Keratin 8/18, a simple epithelia specific keratin pair, is often aberrantly expressed in squamous cell carcinomas (SCC) where its expression is correlated with increased invasion and poor prognosis. Majority of Keratin 8 (K8) functions are governed by its phosphorylation at Serine (head-domain) and Serine (tail-domain) residues. Although, deregulation of K8 phosphorylation is associated with progression of different carcinomas, its role in skin-SCC and the underlying mechanism is obscure. In this direction, we performed tandem mass tag-based quantitative phosphoproteomics by expressing K8 wild type, phosphodead, and phosphomimetic mutants in K8-deficient A431 cells. Further analysis of our phosphoproteomics data showed a significant proportion of total phosphoproteome associated with migratory, proliferative, and invasive potential of these cells to be differentially phosphorylated. Differential phosphorylation of CDK1 , EIF4EBP1 , EIF4B , AKT1S1 , CTTN1 , and CAP1 in K8-S73A/D mutant and CTTN1 , BUB1B , and CARHSP1 in K8-S431A/D mutants as well as some anonymous phosphosites including MYC , ZYX , and PNN could be potential candidates associated with K8 phosphorylation mediated tumorigenicity. Biochemical validation followed by phenotypic analysis further confirmed our quantitative phosphoproteomics data. In conclusion, our study provides the first global picture of K8 site-specific phosphorylation function in neoplastic progression of A431 cells and suggests various potential starting points for further mechanistic studies.

Proteome Xchange Consortium via PRIDE database (dataset identifier PXD007206).

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DOI: https://doi.org/10.1002/pmic.201600254 Phosphorylation is a major regulator of Keratin 8 canonical functions but its role in skin squamous cell carcinoma and the underlying mechanism is still uncertain. Our mutational studies followed by tandem mass tag based global quantitative phosphoproteome profiling provides many new start points to delineate mechanisms governing K8 phosphorylation mediated oncogenic potential of A431 cells. The biochemical and phenotypic validations further strengthen these findings. Altogether this study suggests a much broader regulatory role of K8 phosphorylation in cancer. For more details, see the research article by Richa Tiwari et al., article number 1600254.