为了精细进行功能调节，细胞信号蛋白磷酸化修饰往往发生在多个位点，呈现刺激、时间依赖性变化。因此，研究蛋白整体磷酸化的动态变化具有重要意义。Myh9是一种六聚体非肌肉肌球蛋白（Myosin IIa）的重链，在细胞粘附、分化、信号转导及囊泡分泌等过程中起重要作用。最近我们发现，2个丝氨酸位点的磷酸化修饰通过不同方式调节Myh9介导血管内皮细胞的vWF（一种参与凝血止血等作用的多功能活性分子）分泌功能。本申请将研究Myh9蛋白整体磷酸化的动态变化、功能及其调控机制。我们将利用Zn(II)-Phos-tag SDS聚丙烯酰氨凝胶电泳（SDS-PAGE）技术，结合质谱分析、磷酸化位点突变体表达、化学小分子抑制剂干预等手段进行系统研究。将绘制cAMP激动剂诱导内皮细胞Myh9蛋白磷酸化位点图谱，揭示产生磷酸化责任激酶及上下游途径,还将明确调控内皮细胞分泌vWF的关键磷酸化位点及其作用机制。

In order to precisely adapt to environmental stimulus, the phosphorylation of cellular signaling proteins involves multiple residues in a stimulus- and time-dependent manner. Therefore, it is important to study phosphorylation dynamics on the whole protein level. As a heavy chain of hexameric on-muscle myosin IIA (Myosin IIa), Myh9 participates in a variety of cellular processes such as adhesion, differentiation, signal transduction, and granule exocytosis. Recently, we have identified Myh9/Myosin IIa as a critical regulator of endothelial secretion of von Willebrand factor (vWF, a multi-functional bioactive factor critical for hemostasis and thrombosis) mediated via 2 phosphorylation sites with distinctive functions at different secretory stages. In this application, we will systemically investigate the phosphorylation dynamics of Myh9 protein as a whole, its function and regulatory mechanism. Although mass spectrometry (MS) is a powerful technique capable of detecting protein phosphorylation, it can only determine the sequence and location of phosphorylation sites on peptides, but not on an intact protein. We thus employ a technique called Zn(II)–Phos-tag SDS-PAGE in combination with MS analysis, introduction of point mutation into specific residue, and the interference with kinase specific inhibitor. In addition, we will map the phosphorylation sites of Myh9 via MS analysis of purified phosphor-Myh9 protein. We will also determine the key phosphorylation sites in regulation of endothelial secretion via introduction of point mutation and preparation of site specific anti-phosphor-serine/threonine antibody. Finally, we will elucidate its upstream enzymes via specific chemical inhibitors and gene-specific shRNAs, and reveal their biological significance. In summary, this project will significantly deepen our understanding of the role of dynamic phosphorylation in regulation of Myh9’s function.