title: Phosphorylation of G protein-coupled receptor kinase 2-interacting protein 1 tyrosine 392 is required for phospholipase C-gamma activation and podosome formation in vascular smooth muscle cells.
abstract: OBJECTIVE
  Podosomes, which are actin-rich structures, contribute to cell motility, matrix remodeling, and tissue remodeling. We have shown that G protein-coupled receptor kinase 2-interacting protein 1 (GIT1) colocalizes with podosomes and is important in podosome formation in endothelial cells. Src stimulates GIT1 tyrosine phosphorylation, which is critical for phospholipase C-γ (PLCγ) activation. In this study, we identified specific GIT1 tyrosines required for PLCγ activation and podosome formation in vascular smooth muscle cells (VSMC).
  
  
  RESULTS
  We used phorbol 12,13-dibutyrate (PDBU) to induce podosomes in A7r5 VSMC. GIT1 colocalized with podosomes and GIT1 knockdown using short interfering RNA significantly reduced podosome formation. PDBU stimulated GIT1 tyrosine phosphorylation. GIT1 tyrosine phosphorylation was dramatically decreased in SYF-/- cells, and it was also reduced by pretreatment with the protein kinase C (PKC) and Src inhibitors, suggesting that GIT1 phosphorylation was dependent on PKC and Src. By mutation analysis of multiple tyrosines, we found that PDBU specifically increased GIT1-Y392 phosphorylation. Overexpression of GIT1 (Y392F) but not of GIT1 (Y321F) decreased PDBU-mediated PLCγ activation and podosome formation without effect on extracellular signal-regulated kinase 1/2 activation. Additionally, we provide evidence that GIT1 knockout VSMC have markedly fewer podosomes on PDBU treatment compared with wild-type VSMC. These data show that GIT1 is a key regulator of podosome formation in VSMC.
  
  
  CONCLUSIONS
  In conclusion, our data suggest that GIT1-Y392 phosphorylation is critical for PDBU-induced podosome formation by regulating PLCγ activation. We propose that specific signaling modules are assembled in a GIT1 phosphotyrosine-dependent manner as exemplified by PLCγ activation versus extracellular signal-regulated kinase 1/2 activation.
authors.affiliation: Aab Cardiovascular Research Institute and Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA. bradford_berk@urmc.rochester.edu
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.forename: Guoyong
authors.initials: G
authors.lastname: Yin
authors.forename: Prashanthi
authors.initials: P
authors.lastname: Menon
authors.forename: Jinjiang
authors.initials: J
authors.lastname: Pang
authors.forename: Elaine M
authors.initials: EM
authors.lastname: Smolock
authors.forename: Chen
authors.initials: C
authors.lastname: Yan
authors.forename: Bradford C
authors.initials: BC
authors.lastname: Berk
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D021961:Cell Membrane Structures / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D051966:Phospholipase C gamma / Q000378:metabolism*; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D000199:Actins; D018797:Cell Cycle Proteins; C485648:Git1 protein, rat; D050505:Mutant Proteins; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D015240:Phorbol 12,13-Dibutyrate; D014443:Tyrosine; D019061:src-Family Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D051966:Phospholipase C gamma