Chang Jen-Kuan, Ni Yang, Han Liang, Sinnett-Smith James, Jacamo Rodrigo, Rey Osvaldo, Young Steven H, Rozengurt Enrique
From the Division of Digestive Diseases, Department of Medicine, David Geffen School of Medicine.
CURE: Digestive Diseases Research Center, and.
J Biol Chem. 2017 Jun 9;292(23):9523-9539. doi: 10.1074/jbc.M116.771394. Epub 2017 Apr 13.
Although PKC-mediated phosphorylation of protein kinase D1 (PKD1) has been extensively characterized, little is known about PKD1 regulation by other upstream kinases. Here we report that stimulation of epithelial or fibroblastic cells with G protein-coupled receptor agonists, including angiotensin II or bombesin, induced rapid and persistent PKD1 phosphorylation at Ser, a highly conserved residue located within the PKD1 N-terminal domain. Exposure to PKD or PKC family inhibitors did not prevent PKD1 phosphorylation at Ser, indicating that it is not mediated by autophosphorylation. In contrast, several lines of evidence indicated that the phosphorylation of PKD1 at Ser is mediated by kinases of the class I PAK subfamily, specifically 1) exposing cells to four structurally unrelated PAK inhibitors (PF-3758309, FRAX486, FRAX597, and IPA-3) that act via different mechanisms abrogated PKD1 phosphorylation at Ser, 2) siRNA-mediated knockdown of PAK1 and PAK2 in IEC-18 and Swiss 3T3 cells blunted PKD1 phosphorylation at Ser, 3) phosphorylation of Ser markedly increased when recombinant PKD1 was incubated with either PAK1 or PAK2 in the presence of ATP. PAK inhibitors did not interfere with G protein-coupled receptor activation-induced rapid translocation of PKD1 to the plasma membrane but strikingly prevented the dissociation of PKD1 from the plasma membrane and blunted the phosphorylation of nuclear targets, including class IIa histone deacetylases. We conclude that PAK-mediated phosphorylation of PKD1 at Ser triggers its membrane dissociation and subsequent entry into the nucleus, thereby regulating the phosphorylation of PKD1 nuclear targets, including class IIa histone deacetylases.
尽管蛋白激酶D1（PKD1）的蛋白激酶C（PKC）介导的磷酸化已得到广泛研究，但对于PKD1受其他上游激酶的调控却知之甚少。在此，我们报告，用G蛋白偶联受体激动剂（包括血管紧张素II或蛙皮素）刺激上皮细胞或成纤维细胞，会诱导PKD1在丝氨酸（Ser）位点快速且持续的磷酸化，该丝氨酸是位于PKD1 N端结构域内的一个高度保守的残基。暴露于PKD或PKC家族抑制剂并不能阻止PKD1在Ser位点的磷酸化，这表明该磷酸化不是由自身磷酸化介导的。相反，多条证据表明PKD1在Ser位点的磷酸化是由I类p21激活激酶（PAK）亚家族的激酶介导的，具体如下：1）将细胞暴露于四种结构不相关的通过不同机制起作用的PAK抑制剂（PF - 3758309、FRAX486、FRAX597和IPA - 3）可消除PKD1在Ser位点的磷酸化；2）在IEC - 18和瑞士3T3细胞中，小干扰RNA（siRNA）介导的PAK1和PAK2敲低减弱了PKD1在Ser位点的磷酸化；3）当重组PKD1在ATP存在下与PAK1或PAK2一起孵育时，Ser位点的磷酸化显著增加。PAK抑制剂并不干扰G蛋白偶联受体激活诱导的PKD1向质膜的快速转位，但显著阻止了PKD1从质膜解离，并减弱了包括IIa类组蛋白去乙酰化酶在内的核靶点的磷酸化。我们得出结论，PAK介导的PKD1在Ser位点的磷酸化触发其从膜上解离并随后进入细胞核，从而调节PKD1核靶点（包括IIa类组蛋白去乙酰化酶）的磷酸化。
