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 CDK1T14,Y15, EIF4EBP1T46,T50, EIF4BS422, AKT1S1T246,S247, CTTN1T401,S405,Y421, and CAP1S307/309in K8-S73A/D mutant and CTTN1T401,S405,Y421, BUB1BS1043, and CARHSP1S30,S32in K8-S431A/D mutants as well as some anonymous phosphosites including MYCS176, ZYXS344, and PNNS692could 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.
Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai, India.|Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai, India.
Richa
R
Tiwari
Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai, India.|Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai, India.|Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
Indrajit
I
Sahu
Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai, India.|Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai, India.|Medical College of Wisconsin, Milwaukee, WI, USA.
Bihari Lal
BL
Soni
Institute of Bioinformatics, Bangalore, India.
Gajanan J
GJ
Sathe
Institute of Bioinformatics, Bangalore, India.
Keshava K
KK
Datta
Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai, India.|Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai, India.
Pankaj
P
Thapa
Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai, India.
Shruti
S
Sinha
Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai, India.
Chella Krishna
CK
Vadivel
Institute of Bioinformatics, Bangalore, India.
Bharti
B
Dhaka
Institute of Bioinformatics, Bangalore, India.
Harsha
H
Gowda
Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai, India.|Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai, India.
Milind M
MM
Vaidya
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D016203:CDC2 Protein Kinase; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D051356:Cortactin / Q000235:genetics / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D039642:Eukaryotic Initiation Factors / Q000235:genetics / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D053533:Keratin-8 / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D012867:Skin / Q000378:metabolism / Q000473:pathology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
C484523:AKT1S1 protein, human; D048868:Adaptor Proteins, Signal Transducing; C584279:BUB1B protein, human; C489549:CAP1 protein, human; C114342:CARHSP1 protein, human; C495868:CTTN protein, human; D018797:Cell Cycle Proteins; D051356:Cortactin; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; C087000:EIF4EBP1 protein, human; D039642:Eukaryotic Initiation Factors; D053533:Keratin-8; D010750:Phosphoproteins; D014157:Transcription Factors; C031799:eIF-4B; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; C528899:CDK1 protein, human; D018844:Cyclin-Dependent Kinases
18 Phosphorylation; IPA; Keratin 8; Quantitative phosphoproteomics; Tumorigenic potential