Combined integrin phosphoproteomic analyses and small interfering RNA--based functional screening identify key regulators for cancer cell adhesion and migration.
Integrins interact with extracellular matrix (ECM) and deliver intracellular signaling for cell proliferation, survival, and motility. During tumor metastasis, integrin-mediated cell adhesion to and migration on the ECM proteins are required for cancer cell survival and adaptation to the new microenvironment. Using stable isotope labeling by amino acids in cell culture-mass spectrometry, we profiled the phosphoproteomic changes induced by the interactions of cell integrins with type I collagen, the most common ECM substratum. Integrin-ECM interactions modulate phosphorylation of 517 serine, threonine, or tyrosine residues in 513 peptides, corresponding to 357 proteins. Among these proteins, 33 key signaling mediators with kinase or phosphatase activity were subjected to small interfering RNA-based functional screening. Three integrin-regulated kinases, DBF4, PAK2, and GRK6, were identified for their critical role in cell adhesion and migration possibly through their regulation of actin cytoskeleton arrangement. Altogether, we not only depict an integrin-modulated phosphorylation network during cell-ECM protein interactions but also reveal novel regulators for cell adhesion and migration.
Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.
Yanling
Y
Chen
Bingwen
B
Lu
Qingkai
Q
Yang
Colleen
C
Fearns
John R
JR
Yates
Jiing-Dwan
JD
Lee
D000199:Actins / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology; D018797:Cell Cycle Proteins / Q000378:metabolism; D002465:Cell Movement / Q000502:physiology; D024042:Collagen Type I; D003599:Cytoskeleton / Q000378:metabolism / Q000473:pathology; D054768:G-Protein-Coupled Receptor Kinases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D040901:Proteomics / Q000379:methods*; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection; D054462:p21-Activated Kinases / Q000378:metabolism
D000199:Actins; D018797:Cell Cycle Proteins; D024042:Collagen Type I; C485678:DBF4 protein, human; D016023:Integrins; D034741:RNA, Small Interfering; C515907:PAK2 protein, human; D054462:p21-Activated Kinases; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6