Specific dephosphorylation at tyr-554 of git1 by ptprz promotes its association with paxillin and hic-5.
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 was Tyr-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.
Division of Molecular Neurobiology, National Institute for Basic Biology, Okazaki, Aichi, Japan.
Akihiro
A
Fujikawa
Division of Molecular Neurobiology, National Institute for Basic Biology, Okazaki, Aichi, Japan.
Masahito
M
Matsumoto
Division of Molecular Neurobiology, National Institute for Basic Biology, Okazaki, Aichi, Japan.
Kazuya
K
Kuboyama
Division of Molecular Neurobiology, National Institute for Basic Biology, Okazaki, Aichi, Japan.
Ryoko
R
Suzuki
Division of Molecular Neurobiology, National Institute for Basic Biology, Okazaki, Aichi, Japan; School of Life Science, The Graduate University for Advanced Studies, Okazaki, Aichi, Japan.
Masaharu
M
Noda
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D002465:Cell Movement; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D051379:Mice; D032342:Muscle Cells / Q000502:physiology; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism; D051419:Paxillin / Q000378:metabolism*; D010766:Phosphorylation; D054633:Receptor-Like Protein Tyrosine Phosphatases, Class 5 / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; C485647:GIT1 protein, human; D020690:GTPase-Activating Proteins; C120766:Git1 protein, mouse; D051419:Paxillin; D014443:Tyrosine; D054633:Receptor-Like Protein Tyrosine Phosphatases, Class 5