Riojas Ramon A, Kikani Chintan K, Wang Changhua, Mao Xuming, Zhou Lijun, Langlais Paul R, Hu Derong, Roberts James L, Dong Lily Q, Liu Feng
Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229.
Department of Pharmacology, University of Texas Health Science Center, San Antonio, Texas 78229.
J Biol Chem. 2006 Aug 4;281(31):21588-21593. doi: 10.1074/jbc.M600393200. Epub 2006 Jun 2.
3-Phosphoinositide-dependent protein kinase-1 (PDK1) mediates phosphorylation and activation of members of the AGC protein kinase family and plays an essential role in insulin signaling and action. However, whether and how PDK1 activity is regulated in cells remains largely uncharacterized. In the present study, we show that PDK1 undergoes insulin-stimulated and phosphatidylinositol 3-kinase-dependent phosphorylation at Ser244 in the activation loop and at a novel site: Ser163 in the hinge region between the two lobes of the kinase domain. Sequence alignment studies revealed that the residue corresponding to Ser163 of PDK1 in all other AGC kinases is glutamate, suggesting that a negative charge at this site may be important for PDK1 function. Replacing Ser163 with a negatively charged residue, glutamate, led to a 2-fold increase in PDK1 activity. Molecular modeling studies suggested that phosphorylated Ser163 may form additional hydrogen bonds with Tyr149 and Gln223. In support of this, mutation of Tyr149 to Ala is sufficient to reduce PDK1 activity. Taken together, our results suggest that PDK1 phosphorylation of Ser163 may provide a mechanism to fine-tune PDK1 activity and function in cells.
3-磷酸肌醇依赖性蛋白激酶-1（PDK1）介导AGC蛋白激酶家族成员的磷酸化和激活，在胰岛素信号传导和作用中起重要作用。然而，PDK1活性在细胞中是否以及如何被调节在很大程度上仍不清楚。在本研究中，我们发现PDK1在激活环的Ser244以及一个新位点：激酶结构域两叶之间铰链区的Ser163处发生胰岛素刺激的、磷脂酰肌醇3激酶依赖性磷酸化。序列比对研究表明，在所有其他AGC激酶中，与PDK1的Ser163相对应的残基是谷氨酸，这表明该位点的负电荷可能对PDK1功能很重要。用带负电荷的残基谷氨酸取代Ser163导致PDK1活性增加2倍。分子模拟研究表明，磷酸化的Ser163可能与Tyr149和Gln223形成额外的氢键。支持这一点的是，将Tyr149突变为Ala足以降低PDK1活性。综上所述，我们的结果表明，Ser163的PDK1磷酸化可能提供一种在细胞中微调PDK1活性和功能的机制。
