Abstract 2554: Phosphorylationdependent control of protein synthesis in response to inhibitors of mRNA translation and mitochondrial respiration in HeLa cancer cell line

Abstract Control of protein turnover via regulation of the rates of protein synthesis and degradation is one of the principal means by which cell signaling networks can be modulated. This regulation can also be achieved through alterations in post-translational modifications such as phosphorylation. The human genome encodes ~21,300 proteins, and over 95% of these are subjected to reversible phosphorylation at nearly a million sites by at least 568 protein kinases (PK) and about 156 protein phosphatases (PP). The aim of current study was to use differential treatments of cultured cells with diverse agents to uncover connectivities between protein kinases and their substrates to elucidate the architecture of signaling networks. Western blot analyses showed that the protein synthesis inhibitor anisomycin (Alomone labs, Israel) and the mitochondrial respiratory chain inhibitor arsenate (Sigma, Saint Louis, MO) both increased ribosomal proteins S6 (RPS6) S235+S236+S240 phosphorylation. There were similar patterns of activatory phosphorylations observed for upstream kinases of RPS6, including p70S6K at T255 and RSK1 at S380. Anisomycin and arsenate also enhanced activatory phosphorylation of the kinases p38α at T180+Y182 and CK2α at Y255. We explored whether or not the effect of anisomycin or arsenate on these protein kinases were dependent on mTOR signaling with its inhibitor rapamycin (Calbiochem, EMD Chemicals Inc.), which by itself slightly increased the RPS6 S235+S236+S240 phosphorylation. However, rapamycin completely blocked both anisomycin and arsenate induced RPS6 phosphorylation. Rapamycin did not block phosphorylation of p38α MAPK T180 and Y182 in response to anisomycin or arsenate. The results of CK2α Y255 phosphorylation were particularly interesting, because rapamycin treatment alone increased the phosphorylation at this site as did anisomycin alone, but the combination of these inhibitors blocked this phosphorylation. However, CK2α Y255 phosphorylation in response to arsenate alone and in combination with rapamycin showed that the arsenate induction of phosphorylation was not dependent on mTOR signaling. We hypothesized that this mTOR-independent signaling pathway involves the activation of protein-tyrosine kinases that may target both CK2a and p38 MAPK. Therefore, we designed an in vitro experiment and we found that some protein-tyrosine kinases like Blk, Bmx, Btk, EGFR, Fer, Fes, Frk, Fyn, LynA, Pyk2, Src, Syk, Tec, and TrkB can significantly phosphorylate p38 MAPK at Y182 and CK2α at Y255, whereas some other protein-tyrosine kinases such as Fgr, InsR, Jak2, Met, and Txk primarily targeted CK2α at Y255. We also demonstrated that p38 MAPK is able to directly phosphorylate CK2α near its C-terminus at T360 and S362, and this correlated with activation of its casein phosphotransferase activity. Citation Format: Hamidreza Galavi, Steven Pelech. Phosphorylationdependent control of protein synthesis in response to inhibitors of mRNA translation and mitochondrial respiration in HeLa cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2554.