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

描述（由申请人提供）：本项目的长期目标 应用是为了了解B细胞抗原受体 (BCR)与IG受体（FcgRIIB）同时抑制B细胞 增殖FcgRIIB功能的丧失可导致自身抗体产生， 可能导致自身免疫性疾病因此，了解分子 增长停滞的基础对两者都有重大影响。 与FcgRIIB失调相关的体液免疫应答和病理学。 D型细胞周期蛋白/细胞周期蛋白依赖性激酶（cdk）4-视网膜母细胞瘤（pRb）通路 是BCR信号的主要靶点，其功能是调节G1至S期 进展我们的研究结果表明，BCR-FcgRIIB共交联发挥其作用。 生长抑制作用，部分是通过信号转导Cdc 37的磷酸化。 Cdc 37沿着hsp 90在导致细胞凋亡的途径中起重要作用。 d型细胞周期蛋白-cdk 4复合体组装。本文提出的研究将测试 Cdc 37磷酸化应答BCR-FcgRIIB假说 共结合阻止了hsp 90/Cdc 37与cdk 4的结合， 成熟B淋巴细胞中细胞周期蛋白D2-cdk 4复合物的组装。这些假设 沿着Cdc 37激酶的鉴定，将在三个 具体目的：1）质谱（MS）鉴定BCR-FcgRIIB诱导型 Cdc 37上的磷酸受体位点，结合体外结合测定， 评估磷酸化对hsp 90/Cdc 37靶向cdk 4的作用; 2） 丙氨酸点突变的GST-Cdc 37的异位表达，不能被 磷酸化，将被用来评估磷酸化的作用， hsp 90/Cdc 37对cdk 4的靶向性及天冬氨酸的异位表达 模拟磷酸化的取代FLAG-Cdc 37将用于迫使 破坏细胞周期蛋白D2-cdk 4复合物并促进生长停滞;和3） GST-Cdc 37亲和纯化和MS鉴定Cdc 37磷酸化 激酶。从这些实验中获得的信息将有助于指导 未来的研究旨在治疗自身免疫性疾病的干预措施。

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.