Phosphorylation of cell cycle and apoptosis regulatory protein-1 by stress activated protein kinase P38γ is a novel mechanism of apoptosis signaling by genotoxic chemotherapy

Jaganathan Venkatesh; Magesh Muthu; Indulekha Singaravelu; Vino T Cheriyan; Sreeja C Sekhar; Nuwan C P N Acharige; Edi Levi; Hadeel Assad; Mary Kay H Pflum; Arun K Rishi

doi:10.3389/fonc.2024.1376666

Phosphorylation of cell cycle and apoptosis regulatory protein-1 by stress activated protein kinase P38γ is a novel mechanism of apoptosis signaling by genotoxic chemotherapy

Front Oncol. 2024 May 2:14:1376666. doi: 10.3389/fonc.2024.1376666. eCollection 2024.

Authors

Jaganathan Venkatesh^{1

2

3}, Magesh Muthu^{1

2

3}, Indulekha Singaravelu^{1

2

3}, Vino T Cheriyan^{1

2

3}, Sreeja C Sekhar^{1

2

3}, Nuwan C P N Acharige⁴, Edi Levi^{1

5}, Hadeel Assad^{2

3}, Mary Kay H Pflum⁴, Arun K Rishi^{1

2

3}

Affiliations

¹ John D. Dingell V.A. Medical Center, Wayne State University, Detroit, MI, United States.
² Karmanos Cancer Institute, Wayne State University, Detroit, MI, United States.
³ Department of Oncology, Wayne State University, Detroit, MI, United States.
⁴ Department of Chemistry, Wayne State University, Detroit, MI, United States.
⁵ Department of Pathology, Wayne State University, Detroit, MI, United States.

Abstract

CARP-1, a perinuclear phospho-protein, regulates cell survival and apoptosis signaling induced by genotoxic drugs. However, kinase(s) phosphorylating CARP-1 and down-stream signal transduction events remain unclear. Here we find that CARP-1 Serine (S)⁶²⁶ and Threonine (T)⁶²⁷ substitution to Alanines (AA) inhibits genotoxic drug-induced apoptosis. CARP-1 T⁶²⁷ is followed by a Proline (P), and this TP motif is conserved in vertebrates. Based on these findings, we generated affinity-purified, anti-phospho-CARP-1 T⁶²⁷ rabbit polyclonal antibodies, and utilized them to elucidate chemotherapy-activated, CARP-1-dependent cell growth signaling mechanisms. Our kinase profiling studies revealed that MAPKs/SAPKs phosphorylated CARP-1 T⁶²⁷. We then UV cross-linked protein extracts from Adriamycin-treated HeLa cervical cancer cells with a CARP-1 (614-638) peptide, and conducted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the peptide-bound protein complexes. This experiment revealed SAPK p38γ interaction with CARP-1 (614-638) peptide. Our studies further established that SAPK p38γ, but not other MAPKs, phosphorylates CARP-1 T⁶²⁷ in cancer cells treated with genotoxic drugs. Loss of p38γ abrogates CARP-1 T⁶²⁷ phosphorylation, and results in enhanced survival of breast cancer cells by genotoxic drugs. CARP-1 T⁶²⁷ phosphorylation was also noted in breast tumors from patients treated with radiation or endocrine therapies. We conclude that genotoxic drugs activate p38γ-dependent CARP-1 T⁶²⁷ phosphorylation to inhibit cell growth.

Keywords: CCAR1/CARP-1; P38γ; breast cancer; genotoxic chemotherapy; phosphorylation.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Department of Veterans Affairs Merit Review grant (AR), Department of Veterans Affairs Basic Laboratory Research & Development Research Career Scientist award (AR). We would like to thank the National Institutes of Health (GM079529 and GM131821) and Wayne State University for funding, and the Wayne State University and Karmanos Cancer Center Proteomics Core, which is supported by NIH Grants P30 ES020957, P30 CA022453, and S10 OD010700.