Investigating cellular function and biochemical mechanism for STK24-CCM3 complex

项目描述（申请人提供）：我们的研究重点之一是了解GPCR（G蛋白偶联受体）信号传导机制和功能。在siRNA筛选中，我们意外地发现丝氨酸/苏氨酸激酶（STK）24及其结合伴侣脑海绵状畸形（CCM）3在调节中性粒细胞脱粒中。STK 24与MST 4和STK 25一起属于Sterile-20激酶的生发中心激酶（GCK）III亚家族。已知这些激酶与CCM 3相互作用。CCM 3直接参与CCM疾病，而GCK III激酶间接参与CCM疾病。CCM是一种影响中枢神经系统血管系统并导致中风、癫痫发作和脑出血的血管病理状况。尽管STK-CCM 3复合物具有潜在的重要性，但它们的细胞功能和潜在的生化机制仍然不清楚。在我们发现它们在脱粒中的作用之后，我们通过质谱（MS）分析鉴定了CCM 3和STK 24与胞吐调节蛋白UNC 13 D的相互作用。此外，我们已经收集了可靠的初步结果，以帮助我们了解STK 24和CCM 3如何机械地调节脱粒以及细胞外刺激物（包括GPCR配体）如何通过Ca 2+调节STK 24/CCM 3控制的颗粒池的释放。因此，我们的发现提供了对具有明确生物化学基础的STK 24-CCM 3复合物的细胞功能的第一次了解。此外，我们目前的研究揭示了STK 24和CCM 3在调节中性粒细胞脱粒中的先前未知的作用，这是一个对慢性和全身性炎症和缺血再灌注中发生的急性先天免疫应答和组织损伤很重要的过程，但研究不足。此外，我们的新机制，调控脱粒将促进配体刺激的胞吐作用的一般理解，脱粒是一种形式，并发生在许多细胞，包括内皮细胞。 因此，我们的研究可能为最终了解CCM疾病的致病基础铺平道路，并对许多领域（基础细胞生物学，先天免疫和CCM疾病）产生潜在的重大影响。在本研究中，我们将通过检验STK 24和CCM 3复合物是中性粒细胞脱粒的关键负调节剂的假设来关注中性粒细胞脱粒。我们将研究CCM 3和STK 24调节脱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.