Abstract 1550 Studying the interaction of PYK2 with Cas scaffold proteins to understand tumor infiltration by macrophages

Evidence suggests that the anti-tumor activity of cytotoxic T-cells is inhibited by tumor-associated macrophages (TAMs) when these macrophages infiltrate tumors and adopt an immunosuppressive state. TAM infiltration of tumors is suppressed when PYK2 (proline-rich tyrosine kinase 2) activity is inhibited; thus, we hypothesize that PYK2 is a key player in TAM infiltration of tumors. We seek to better understand the role of PYK2 within TAMs by expanding our knowledge of its interactions with the Cas scaffold proteins BCAR1 (breast cancer anti-estrogen resistance protein 1) and BCAR1's homologous protein CASS4 (Cas scaffold protein family member 4). We studied these interactions by conducting co-transfection/co-immunoprecipitation experiments using tagged versions of PYK2, Focal Adhesion Kinase (FAK, a homolog of PYK2), BCAR1, and CASS4 and discovered that all four kinase-scaffold pairs co-purify. Further studies were used to investigate the previously unidentified interaction between PYK2 and CASS4. We found that simulation of tyrosine kinase activation using orthovanadate, a tyrosine phosphatase inhibitor, increased the interaction between PYK2 and CASS4. We then performed co-transfection/co-immunoprecipitation studies using truncated versions of PYK2 and CASS4 paired with full length PYK2 or CASS4 to identify the domains involved in their interaction. These experiments revealed that additional domains besides the SH3 domain of CASS4 and the proline-rich region of PYK2 were sufficient to maintain the interaction. In summary, we identified a novel interaction between PYK2 and CASS4 that is enhanced by tyrosine phosphorylation and involves multiple domains of each protein. Across myeloid cell lines, mRNA expression levels for PYK2 and CASS4 are notably higher than those of FAK and BCAR1, suggesting that the PYK2 / CASS4 interaction may be critical for tumor infiltration by macrophages.