Abstract 4144: PTPTF negatively regulates c-Src kinase through the dephosphorylation of the activation loop

Kinases and phosphatases play a dynamic role in the regulation of signaling propagation. c-Src is an oncogenic kinase that plays multifunctional roles in the initiation and progression of many cancers, including breast, colorectal and lung cancers. The activity of c-Src kinase is tightly regulated by the phosphorylation status of two key tyrosine residues (Tyr419 and Tyr530 in human c-Src) that controls its propensity for entering an active conformation. Phosphorylation of Tyr530 at the C-terminus of c-Src locks the kinase in an inactive confirmation whereas phosphorylation of Tyr419 in the activation loop is required to activate c-Src. A number of tyrosine phosphatases have been identified to activate c-Src by dephosphorylating Tyr530. However, the phosphatases controlling the Tyr419 site remain elusive. In this study, we investigated the role of protein tyrosine phosphatase receptor type F (PTPRF) in regulating the phosphorylation of c-Src. PTPRF is characterized by an extracellular domain, a single transmembrane motif, and two tandem intracellular phosphatase domains (including a catalytically active D1 domain and a catalytically inactive D2 domain). We show that overexpression of PTPRF, but not a phosphatase inactive mutant, results in a decrease in Tyr419 phosphorylation of both wild-type and Y530F constitutively active c-Src in 293T cells. On the other hand, CRISPR-mediated knockout of PTPRF increases the phosphorylation of c-Src and signaling downstream. Co-immunoprecipitation experiments indicate that PTPRF interacts with c-Src through its D2 pseudo phosphatase domain. Consistent with the notion that the kinase activation reduces c-Src protein stability, we find that the expression of c-Src is increased when co-expressed with PTPRF, suggesting that PTPRF-mediated dephosphorylation stabilizes c-Src protein. Taken together, this study identifies PTPRF as a novel phosphatase that negatively regulates c-Src kinase activity through the dephosphorylation of Tyr419 in the activation loop. Citation Format: Haley Stanczyk, Carolina Galeano-Naranjo, Tianyan Gao. PTPTF negatively regulates c-Src kinase through the dephosphorylation of the activation loop [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4144.