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Different modes and qualities of tyrosine phosphorylation of Fak and Pyk2 during epithelial-mesenchymal transdifferentiation and cell migration: analysis of specific phosphorylation events using site-directed antibodies

Oncogene volume 20, pages 2626–2635 (2001)Cite this article

Abstract

Integrin signaling is activated during epithelial-mesenchymal transdifferentiation (EMT) and cell migration, processes serving as models for carcinogenesis. We have shown that paxillin and p130Cas become highly tyrosine phosphorylated during these processes in NMuMG cells. Here, we examined the regulation of Fak and Pyk2, kinases implicated in this phosphorylation. Pyk2 became phosphorylated at the major autophosphorylation site (Tyr-402) and the potential Grb2-binding site (Tyr-881) during EMT. In contrast, phosphorylation of Fak at the corresponding autophosphorylation site (Tyr-397) occurred even in sedentary epithelial cells, whereas phosphorylation at Tyr-407 and Tyr-861 was induced during EMT. During cell migration, these phosphorylation events, except Fak Tyr-397, were augmented further, and phosphorylation of Fak Tyr-577 and the corresponding Pyk2 Tyr-580, both within the kinase activation loops, was also induced. In all cases, phosphorylation of the putative Grb2-binding site in Fak (Tyr-925) was almost undetectable. Although Fak and Pyk2 have several phosphorylation sites in common, Tyr-407 and Tyr-861 are unique to Fak. Our results revealed that Fak and Pyk2 are non-equivalent in the tyrosine phosphorylation events and thereby likely to evoke different downstream signaling cascades during EMT and cell migration of NMuMG cells. We also show that Fak Tyr-397 phosphorylation occurs exclusively at the cytoplasm, but not at focal contacts, in the sedentary epithelial cells. In contrast, all other tyrosine phosphorylated forms of Fak and Pyk2 are predominantly localized to focal adhesions and the cell periphery in motile cells, all colocalized with paxillin and p130Cas.

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Acknowledgements

We are grateful to Manami Hiraishi and Mihoko Sato for their technical assistance, and Mayumi Yoneda for her secretarial work. This work was supported in part by Grants-in-aid from the Ministry of Education, Science, Sports and Culture of Japan; Grants from Takeda Medical Foundation, Mitsubishi Foundation, Ciba-Geigy Foundation (Japan) for the Promotion of Science, the Mochida Memorial Foundation for Medical and Pharmaceutical Research, and Novartis Foundation for the Promotion of Science. OBI was founded as a commemoration of the one hundredth year of the anniversary of the municipal government of Osaka City, and is supported by Osaka City.

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  1. Department of Molecular Biology, Osaka Bioscience Institute, Suita, 565-0874, Osaka, Japan

    Kuniaki Nakamura, Hajime Yano & Hisataka Sabe

  2. BioSource International, Hopkinton, 01748, Massachusetts, MA, USA

    Erik Schaefer

  3. Graduate School of Biostudies, Kyoto University, Sakyoku, 606-8502, Kyoto, Japan

    Hisataka Sabe

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Nakamura, K., Yano, H., Schaefer, E. et al. Different modes and qualities of tyrosine phosphorylation of Fak and Pyk2 during epithelial-mesenchymal transdifferentiation and cell migration: analysis of specific phosphorylation events using site-directed antibodies. Oncogene 20, 2626–2635 (2001). https://doi.org/10.1038/sj.onc.1204359

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