Institute of Clinical Biochemistry and Pathobiochemistry, University of Wuerzburg, Wuerzburg, Germany.
J Thromb Haemost. 2013 Aug;11(8):1574-82. doi: 10.1111/jth.12271.
Signaling via protein kinase A (PKA) and protein kinase G (PKG) is critical for maintaining platelets in the resting state. Both kinases down-regulate the activity of the small GTPase Rap1b, a critical signaling switch for integrin activation and platelet aggregation. However, the mechanism of Rap1b regulation by PKA and PKG is largely unknown.
To identify the PKA phosphorylation sites in calcium and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), the main GEF for Rap1b in platelets, and the effect of CalDAG-GEFI phosphorylation in Rap1b activation.
The phosphorylation sites in CalDAG-GEFI were identified by radio-active phosphate incorporation assay and mass spectrometry. Phospho-antibody was developed to detect CalDAG-GEFI phosphorylation in Western blots. Rap1b activation was detected by Rap1-GTP pull-down assay.
S587 was identified as the major PKA phosphorylation site in CalDAG-GEFI, while S116/117 was weakly phosphorylated. Phosphorylation of S587 correlated with the inhibitory effect of PKA on Rap1b activation in platelets. In HEK293 cells, expression of a phospho-mimetic mutant of CalDAG-GEFI (S587D) abolished agonist-induced Rap1b activation. Mutation of S587 to alanine partially reversed the inhibitory effect of PKA signaling on Rap1b activation, while mutation of S116, S117 and S587 to alanine completely abolished the inhibitory effect of PKA on Rap1b activation.
Our study strongly suggests that phosphorylation of CalDAG-GEFI is a critical mechanism by which PKA controls Rap1b-dependent platelet aggregation.
蛋白激酶 A（PKA）和蛋白激酶 G（PKG）的信号传递对于维持血小板的静止状态至关重要。这两种激酶都下调小 GTPase Rap1b 的活性，Rap1b 是整合素激活和血小板聚集的关键信号开关。然而，PKA 和 PKG 调节 Rap1b 的机制在很大程度上尚不清楚。
鉴定钙和二酰基甘油调节鸟嘌呤核苷酸交换因子 I（CalDAG-GEFI）中 PKA 磷酸化位点，CalDAG-GEFI 是血小板中 Rap1b 的主要 GEF，以及 CalDAG-GEFI 磷酸化对 Rap1b 激活的影响。
通过放射性磷酸掺入测定和质谱法鉴定 CalDAG-GEFI 中的磷酸化位点。开发磷酸抗体检测 Western blot 中的 CalDAG-GEFI 磷酸化。通过 Rap1-GTP 下拉测定检测 Rap1b 激活。
鉴定出 S587 是 CalDAG-GEFI 中的主要 PKA 磷酸化位点，而 S116/117 则较弱地磷酸化。S587 的磷酸化与 PKA 对血小板中 Rap1b 激活的抑制作用相关。在 HEK293 细胞中，表达 CalDAG-GEFI 的磷酸模拟突变体（S587D）消除了激动剂诱导的 Rap1b 激活。将 S587 突变为丙氨酸部分逆转了 PKA 信号对 Rap1b 激活的抑制作用，而将 S116、S117 和 S587 突变为丙氨酸则完全消除了 PKA 对 Rap1b 激活的抑制作用。
我们的研究强烈表明，CalDAG-GEFI 的磷酸化是 PKA 控制 Rap1b 依赖性血小板聚集的关键机制。
