Investigating cellular function and biochemical mechanism for STK24-CCM3 complex

描述(申请人提供)：我们的研究重点之一是了解G蛋白偶联受体(G蛋白偶联受体)的信号机制和功能。在siRNA筛选中，我们意外地发现丝氨酸/苏氨酸激酶(STK)24及其结合伙伴脑海绵畸形(CCM)3在调节中性粒细胞脱颗粒中起作用。STK24与MST4、STK25同属生发中心激酶(GCK)III亚家族的20个亚家族。已知这些激酶与CCM3相互作用。CCM3直接参与CCM的发病，而GCK III则间接参与CCM的发病。CCM是一种血管病理状态，影响中枢神经系统的血管系统，导致中风、癫痫和脑出血。尽管STK-CCM3复合体具有潜在的重要性，但它们的细胞功能和潜在的生化机制仍然不清楚。在我们发现CCM3和STK24在脱颗粒中的作用后，我们通过质谱仪(MS)分析确定了CCM3和STK24与胞吐调节蛋白UN13D的相互作用。此外，我们收集了坚实的初步结果，以帮助我们了解STK24和CCM3如何机械地调节脱颗粒，以及细胞外刺激，包括GPCR配体，如何通过钙调节STK24/CCM3控制的颗粒池的释放。因此，我们的发现首次为STK24-CCM3复合体的细胞功能提供了明确的生化基础。此外，我们目前的研究揭示了STK24和CCM3在中性粒细胞脱颗粒调节中的未知作用，中性粒细胞脱颗粒是一个对急性先天性免疫反应和组织损伤很重要的过程，发生在慢性和全身炎症和缺血-再灌注中，但研究不足。此外，我们调节脱颗粒的新机制将有助于对配体刺激的胞吐作用的一般理解，脱颗粒是许多细胞的一种形式，并且发生在许多细胞中，包括内皮细胞。 因此，我们的研究可能为最终了解CCM病的致病基础铺平道路，并在许多领域(基础细胞生物学、先天免疫和CCM病)产生潜在的高影响。在本研究中，我们将通过检验STK24和CCM3复合体是中性粒细胞脱颗粒的关键负调节因子的假设来关注中性粒细胞脱颗粒。我们将研究CCM3和STK24调节脱颗粒的生化和分子机制。

DESCRIPTION (provided by applicant): One of our research emphases is to understand GPCR (G protein-coupled receptor) signaling mechanisms and functions. In a siRNA screen, we unexpectedly discovered that Serine/Threonine Kinase (STK) 24 and its binding partner Cerebral Cavernous Malformation (CCM) 3 in regulation of neutrophil degranulation. STK24, together with MST4 and STK25, belongs to the germinal center kinase (GCK) III sub-family of Sterile-20 kinases. These kinases are known to interact with CCM3. CCM3 has been directly, whereas the GCK III kinases indirectly, implicated in the CCM disease. CCM is a vascular pathological condition that affects the vasculature of the central nervous system and results in stroke, seizure and cerebral hemorrhage. Despite the potential importance of the STK-CCM3 complex, their cellular functions and underlying biochemical mechanisms remain obscure. Following our discovery of their role in degranulation, we identified CCM3 and STK24 interaction with an exocytosis regulatory protein, UNC13D, through the mass spectrometry (MS) analysis. Furthermore, we have gathered solid preliminary results to help us understand how STK24 and CCM3 mechanistically regulate the degranulation and how extracellular stimuli, including GPCR ligands, regulate the release of STK24/CCM3-controlled granule pools via Ca2+. Thus, our findings provide the first insights into a cellular function of the STK24-CCM3 complex with well- defined biochemical basis. Moreover, our present study has unraveled previously unknown roles of STK24 and CCM3 in the regulation of neutrophil degranulation, a process important for acute innate immune responses and tissue damage that occurs in chronic and systemic inflammation and ischemia-reperfusion, but understudied. Furthermore, our new mechanism for regulation of degranulation would facilitate the general understanding of ligand-stimulated exocytosis, which degranulation is a form of and occurs in many cells including endothelial cells. Thus our study may pave the way for the eventual understanding of the pathogenic basis for the CCM disease and exert potentially high impact on a number of fields (basic cell biology, innate immunity, and CCM disease). In the present study, we will focus on neutrophil degranulation by testing the hypothesis that the STK24 and CCM3 complex is a key negative regulator of neutrophil degranulation. We will investigate the biochemical and molecular mechanisms by which CCM3 and STK24 regulate degranulation.