GIT1 is a scaffold for ERK1/2 activation in focal adhesions.
GIT1 (G protein-coupled receptor kinase-interacting protein 1) has been shown to regulate focal adhesion disassembly. We previously reported that GIT1 associates with MEK1 and acts as a scaffold to enhance ERK1/2 activation. Here, we show that GIT1 co-localizes with ERK1/2 in focal adhesions and regulates cell migration in vascular smooth muscle cells, HEK293 cells, and HeLa cells. Immunofluorescence showed that GIT1 co-localized with phospho-ERK1/2 in focal adhesions after epidermal growth factor stimulation. Because Src is required for both GIT1 tyrosine phosphorylation and focal adhesion disassembly, we studied the effects of Src on GIT1-ERK1/2 interactions. PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) inhibited association of GIT1 with ERK1/2, and their co-localization in focal adhesions was dramatically decreased in SYF-/- cells. GIT1 small interfering RNA significantly inhibited ERK1/2 recruitment to and activation in focal adhesions. GIT1 small interfering RNA and mutated GIT1 lacking the MEK1 binding domain significantly decreased epidermal growth factor-stimulated cell spreading and migration, suggesting that GIT1-mediated events such as ERK1/2 activation are required for spreading and migration. In summary, the present study further supports a key role for GIT1 (a MEK1-binding protein) as a scaffold for signal transduction in focal adhesions.
Yin
Guoyong
G
Center for Cardiovascular Research and Department of Medicine, University of Rochester, Rochester, New York 14642, USA.
Zheng
Qinlei
Q
Yan
Chen
C
Berk
Bradford C
BC
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D002465:Cell Movement; D002633:Chemotaxis; D003599:Cytoskeleton / Q000378:metabolism; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism; D022001:Focal Adhesions / Q000378:metabolism; D020690:GTPase-Activating Proteins; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D051379:Mice; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D009154:Mutation; D010750:Phosphoproteins / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; C485647:GIT1 protein, human; D020690:GTPase-Activating Proteins; C120766:Git1 protein, mouse; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D014443:Tyrosine; D004815:Epidermal Growth Factor; D019061:src-Family Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3