Crk adaptor protein-induced phosphorylation of Gab1 on tyrosine 307 via Src is important for organization of focal adhesions and enhanced cell migration

Takuya Watanabe, Masumi Tsuda, Yoshinori Makino, Tassos Konstantinou, Hiroshi Nishihara, Tokifumi Majima, Akio Minami, Stephan M. Feller, Shinya Tanaka

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Upon growth factor stimulation, the scaffold protein, Gab1, is tyrosine phosphorylated and subsequently the adaptor protein, Crk, transmits signals from Gab1. We have previously shown that Crk overexpression, which is detectable in various human cancers, induces tyrosine phosphorylation of Gab1 without extracellular stimuli. In the present study, the underlying mechanisms were further investigated. Mutational analyses of CrkII demonstrated that the SH2 domain, but not the SH3(N) or the regulatory Y221 residue of CrkII, is critical for the induction of Gab1-Y307 phosphorylation. SH2 mutation of CrkII also decreased the interaction with Gab1. In GST pull-down assay, Crk-SH2 bound to wild-type Gab1, whereas Crk-SH3(N) interacted with the Gab1 mutant, which lacks the clustered tyrosine region (residues 242-410). Tyrosine phosphorylation of Gab1 was induced by all Crk family proteins, but not other SH2-containing signalling adaptors. Src-family kinase inhibitor, PP2, abrogates Crk-induced tyrosine phosphorylations of Gab1. Y307 phosphorylation was undetectable in fibroblasts lacking Src, Yes, and Fyn, even upon overexpression of Crk, whereas cells lacking only Yes and Fyn still contained Gab1 with phosphorylated Y307. Furthermore, Crk induced the phosphorylation of Src-Y416; accordingly the interaction between Crk and Csk was increased. The Gab1-Y307F mutant failed to localize near the plasma membrane even upon HGF stimulation and decreased cell migration. Moreover, Gab1-Y307F disturbed the localization of Crk, FAK, and paxillin, which are the typical components of focal adhesions. Taken together, these results indicate that Crk facilitates tyrosine phosphorylation of Gab1-Y307 through Src, contributing to the organization of focal adhesions and enhanced cell migration, thereby possibly promoting human cancer development.

Original languageEnglish
Pages (from-to)638-650
Number of pages13
JournalCell Research
Volume19
Issue number5
DOIs
Publication statusPublished - 2009 May 1
Externally publishedYes

Fingerprint

Focal Adhesions
Cell Movement
Tyrosine
Phosphorylation
Proteins
Paxillin
src Homology Domains
Human Development
Neoplasms
Intercellular Signaling Peptides and Proteins
Fibroblasts
Cell Membrane
Mutation

Keywords

  • Crk
  • Gab1
  • Src
  • Tyrosine 307 phosphorylation

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

Cite this

Crk adaptor protein-induced phosphorylation of Gab1 on tyrosine 307 via Src is important for organization of focal adhesions and enhanced cell migration. / Watanabe, Takuya; Tsuda, Masumi; Makino, Yoshinori; Konstantinou, Tassos; Nishihara, Hiroshi; Majima, Tokifumi; Minami, Akio; Feller, Stephan M.; Tanaka, Shinya.

In: Cell Research, Vol. 19, No. 5, 01.05.2009, p. 638-650.

Research output: Contribution to journalArticle

Watanabe, Takuya ; Tsuda, Masumi ; Makino, Yoshinori ; Konstantinou, Tassos ; Nishihara, Hiroshi ; Majima, Tokifumi ; Minami, Akio ; Feller, Stephan M. ; Tanaka, Shinya. / Crk adaptor protein-induced phosphorylation of Gab1 on tyrosine 307 via Src is important for organization of focal adhesions and enhanced cell migration. In: Cell Research. 2009 ; Vol. 19, No. 5. pp. 638-650.
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abstract = "Upon growth factor stimulation, the scaffold protein, Gab1, is tyrosine phosphorylated and subsequently the adaptor protein, Crk, transmits signals from Gab1. We have previously shown that Crk overexpression, which is detectable in various human cancers, induces tyrosine phosphorylation of Gab1 without extracellular stimuli. In the present study, the underlying mechanisms were further investigated. Mutational analyses of CrkII demonstrated that the SH2 domain, but not the SH3(N) or the regulatory Y221 residue of CrkII, is critical for the induction of Gab1-Y307 phosphorylation. SH2 mutation of CrkII also decreased the interaction with Gab1. In GST pull-down assay, Crk-SH2 bound to wild-type Gab1, whereas Crk-SH3(N) interacted with the Gab1 mutant, which lacks the clustered tyrosine region (residues 242-410). Tyrosine phosphorylation of Gab1 was induced by all Crk family proteins, but not other SH2-containing signalling adaptors. Src-family kinase inhibitor, PP2, abrogates Crk-induced tyrosine phosphorylations of Gab1. Y307 phosphorylation was undetectable in fibroblasts lacking Src, Yes, and Fyn, even upon overexpression of Crk, whereas cells lacking only Yes and Fyn still contained Gab1 with phosphorylated Y307. Furthermore, Crk induced the phosphorylation of Src-Y416; accordingly the interaction between Crk and Csk was increased. The Gab1-Y307F mutant failed to localize near the plasma membrane even upon HGF stimulation and decreased cell migration. Moreover, Gab1-Y307F disturbed the localization of Crk, FAK, and paxillin, which are the typical components of focal adhesions. Taken together, these results indicate that Crk facilitates tyrosine phosphorylation of Gab1-Y307 through Src, contributing to the organization of focal adhesions and enhanced cell migration, thereby possibly promoting human cancer development.",
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T1 - Crk adaptor protein-induced phosphorylation of Gab1 on tyrosine 307 via Src is important for organization of focal adhesions and enhanced cell migration

AU - Watanabe, Takuya

AU - Tsuda, Masumi

AU - Makino, Yoshinori

AU - Konstantinou, Tassos

AU - Nishihara, Hiroshi

AU - Majima, Tokifumi

AU - Minami, Akio

AU - Feller, Stephan M.

AU - Tanaka, Shinya

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AB - Upon growth factor stimulation, the scaffold protein, Gab1, is tyrosine phosphorylated and subsequently the adaptor protein, Crk, transmits signals from Gab1. We have previously shown that Crk overexpression, which is detectable in various human cancers, induces tyrosine phosphorylation of Gab1 without extracellular stimuli. In the present study, the underlying mechanisms were further investigated. Mutational analyses of CrkII demonstrated that the SH2 domain, but not the SH3(N) or the regulatory Y221 residue of CrkII, is critical for the induction of Gab1-Y307 phosphorylation. SH2 mutation of CrkII also decreased the interaction with Gab1. In GST pull-down assay, Crk-SH2 bound to wild-type Gab1, whereas Crk-SH3(N) interacted with the Gab1 mutant, which lacks the clustered tyrosine region (residues 242-410). Tyrosine phosphorylation of Gab1 was induced by all Crk family proteins, but not other SH2-containing signalling adaptors. Src-family kinase inhibitor, PP2, abrogates Crk-induced tyrosine phosphorylations of Gab1. Y307 phosphorylation was undetectable in fibroblasts lacking Src, Yes, and Fyn, even upon overexpression of Crk, whereas cells lacking only Yes and Fyn still contained Gab1 with phosphorylated Y307. Furthermore, Crk induced the phosphorylation of Src-Y416; accordingly the interaction between Crk and Csk was increased. The Gab1-Y307F mutant failed to localize near the plasma membrane even upon HGF stimulation and decreased cell migration. Moreover, Gab1-Y307F disturbed the localization of Crk, FAK, and paxillin, which are the typical components of focal adhesions. Taken together, these results indicate that Crk facilitates tyrosine phosphorylation of Gab1-Y307 through Src, contributing to the organization of focal adhesions and enhanced cell migration, thereby possibly promoting human cancer development.

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