Examining the Potential Modification of the Protein Tyrosine Kinase Pyk2 by SUMO-1

Abstract

Proline-rich tyrosine kinase 2 (Pyk2) is a non-receptor protein tyrosine kinase that is highly expressed in hematopoietic cells. Our lab generated two polyclonal antibodies to investigate the regulation of Pyk2 in macrophages and T cells. In macrophages the N-terminal antibody immunoprecipitated a higher molecular weight form of Pyk2. This shift was not due to differential phosphorylation or isoform expression. Since FAK, a close relative to Pyk2 undergoes a molecular weight shift due to SUMOylation, my thesis project was to investigate the potential SUMOylation of Pyk2. This study demonstrates that endogenous and exogenous Pyk2 associates with SUMO-1. The E3 ligase PIAS1 was shown to promote the association of Pyk2 with SUMO-1. Lysines 35, 145, and 646 were not the sites of Pyk2 SUMOylation, although SUMO-1 does associate with Pyk2 in the FERM domain. Direct Pyk2 SUMOylation was not confirmed, although SUMO-1 and PIAS1 overexpression increases Pyk2 protein levels.