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Specific Dephosphorylation at Tyr-554 of Git1 by Ptprz Promotes Its Association with Paxillin and Hic-5

Significance Statement

GIT1 is a member of the GTPase-activating protein for ADP-ribosylation factor (Arf GAP) family.  It consists of an Arf GAP domain at the N-terminus, ankyrin repeats, a Spa2-homology domain (SHD), and a focal adhesion targeting-homology (FAH).  The SHD is important for forming stable molecular complexes with the p21-activated kinases (PAKs) interacting exchange factor (PIX) family of Rho guanine nucleotide exchange factors.  GIT1-PIX complexes are known to be recruited to focal complexes through an interaction between the FAH domain of GIT1 and paxillin, which promotes membrane protrusions at the leading edge of migrating cells. Although GIT1 is tyrosine-phosphorylated in a Src-dependent manner, the involvement of respective tyrosine phosphorylation in GIT1 in its binding to focal adhesion proteins has not yet been elucidated in detail.  We previously found that GIT1 is multiply tyrosine phosphorylated, in which Tyr554 is the primary phosphorylation site by Src and dephosphorylated by protein tyrosine phosphatase receptor type Z (PTPRZ) (Fujikawa, A. et al., JBC, 286, 37137-37146, 2011).  Here it should be noted that paxillin at Tyr118 is also dephosphorylated by PTPRZ. In the present study, we revealed that cyclic phosphorylation-dephosphorylation at Tyr554 of GIT1 is crucial for its dynamic interaction with paxillin and Hic-5 to control cell motility.  A stable complex of GIT1-PIX containing PAK may reversibly associate with paxillin or Hic-5 at focal complexes by cyclic phosphorylation-dephosphorylation at Tyr554, which is required to ensure the appropriate activation of Cdc42/Rac1 for localized protrusion activity in the front of the cells and co-ordinated cell movement.  Focal adhesion kinase (FAK) is activated in response to integrin engagement.  There is a possibility that FAK directly phosphorylates the Tyr554 site.

Specific Dephosphorylation at Tyr-554 of Git1 by Ptprz Promotes Its Association with Paxillin and Hic-5. Global Medical Discovery

 

 

 

 

 

 

 

 

 

Journal Reference

Fujikawa A1, Matsumoto M1, Kuboyama K1, Suzuki R1, Noda M2. PLoS One. 2015 ;10(3):e0119361.

Show Affiliations

1Division of Molecular Neurobiology, National Institute for Basic Biology, Okazaki, Aichi, Japan.

2Division of Molecular Neurobiology, National Institute for Basic Biology, Okazaki, Aichi, Japan; School of Life Science, The Graduate University for Advanced Studies, Okazaki, Aichi, Japan.

Abstract

G protein-coupled receptor kinase-interactor 1 (Git1) is involved in cell motility control by serving as an adaptor that links signaling proteins such as Pix and PAK to focal adhesion proteins. We previously demonstrated that Git1 was a multiply tyrosine-phosphorylated protein, its primary phosphorylation site wasTyr-554 in the vicinity of the focal adhesion targeting-homology (FAH) domain, and this site was selectively dephosphorylated by protein tyrosine phosphatase receptor type Z (Ptprz). In the present study, we showed that Tyr-554 phosphorylation reduced the association of Git1 with the FAH-domain-binding proteins, paxillin and Hic-5, based on immunoprecipitation experiments using the Tyr-554 mutants of Git1. The Tyr-554 phosphorylation of Git1 was higher, and its binding to paxillin was consistently lower in the brains of Ptprz-deficient mice than in those of wild-type mice. We then investigated the role of Tyr-554 phosphorylation in cell motility control using three different methods: random cell motility, wound healing, and Boyden chamber assays. The shRNA-mediated knockdown of endogenous Git1 impaired cell motility in A7r5 smooth muscle cells. The motility defect was rescued by the exogenous expression of wild-type Git1 and a Git1 mutant, which only retained Tyr-554 among the multiple potential tyrosine phosphorylation sites, but not by the Tyr-554 phosphorylation-defective or phosphorylation-state mimic Git1 mutant. Our results suggested that cyclic phosphorylation-dephosphorylation at Tyr-554 of Git1 was crucial for dynamic interactions between Git1 and paxillin/Hic-5 in order to ensure coordinated cell motility.

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