Mast cell regulation by PKC and Akt

描述(由申请人提供)：肥大细胞在依赖IgE的过敏反应和宿主对某些寄生虫的防御中发挥关键作用。高亲和力的IgE受体(FcepsilonRI)与IgE和多价变应原的交联会引起肥大细胞的激活，最终导致一系列促炎介质的释放。PKC在肥大细胞激活中起关键作用。在FcepsilonRI交联激活的各种PKC亚型中，PKCaI活性受蛋白酪氨酸激酶(PTKs)、Lyn和Syk以BTK依赖的方式特异性调节。我们已经证明，在FcnRI刺激下，PKCbetaI和PKCalpha的C端酪氨酸残基Tyr-662和Tyr-658分别被质膜上的Syk磷酸化。这种磷酸化为GRB-2的Src同源2(SH2)结构域创建了结合位点，GRB-2是一种适配器蛋白。GRB-2/SOS复合体在RAS附近的募集有助于RAS/ERK通路的激活。FcepsilonRI交联后，AKT也被激活并参与细胞因子的产生。我们的初步数据表明，Akt的活性受肥大细胞中传统的PKC亚型(阿尔法、贝塔和贝塔)调节。根据这些数据，我们假设Syk对几种PKC亚型(a、Betal、Zeta和lambda/I)的C末端酪氨酸磷酸化招募GRB-2/SOS复合体来激活RAS(假设1)，而传统的PKC亚型磷酸化关键残基Ser-473以激活Akt(假设2)。我们也有初步的数据表明PKCA的C末端疏水基序在底物识别中的作用(假设3)。为了深入研究PKC亚型和Akt在肥大细胞激活中的作用，我们将在体外和体内实验中对这些假说进行评估。提出的研究将为我们对肥大细胞信号转导的理解带来新的见解。鉴于PKC在脱颗粒和其他激活事件中的关键重要性，这些研究也可能为针对过敏性疾病的新的治疗方式提供机会。

DESCRIPTION (provided by applicant): Mast cells play a critical role in IgE-dependent allergic hypersensitivity and the host defense against certain parasites. Cross-linking of the high-affinity IgE receptor (FcepsilonRI) with IgE and multivalent allergen elicits mast cell activation, culminating in the release of a panel of proinflammatory mediators. PKC plays critical roles in mast cell activation. Among various PKC isoforms that are activated by FcepsilonRI cross-linking, PKCaI activity was shown to be specifically regulated by protein-tyrosine kinases (PTKs), Lyn and Syk, in a Btk-dependent manner. We have demonstrated that C-terminal tyrosine residues, Tyr-662 and Tyr-658 of PKCbetaI and PKCalpha, respectively, are phosphorylated by Syk at the plasma membrane upon FcnRI stimulation. This phosphorylation creates the binding site for the Src homology 2 (SH2) domain of Grb-2, an adaptor protein. Recruitment of Grb-2/Sos complexes to the vicinity of Ras contributes to activation of the Ras/ERK pathway. Akt was also shown to be activated and involved in cytokine production upon FcepsilonRI cross-linking. Our preliminary data suggest that Akt activity is regulated by conventional PKC isoforms (alpha, betal and betall) in mast cells. Based on these data, we hypothesize that C-terminal tyrosine phosphorylation of several PKC isoforms (a, betal, zeta, and lambda/I) by Syk recruits Grb-2/Sos complexes to activate Ras (Hypothesis 1) and that the conventional PKC isoforms phosphorylate the critical residue Ser-473 for Akt activation (Hypothesis 2). We also have preliminary data suggesting the role of C-terminal hydrophobic motif of PKCa in substrate recognition (Hypothesis 3). To characterize in depth the roles of PKC isoforms and Akt in mast cell activation, we will evaluate these hypotheses in in vitro and in vivo experiments. The proposed studies will bring novel insight into our understanding of mast cell signal transduction. Given the critical importance of PKC in degranulation and other activation events, these studies are also likely to provide an opportunity for novel therapeutic modalities aimed at allergic diseases.