PKC-Mediated Phosphorylation Governs the Stability and Function of CELF1 as a Driver of EMT in Breast Epithelial Cells

Authors

Shebna Massey
Natee Kongchan
Yang Gao
Arindam Chaudhury
Emuejevoke Olokpa
Jason Karch
Anna Malovannaya
Chonghui Cheng
Xiang Zhang
Joel R Neilson

Publication Date

11-1-2024

Journal

Journal of Biological Chemistry

DOI

10.1016/j.jbc.2024.107826

PMID

39343007

PMCID

PMC11585768

PubMedCentral® Posted Date

9-27-2024

PubMedCentral® Full Text Version

Post-print

Published Open-Access

yes

Keywords

Humans, Epithelial-Mesenchymal Transition, Phosphorylation, Female, CELF1 Protein, Animals, Breast Neoplasms, Mice, Epithelial Cells, Protein Stability, Cell Line, Tumor, Protein Kinase C, Ubiquitination, epithelial–mesenchymal transition (EMT), metastasis, RNA binding protein, CELF1, protein phosphorylation, post-translational regulation, PKCα, PKCε

Abstract

Epithelial to mesenchymal transition (EMT) is believed to be a principal factor contributing to cancer metastasis. The post-transcriptional and post-translational mechanisms underlying EMT are comparatively underexplored. We previously demonstrated that the CELF1 RNA binding protein is necessary and sufficient to drive the EMT of breast epithelial cells, and that the relative protein expression of CELF1 in this context was dictated at the post-translational level. Here, we elucidate the mechanism of this regulation. Mass spectrometric analysis of CELF1 isolated from mesenchymal MCF-10A cells identified multiple sites of serine and threonine phosphorylation on the protein, correlating with the increased stability of this protein in this cellular state. Analysis of phosphomimetic and serine/threonine-to-alanine phosphomutant variants of CELF1 revealed that these phosphorylation sites indeed dictate CELF1 stability, ubiquitination state, and function in vitro. Via co-immunoprecipitation and in vitro kinase assays, we identified the protein kinase C alpha and epsilon isozymes as the kinases responsible for CELF1 phosphorylation in a breast cell line. Genetic epistasis experiments confirmed that these PKCs function upstream of CELF1 in this EMT program, and CELF1 phosphorylation impacts tumor metastasis in a xenograft model. This work is the first to formally establish the mechanisms underlying post-translational control of CELF1 expression and function during EMT of breast epithelial cells. Given the broad dysregulation of CELF1 expression in human breast cancer, our results may ultimately provide knowledge that may be leveraged for novel therapeutic interventions in this context.

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