Regulation of Keratin 8 Phosphorylation during cancer cell division

Keratins, distributed within epithelial cells, play a crucial role in providing tissue strength and are utilized as diagnostic markers in tumor pathology due to their varied expression patterns. Despite their robust structure, their regulation during dynamic processes such as cell division, particularly cytokinesis, remains poorly understood. Phosphorylation has been identified as a mechanism to regulate Keratin solubility and phenotype. Our study focuses on Keratin 8, aiming to elucidate its plasticity during cancer cell division through an investigation of its phosphorylation profile.

Methodologically, cancer cell lines including MCF7, HeLa.S3, MDA-MB-231, and CAKI-2 are arrested at different cell cycle stages (interphase, mitosis, and cytokinesis). Phosphorylation profiles are examined using stable isotope dimethyl labeling-based quantitative phosphoproteomics, with samples from various cell cycle stages combined for each cell line. To simplify analysis, labeled peptides are fractionated via Strong Cation Exchange Chromatography and low-abundance phospho-peptides are enriched using TiO2 beads. LC-MS/MS analysis is then performed on the enriched phospho-peptides, with raw data processed using PD 2.3, MaxQuant and Perseus software.

To comprehend Keratin 8 phosphorylation dynamics during cancer cell division, triplex dimethyl labeling is conducted, and cell cycle stage-specific phospho-sites are analyzed for each cell line. Inter-cell type comparisons reveal commonly regulated phospho-sites in the head domain of Keratin 8 across all cell lines. Intra-cellular phosphorylation analysis indicates that MCF7 cells exhibit the highest number of phospho-sites, correlating with elevated Keratin 8 expression levels. The number of phospho-sites decreases as Keratin 8 expression levels decline from epithelial to epithelial-mesenchymal cells. Our findings uncover upregulated phospho-sites of Keratin 8 at specific cell cycle stages, including both commonly regulated and cell-type unique phospho-sites. These findings present potential marker candidates for cell division and tumor diagnosis.