Mapping the integrin-linked kinase interactome using SILAC.
Protein-protein interactions play an essential role in the regulation of vital biological functions. Through a network of interactions, integrin-linked kinase (ILK) functions downstream of integrin receptors to control cell spreading, migration, growth, survival, and cell cycle progression. Despite many reports on the role of ILK in the regulation of multiple signaling pathways, it is still not understood how ILK integrates and controls complex cellular signals. A more global analysis of ILK-protein complexes will give important insights in the complexity of ILK-dependent signal transduction. Here, we applied a SILAC (stable isotope labeling with amino acids in cell culture)-based proteomics approach to discover novel ILK-interacting proteins. Of 752 proteins identified in ILK immunoprecipitates, 24 proteins had SILAC ratios higher than PINCH, previously identified as direct ILK-binding partner. Some of the newly identified proteins specifically enriched in ILK immunoprecipitates, with potentially interesting roles in ILK biology, include rapamycin-insensitive companion of mTOR (Rictor), alpha- and beta-tubulin, RuvB-like 1 and 2, HS1-associating protein 1 (HAX-1), T-complex protein 1 subunits, and Ras-GTP-ase activating-like protein 1 (IQ-GAP1). Functional interactions between ILK and several of the new binding partners were confirmed by coimmunoprecipitation/Western blot and colocalization experiments. Detailed analysis showed that when ILK is found in a complex with alpha-tubulin and RuvB-like 1, alpha-parvin and PINCH are not present, suggesting that ILK has the ability to form distinct protein complexes throughout the cell. Inhibition of ILK activity with an ILK-kinase inhibitor QLT0267 or downregulation of its expression impaired the ability of RuvB-like 1 to bind to tubulin pointing toward a possible role of ILK in the regulation of RuvB-like 1/tubulin interaction. Using the power of quantitative proteomics to resolve specific from nonspecific protein interactions, we identified several novel ILK-binding proteins, which sheds light on the molecular mechanisms of regulation of ILK-dependent signal transduction.
Dobreva
Iveta
I
Department of Cancer Genetics, British Columbia Cancer Research Centre, 675 West 10th Avenue, V5Z 1L3, Vancouver, BC, Canada.
Fielding
Andrew
A
Foster
Leonard J
LJ
Dedhar
Shoukat
S
D000074183:ATPases Associated with Diverse Cellular Activities; D000185:Actinin / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism; D004265:DNA Helicases / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D047468:Immunoprecipitation; D007553:Isotope Labeling / Q000379:methods*; D060588:LIM Domain Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D025941:Protein Interaction Mapping / Q000379:methods*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D053719:Tandem Mass Spectrometry; D014162:Transfection; D014404:Tubulin / Q000032:analysis / Q000378:metabolism; D000099200:Scaffold Protein ILK; D000096985:Calponins
D000074183:ATPases Associated with Diverse Cellular Activities; D000185:Actinin; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004265:DNA Helicases; D004268:DNA-Binding Proteins; D060588:LIM Domain Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009842:Oligopeptides; D010455:Peptides; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011506:Proteins; D034741:RNA, Small Interfering; D014404:Tubulin; D000099200:Scaffold Protein ILK; C088088:LIMS1 protein, human; C486590:PARVA protein, human; D000096985:Calponins; C085706:FLAG peptide; C486549:RUVBL1 protein, human