Quantitative phosphoproteomic analysis reveals system-wide signaling pathways regulated by site-specific phosphorylation of Keratin-8 in skin squamous cell carcinoma derived cell line

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 Serine⁷³ (head-domain) and Serine⁴³¹ (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^T14,Y15, EIF4EBP1^T46,T50, EIF4B^S422, AKT1S1^T246,S247, CTTN1^{T401,S405,Y421}, and CAP1^S307/309 in K8-S73A/D mutant and CTTN1^{T401,S405,Y421}, BUB1B^S1043, and CARHSP1^S30,S32 in K8-S431A/D mutants as well as some anonymous phosphosites including MYC^S176, ZYX^S344, and PNN^S692 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.