Dutil E M, Toker A, Newton A C
Department of Pharmacology, University of California at San Diego, La Jolla, California 92093-0640, USA.
Curr Biol. 1998;8(25):1366-75. doi: 10.1016/s0960-9822(98)00017-7.
Phosphorylation critically regulates the catalytic function of most members of the protein kinase superfamily. One such member, protein kinase C (PKC), contains two phosphorylation switches: a site on the activation loop that is phosphorylated by another kinase, and two autophosphorylation sites in the carboxyl terminus. For conventional PKC isozymes, the mature enzyme, which is present in the detergent-soluble fraction of cells, is quantitatively phosphorylated at the carboxy-terminal sites but only partially phosphorylated on the activation loop.
This study identifies the recently discovered phosphoinositide-dependent kinase 1, PDK-1, as a regulator of the activation loop of conventional PKC isozymes. First, studies in vivo revealed that PDK-1 controls the amount of mature (carboxy-terminally phosphorylated) conventional PKC. More specifically, co-expression of the conventional PKC isoform PKC betaII with a catalytically inactive form of PDK-1 in COS-7 cells resulted in both the accumulation of non-phosphorylated PKC and a corresponding decrease in PKC activity. Second, studies in vitro using purified proteins established that PDK-1 specifically phosphorylates the activation loop of PKC alpha and betaII. The phosphorylation of the mature PKC enzyme did not modulate its basal activity or its maximal cofactor-dependent activity. Rather, the phosphorylation of non-phosphorylated enzyme by PDK-1 triggered carboxy-terminal phosphorylation of PKC, thus providing the first step in the generation of catalytically competent (mature) enzyme.
We have shown that PDK-1 controls the phosphorylation of conventional PKC isozymes in vivo. Studies performed in vitro establish that PDK-1 directly phosphorylates PKC on the activation loop, thereby allowing carboxy-terminal phosphorylation of PKC. These data suggest that phosphorylation of the activation loop by PDK-1 provides the first step in the processing of conventional PKC isozymes by phosphorylation.
磷酸化对蛋白激酶超家族大多数成员的催化功能起着关键的调节作用。蛋白激酶C（PKC）就是其中一员，它含有两个磷酸化开关：一个位于激活环上，可被另一种激酶磷酸化；另一个位于羧基末端，有两个自身磷酸化位点。对于传统的PKC同工酶，存在于细胞去污剂可溶部分的成熟酶在羧基末端位点被定量磷酸化，但在激活环上仅部分磷酸化。
本研究确定最近发现的磷酸肌醇依赖性激酶1（PDK-1）是传统PKC同工酶激活环的调节因子。首先，体内研究表明PDK-1控制成熟（羧基末端磷酸化）传统PKC的量。更具体地说，在COS-7细胞中，将传统PKC同工型PKCβII与催化无活性形式的PDK-1共表达，导致未磷酸化的PKC积累以及PKC活性相应降低。其次，使用纯化蛋白进行的体外研究证实，PDK-1特异性磷酸化PKCα和βII的激活环。成熟PKC酶的磷酸化并未调节其基础活性或其最大辅因子依赖性活性。相反，PDK-1对未磷酸化酶的磷酸化引发了PKC的羧基末端磷酸化，从而为产生具有催化活性（成熟）的酶提供了第一步。
我们已经表明，PDK-1在体内控制传统PKC同工酶的磷酸化。体外研究证实，PDK-1直接在激活环上磷酸化PKC，从而使PKC的羧基末端磷酸化。这些数据表明，PDK-1对激活环的磷酸化是通过磷酸化处理传统PKC同工酶的第一步。
