Role of Cdc37 in FcyR-induced Growth Arrest in B Cells

描述(由申请人提供)：这项计划的长期目标 其应用是了解B细胞抗原受体如何参与 (Bcr)与Ig受体(FcgRIIB)同时抑制B细胞 扩散。FcgRIIB功能丧失可导致自身抗体产生和 可能导致自身免疫性疾病。因此，理解分子 增长停滞的基础对这两个国家都有重大影响 与FcgRIIB失调相关的体液免疫反应和病理学。 D型细胞周期蛋白/细胞周期蛋白依赖性激酶4-视网膜母细胞瘤(PRB)通路 是bcr信号的主要靶点，具有调节G1-S期的功能 进步。我们的结果表明，BCR-FcgRIIB共交联能发挥其 生长抑制作用，部分是通过信号传递CDC37的磷酸化来实现的。 Cdc37和hsp90一起，在导致 D型细胞周期蛋白-CDK4复合体组装。这里提出的研究将检验 BCR-FcgRIIB对CDC37磷酸化反应的假说 共结合阻止HSP90/CDC37与CDK4结合，进而破坏 成熟B淋巴细胞中细胞周期蛋白D2-CDK4复合体的组装这些假设 将在三年内进行检测，并鉴定出cdc37激酶。 具体目标：1)质谱学鉴定bcr-FcgRIIB诱导 结合体外结合试验，在CDC37上的磷受体位点 评价磷酸化在HSP90/CDC37靶向CDK4中的作用； 丙氨酸点突变的GST-CDC37的异位表达 磷酸化，将被用来评估磷酸化在 靶向CDK4的HSP90/CDC37与天冬氨酸的异位表达 将使用模拟磷酸化的替代FLAG-CDC37来强制 破坏细胞周期蛋白D2-CDK4复合体，促进生长停滞； GST-CDC37亲和纯化及质谱法鉴定CDC37磷酸化 激活剂。从这些实验中获得的信息将有助于指导 未来的研究旨在对自身免疫性疾病进行治疗干预。

DESCRIPTION (provided by the applicant): The long-term objective of this application is to understand how engagement of the B cell antigen receptor (BCR), simultaneously with the Ig receptor (FcgRIIB), inhibits B cell proliferation. Loss of FcgRIIB function can lead to autoantibody production and may contribute to autoimmune disease. Therefore, understanding the molecular basis that underlies growth arrest has significant implications for both humoral immune responses and pathology associated with FcgRIIB dysregulation. The D-type cyclin/cyclin-dependent kinase(cdk) 4-retinoblastoma (pRb) pathway is a primary target for BCR signals and functions to regulate G1-to-S phase progression. Our results indicate that BCR-FcgRIIB co-cross-linking exerts its growth inhibitory effect, in part, by signaling the phosphorylation of Cdc37. Cdc37, along with hsp90, plays an essential role in the pathway leading to D-type cyclin-cdk4 complex assembly. The research proposed herein will test the hypotheses that phosphorylation of Cdc37 in response to BCR-FcgRIIB coengagement prevents hsp90/Cdc37 from binding to cdk4 and in turn, disrupts assembly of cyclin D2-cdk4 complexes in mature B lymphocytes. These hypotheses will be tested, along with identification of the Cdc37 kinase, in three specific aims: 1) mass spectrometry (MS) to identify BCR-FcgRIIB-inducible phosphoacceptor sites on Cdc37, in conjunction with in vitro binding assays, to evaluate the role of phosphorylation on targeting of hsp90/Cdc37 to cdk4; 2) ectopic expression of alanine point-mutated GST-Cdc37, that cannot be phosphorylated, will be used to evaluate the role of phosphorylation on targeting of hsp90/Cdc37 to cdk4 and ectopic expression of aspartic acid substituted FLAG-Cdc37 that mimics phosphorylation will be used to force disruption of cyclin D2-cdk4 complexes and promote growth arrest; and 3) GST-Cdc37 affinity purification and MS to identify the Cdc37 phosphorylating kinase. The information obtained from these experiments will serve to direct future studies aimed at therapeutic interventions in autoimmune disease.