Cdc37 in Fcgamma R-induced Growth Arrest in B Cells

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DESCRIPTION (provided by applicant): The long-term objective of this new application is to understand how engagement of the B-cell antigen receptor (BCR), simultaneously with the Ig receptor (FcgammaRIIB), inhibits B-cell proliferation. Loss of FcgammaRIIB 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 FcgammaRIIB dysregulation. The D-type cyclin/cyclin dependent kinase(cdk) 4-retinoblastoma (pRb) pathway is a primary target for growth factor signals and functions to regulate G1-to-S phase progression. Our results indicate that BCR-FcgammaRIIB co-cross-linking exerts its growth inhibitory effect, in part, by signaling the phosphorylation of Cdc37. Cdc37, along with hsp9O, 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-FcgammaRIIB coengagement: a) prevents targeting of hsp9O/Cdc37 to cdk4; b) blocks assembly of D-type cyclin kinase complexes; and c) promotes growth arrest in mature B lymphocytes. These hypotheses will be tested in three specific aims that include: 1) mass spectrometry to identify BCR-FcgammaRIIB-inducible phosphoacceptor sites on Cdc37; 2) in vitro binding assays and in vivo ectopic expression of alanine point-mutated GSTCdc37, that cannot be phosphorylated by negative signals, will be used to evaluate the role of phosphorylation on targeting of hsp9O/Cdc37 to cdk4 and in D-type cyclin kinase assembly; and 3) ectopic expression of aspartic acid substituted FLAG-Cdc37 will be used to mimic phosphorylation and force the disruption of D-type cyclin kinase complexes and promote growth arrest. The information obtained from these studies will serve to direct future studies aimed at therapeutic interventions in autoimmune disease.