MAPKAP Kinase-2 phosphorylation of PABPC1 controls its interaction with 14-3-3 proteins after DNA damage: A combined kinase and protein array approach

Justine R Stehn; Scott R Floyd; Erik W Wilker; H Christian Reinhardt; Scott M Clarke; Qiuying Huang; Roberto D Polakiewicz; Nahum Sonenberg; Yi Wen Kong; Michael B Yaffe

doi:10.3389/fmolb.2023.1148933

MAPKAP Kinase-2 phosphorylation of PABPC1 controls its interaction with 14-3-3 proteins after DNA damage: A combined kinase and protein array approach

Front Mol Biosci. 2023 Apr 6:10:1148933. doi: 10.3389/fmolb.2023.1148933. eCollection 2023.

Authors

Justine R Stehn¹, Scott R Floyd¹, Erik W Wilker¹, H Christian Reinhardt¹, Scott M Clarke¹, Qiuying Huang¹, Roberto D Polakiewicz², Nahum Sonenberg³, Yi Wen Kong^{1

4}, Michael B Yaffe^{1

4

5

6

7

8}

Affiliations

¹ David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, United States.
² Cell Signaling Technology, Danvers, MA, United States.
³ Rosalind and Morris Goodman Cancer Centre, Department of Biochemistry, McGill University, Montreal, QC, Canada.
⁴ Center for Precision Cancer Medicine, Massachusetts Institute of Technology, Cambridge, MA, United States.
⁵ Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, United States.
⁶ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, United States.
⁷ Divisions of Surgical Oncology, Trauma, and Surgical Critical Care, Beth Israel Deaconess Medical Center, Department of Surgery, Harvard Medical School, Boston, MA, United States.
⁸ Surgical Oncology Program, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States.

Abstract

14-3-3 proteins play critical roles in controlling multiple aspects of the cellular response to stress and DNA damage including regulation of metabolism, cell cycle progression, cell migration, and apoptotic cell death by binding to protein substrates of basophilic protein kinases following their phosphorylation on specific serine/threonine residues. Although over 200 mammalian proteins that bind to 14-3-3 have been identified, largely through proteomic studies, in many cases the relevant protein kinase responsible for conferring 14-3-3-binding to these proteins is not known. To facilitate the identification of kinase-specific 14-3-3 clients, we developed a biochemical approach using high-density protein filter arrays and identified the translational regulatory molecule PABPC1 as a substrate for Chk1 and MAPKAP Kinase-2 (MK2) in vitro, and for MK2 in vivo, whose phosphorylation results in 14-3-3-binding. We identify Ser-470 on PABPC1 within the linker region connecting the RRM domains to the PABC domain as the critical 14-3-3-binding site, and demonstrate that loss of PABPC1 binding to 14-3-3 results in increased cell proliferation and decreased cell death in response to UV-induced DNA damage.

Keywords: 14-3-3; DNA damage; MAPKAP Kinase-2; polyA-binding protein; signal transduction.

Grants and funding

This work was supported by grants from the National Institutes of Health (R01-ES015339, R35-ES028374, R01-CA226898, and R01-GM104047 to MY), the Ovarian Cancer Research Foundation (MY), the Charles and Marjorie Holloway Foundation (MY), the STARR Cancer Consortium (MY), the Misrock Foundation (YK), the MIT Center for Precision Cancer Medicine (MY, YK), a Burroughs-Wellcome Career Award for Medical Scientists (SF) and a joint Cancer Research United Kingdom and Brain Tumour Charity funded Brain Tumour Award C42454/A28596 (MY, YK). Support was provided in part by the Koch Institute Support Grant (P30-CA14051) from the National Cancer Institute, and the MIT MRSEC Shared Experimental Facilities Grant (DMR-0819762) from the National Science Foundation.