1. PKC-mediated phosphorylation governs the stability and function of CELF1 as a driver of EMT in breast epithelial cells.
- Author
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Massey S, Kongchan N, Gao Y, Chaudhury A, Olokpa E, Karch J, Malovannaya A, Cheng C, Zhang X, and Neilson JR
- Subjects
- Humans, Phosphorylation, Female, Animals, Mice, Epithelial Cells metabolism, Epithelial Cells pathology, Protein Stability, Cell Line, Tumor, Protein Kinase C metabolism, Protein Kinase C genetics, Ubiquitination, Epithelial-Mesenchymal Transition, CELF1 Protein metabolism, CELF1 Protein genetics, Breast Neoplasms metabolism, Breast Neoplasms pathology, Breast Neoplasms genetics
- 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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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