Phosphorylation of GIT1 tyrosine 321 is required for association  with FAK at focal adhesions and for PDGF-activated migration  of osteoblasts.
Osteoblast migration and proliferation are fundamental processes in bone healing. We demonstrated that the G-protein-coupled receptor kinase interacting protein 1(GIT1) is a key regulator of bone mass and osteoblast cell migration, but little is known about GIT1 regulation by upstream signaling systems or the impact of GIT1 on downstream effectors. We found that platelet-derived growth factor (PDGF) stimulated the GIT1 tyrosine phosphorylation in osteoblast cells and increased the association of GIT1 with focal adhesion kinase (FAK) at osteoblast focal adhesions. The Src inhibitor PP2 and FAK siRNA inhibited GIT1 tyrosine phosphorylation and the increased association between GIT1 and FAK following stimulation with PDGF. The spa2 homology domain (SHD) of GIT1 was required for association with FAK. Furthermore, phosphorylation of tyrosine 321 of GIT1, which is localized within the SHD, was critical for association with FAK. Mutagenesis analysis revealed that GIT1Y321F inhibited the increased association between GIT1 and FAK. Immunofluorescent staining revealed that GIT1Y321F inhibited FAK activation in focal adhesions after PGDF stimulation. A cell spreading assay demonstrated that GIT1Y321F also inhibited osteoblast cell motility, while the Boyden chamber assay demonstrated that the GIT1Y321F mutation inhibited PDGF-induced osteoblastic cell migration. Phosphorylation of tyrosine 321 of GIT1 is necessary for PDGF-induced association with FAK, FAK activation in focal adhesions, and for osteoblastic cell migration.
The First Affiliated Hospital of Nanjing Medical University, 300 Guang Zhou Road, Jiangsu 210029, China.
Yongxin
Y
Ren
Lipeng
L
Yu
Jin
J
Fan
Ze
Z
Rui
Zhengzhe
Z
Hua
Zitao
Z
Zhang
Ning
N
Zhang
Guoyong
G
Yin
D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002465:Cell Movement; D048429:Cell Size; D002478:Cells, Cultured; D004789:Enzyme Activation; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism*; D022001:Focal Adhesions / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010006:Osteoblasts / Q000201:enzymology / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000502:physiology*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011499:Protein Processing, Post-Translational; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
D018797:Cell Cycle Proteins; D020690:GTPase-Activating Proteins; C120766:Git1 protein, mouse; D010982:Platelet-Derived Growth Factor; D014443:Tyrosine; D051417:Focal Adhesion Kinase 1; C496051:Ptk2 protein, mouse; D019061:src-Family Kinases