Abstract 3461: PID1, a novel brain tumor growth suppressor, is a target for phosphorylation by a newly identified binding partner
Authors: Anup S. Pathania, Xiuhai Ren, Min Mahdi, Eslam Nouri Nigjeh, Yang Fu, Ebrahim Zandi, Xiaojiang Chen, Gregory M. Shackleford, Anat Erdreich-Epstein
Journal: Cancer Research (2018)

Abstract Background: Our group were first to establish a link between PID1 (Phosphotyrosine Interaction Domain containing 1) and cancer, showing PID1's tumor inhibitory effect in medulloblastoma, glioblastoma and atypical teratoid rhabdoid tumor (ATRT) brain tumor cell lines and correlation of higher PID1 mRNA with longer patient survival. PID1 was first identified in 2006 and is associated with obesity-related insulin resistance and mitochondrial dysfunction. Here we present a novel interacting partner of PID1 which phosphorylates and modulates its growth suppressive functions. Methods: In silico predictions, in vitro kinase assay, phosphoproteomic analysis, co-immunoprecipitation, SDS-PAGE, western blotting, flow cytometry. Cells: CHLA-07-BSGBM pediatric glioblastoma, U87 glioblastoma and D283 medulloblastoma cell lines, HEK293FT, primary pooled mouse embryo fibroblasts (MEFs). Results: Using prediction algorithms we found that PID1 was a likely phosphorylation target of a member of the G protein-coupled receptor kinase (GRK) family. In vitro kinase assays coupled with phosphoproteomic analysis confirmed this phosphorylation. Overexpressed HA-PID1 and this GRK could be reciprocally co-immunoprecipitated in 293FT cells and endogenous GRK was co-IPd by endogenous PID1 in CHLA-07-BSGBM. A kinase-dead GRK mutant co-IP'd with PID1 similar to the wildtype GRK indicating that the GRK-PID1 interaction did not require the kinase function of this GRK. Deletion mutants of PID1 differentially bound the GRK in co-IPs in a manner that corresponded to the differential growth inhibition these PID1 deletion mutants exerted in glioblastoma and medulloblastoma cell lines. Functionally, siRNA silencing of this GRK caused apoptosis (annexin-PI positive cells and PARP-1 cleavage) in Pid1wt MEFs suggesting that this GRK mediates survival in MEFs. Apoptosis by the GRK siRNA was mitigated in Pid1ko compared to Pid1wt MEFs, suggesting that PID1 functions downstream of the GRK and negatively regulates this GRK's pro-survival signaling. Importantly, we found similar results in glioblastoma cells using gain/loss of function of the two proteins. Conclusions: We found that PID1 is phosphorylated by a member of the GRK family and interacts with it, affecting this GRK's pro-survival activity. Experiments are now uncovering the molecular ordering of PID1 and this GRK in mediating the growth-suppressive effect of PID1 in brain tumors. Citation Format: Anup S. Pathania, Xiuhai Ren, Min Mahdi, Eslam Nouri Nigjeh, Yang Fu, Ebrahim Zandi, Xiaojiang Chen, Gregory M. Shackleford, Anat Erdreich-Epstein. PID1, a novel brain tumor growth suppressor, is a target for phosphorylation by a newly identified binding partner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3461.