Identification of Tyrosine Phosphorylation Sites on 3-Phosphoinositide-dependent Protein Kinase-1 and Their Role in Regulating Kinase Activity*
Identification of Tyrosine Phosphorylation Sites on 3-Phosphoinositide-dependent Protein Kinase-1 and Their Role in Regulating Kinase Activity*
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DOI：
10.1074/jbc.m105916200
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发表时间：
2001-10
期刊：
影响因子：
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通讯作者：
Jongsun Park;M. Hill;D. Hess;D. Brazil;J. Hofsteenge;B. Hemmings
中科院分区：
文献类型：
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作者：
Jongsun Park;M. Hill;D. Hess;D. Brazil;J. Hofsteenge;B. Hemmings
3-磷酸肌醇依赖性蛋白激酶-1（PDK 1）在激活磷酸肌醇3-激酶的信号转导途径中起着核心作用。尽管其作为酶如蛋白激酶B和p70核糖体蛋白S6激酶的上游激活剂的关键作用，但控制PDK 1活性的调节机制知之甚少。据报道，PDK 1在静息细胞中具有组成性活性，并且不被生长因子刺激进一步激活（Casamayor，A.，Morrice，N.一、和Alessi，D. R.（1999）Biochem.J.342，287-292）。在这里，我们报告说，PDK 1成为酪氨酸磷酸化和易位到质膜响应过钒酸盐和胰岛素。在过钒酸盐处理后，PDK 1激酶活性增加1.5- 3倍，而与质膜相关的PDK 1活性增加约6倍。定位于质膜的PDK 1的活性也通过胰岛素处理而增加。使用体内标记和质谱法鉴定了PDK 1的三个酪氨酸磷酸化位点（Tyr-9和Tyr-373/376）。使用定点突变体，我们表明，虽然磷酸化酪氨酸-373/376是重要的PDK 1的活性，磷酸化酪氨酸-9激酶的活性没有影响。这两个残基都可以在体外被v-Src酪氨酸激酶磷酸化，v-Src的共表达导致PDK 1的酪氨酸磷酸化和激活。因此，这些数据表明，PDK 1活性是由可逆的磷酸化，可能是由Src激酶家族的成员。
3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in signal transduction pathways that activate phosphoinositide 3-kinase. Despite its key role as an upstream activator of enzymes such as protein kinase B and p70 ribosomal protein S6 kinase, the regulatory mechanisms controlling PDK1 activity are poorly understood. PDK1 has been reported to be constitutively active in resting cells and not further activated by growth factor stimulation (Casamayor, A., Morrice, N. A., and Alessi, D. R. (1999) Biochem. J. 342, 287–292). Here, we report that PDK1 becomes tyrosine-phosphorylated and translocates to the plasma membrane in response to pervanadate and insulin. Following pervanadate treatment, PDK1 kinase activity increased 1.5- to 3-fold whereas the activity of PDK1 associated with the plasma membrane increased ∼6-fold. The activity of PDK1 localized to the plasma membrane was also increased by insulin treatment. Three tyrosine phosphorylation sites of PDK1 (Tyr-9 and Tyr-373/376) were identified using in vivo labeling and mass spectrometry. Using site-directed mutants, we show that, although phosphorylation on Tyr-373/376 is important for PDK1 activity, phosphorylation on Tyr-9 has no effect on the activity of the kinase. Both of these residues can be phosphorylated by v-Src tyrosine kinasein vitro, and co-expression of v-Src leads to tyrosine phosphorylation and activation of PDK1. Thus, these data suggest that PDK1 activity is regulated by reversible phosphorylation, possibly by a member of the Src kinase family.