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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Acknowledgements
We thank M Hamaguchi (Nagoya Univ., Japan) and T Iwahara (Osaka Bioscience Institute, Japan) for fak null MEFs, and N Gotoh (Tokyo Univ., Japan), H Higashi (Hokkaido Univ., Japan), N Mochizuki (National Cardiovascular Cent. Res. Inst., Japan), H Hanafusa (Prof. emeritus, The Rockefeller Univ., USA; and Director em., OBI, Japan), SK Hanks (Vanderbilt Univ., USA), and M Matsuda (Kyoto Univ., Japan) for plasmids. We also thank K Sasai (Hokkaido Univ., Japan) and Y Ohba (Hokkaido Univ., Japan) for valuable discussion. This work was supported in part by grants-in-aid from the Ministry of Education, Science, Culture, and Sports, and the Ministry of Health, Labor, and Welfare, Japan, as well as Suhara Memorial Foundation (Sapporo, Japan), and the Mochida Medical Science Foundation (Tokyo, Japan). The work of SF and TK is supported by grants from Cancer Research UK and the British Cancer Charity “Heads Up”.
We dedicate this work to our great mentor Hidesaburo Hanafusa, professor emeritus of the Rockefeller University, who passed away on March 15, 2009 at the age of 79. He devoted his life to science and in particular to creating the oncogene research field, to teaching and to providing profound affection to his students and postdocs. All of the alumni of Saburo's laboratory pride themselves in having been his apprentices and we would like to hereby express our deeply felt gratitude to Saburo.
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Supplementary information, Figure S1
293T cells expressing Flag-tagged-wild-type Gab1 (WT) or -Gab1 Y307F were lysed and 0.3, 0.5, or 1.0 mg of cell lysate were subjected to immunoprecipitation with antibody to Flag, followed by immunoblot analysis with antibody to phospho-Gab1 Y307. (PDF 46 kb)
Supplementary information, Figure S2
Flag-tagged wild-type Gab1 was expressed into 293T cells with the individual SH2 domain-containing signaling proteins as indicated. (PDF 83 kb)
Supplementary information, Figure S3
293T cells expressing wild-type or the mutant form of CrkII upon Flag-tagged Gab1 Y307F expression were lysed and the cell lysate was subjected to immunoblot analysis with antibodies to phosphorylated form of Src Y416 or to total Src. (PDF 41 kb)
Supplementary information, Figure S4
Flag-tagged wild-type Gab1 was expressed into 293T cells with the individual Crk-family of adaptor proteins as indicated. (PDF 44 kb)
Supplementary information, Figure S5
MDCK cells were incubated in the absence (upper panels) or presence (lower panels) of HGF (50 ng/ml) for 30 min. (PDF 228 kb)
Supplementary information, Figure S6
MDCK cells were stably transfected with vectors for EGFP-fused-wild-type Gab1 (WT), EGFP-Gab1 Y307F, or with the corresponding empty vector (control). (PDF 62 kb)
Supplementary information, Figure S7
The established MDCK cells stably expressing EGFP-fused wild-type Gab1 (WT), EGFP-Gab1 Y307F, or its control cells transfected with the corresponding empty vector (C) were plated in 60 mm-dishes. (PDF 79 kb)
Supplementary information, Figure S8
Wound healing assay was performed using the established MDCK cells stably expressing EGFP-fused wild-type Gab1 (WT), EGFP-Gab1 Y307F, or its control cells. (PDF 316 kb)
Supplementary information, Figure S9
MDCK cells were transiently co-transfected with expression vectors for RFP-fused CrkI and either GFP-fused wild-type Gab1 (WT) or GFP-fused Gab1 Y307F, and the fluorescence images were acquired with a fluorescence microscope. (PDF 244 kb)
Supplementary information, Figure S10
293T cells were transiently transfected with vectors for Flag-Gab1 and Flag-CrkII as indicated, and their expression levels were examined using anti-Flag antibody. (PDF 77 kb)
Supplementary information, Video S1A
Time-lapse imagings of Figure 5A. MDCK cells expressing either GFP-fused-wild-type Gab1 or –Gab1 Y307F were replaced onto glass-based dishes. Immediately after 50 ng/ml HGF stimulation, fluorescent images were obtained every 5 min with the use of a time-lapse imaging system. (A) MDCK EGFP-Gab1 WT; (B) MDCK EGFP-Gab1 Y307F. (MOV 4203 kb)
Supplementary information, Video S2A
Time-lapse imagings of Figure 6A and 6E. RFP-fused CrkI and either GFP-fused wild-type Gab1 or its Y307F mutant was overexpressed into MDCK cells. Immediately after 50 ng/ml HGF stimulation, fluorescent images were obtained every 1 min using a time-lapse system. Green color indicates Gab1, whereas Red exhibits CrkI. In the context examining the implication of Src family kinases on Gab1 phosphorylation, the cells were preincubated with PP2 (10 μM) for 2 h. (A) MDCK GFP-Gab1 WT, RFP-CrkI; (B) MDCK GFP-Gab1 Y307F, RFP-CrkI; (C) MDCK, GFP-Gab1 WT, RFP-CrkI, PP2; (D) MDCK, GFP-Gab1 Y307F, RFP-CrkI, PP2. (MOV 3039 kb)
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Watanabe, T., Tsuda, M., Makino, Y. et al. Crk adaptor protein-induced phosphorylation of Gab1 on tyrosine 307 via Src is important for organization of focal adhesions and enhanced cell migration. Cell Res 19, 638–650 (2009). https://doi.org/10.1038/cr.2009.40
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DOI: https://doi.org/10.1038/cr.2009.40
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