinositide-dependent protein kinase (PDK1) activates many members of the AGC subfamily of kinases in vitro, including PKC zeta and PRK2 by phosphorylating the T-loop residue. In the present study we demonstrate that the hydrophobic motifs of PKC zeta and PKC iota, as well as PRK1 and PRK2, interact with the kinase domain of PDK1. Mutation of the conserved residues of the hydrophobic motif of full-length PKC zeta, fulllength PRK2, or PRK2 lacking its N-terminal regulatory domain abolishes or significantly reduces the ability of these kinases to interact with PDK1 and to become phosphorylated at their T-loop sites in rtiao. Furthermore, overexpression of the hydrophobic motif of PRK2 in cells prevents the T-loop phosphorylation and thus inhibits the activation of PRK2 and PKC zeta. These findings indicate that the hydrophobic motif of PRK2 and PKC zeta acts as a "docking site" enabling the recruitment of PDK1 to these substrates. This is essential for their phosphorylation by PDK1 in cells.