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Phosphorylation of GIT1 tyrosine 321 is required for association with FAK at focal adhesions and for PDGF-activated migration of osteoblasts

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Abstract

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.

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Acknowledgment

This study was supported by National Natural and Science Foundation (81071481), Talent Person in Medicine Foundation (RC2007059) (all foundations to GY).

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Authors and Affiliations

  1. The First Affiliated Hospital of Nanjing Medical University, 300 Guang Zhou Road, Jiangsu, 210029, China

    Yongxin Ren, Lipeng Yu, Jin Fan, Ze Rui, Zhengzhe Hua, Zitao Zhang, Ning Zhang & Guoyong Yin

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  1. Yongxin Ren
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  2. Lipeng Yu
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  3. Jin Fan
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  4. Ze Rui
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  5. Zhengzhe Hua
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  6. Zitao Zhang
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  7. Ning Zhang
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  8. Guoyong Yin
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Correspondence to Guoyong Yin.

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Yongxin Ren and Lipeng Yu contributed equally to this study.

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Ren, Y., Yu, L., Fan, J. et al. Phosphorylation of GIT1 tyrosine 321 is required for association with FAK at focal adhesions and for PDGF-activated migration of osteoblasts. Mol Cell Biochem 365, 109–118 (2012). https://doi.org/10.1007/s11010-012-1249-3

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  • DOI: https://doi.org/10.1007/s11010-012-1249-3

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