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

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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.