In addition to tyrosine sites, FAK (focal adhesion kinase) is phosphorylated on multiple serine residues. In the present study, the regulation of two of these sites, Ser-722 (S1) and Ser-911 (S4), was investigated. Phosphorylation of S1 (but not S4) decreased in resuspended cells, and recovered during spreading on fibronectin, indicating adhesion-dependent regulation. GSK3 (glycogen synthase kinase 3) inhibitors decreased S1 phosphorylation, and siRNA (short interfering RNA) silencing indicated further the involvement of GSK3β. Furthermore, GSK3β was found to become activated during cell spreading on fibronectin, and to physically associate with FAK. S1 phosphorylation was observed to decrease in wounded cell monolayers, while GSK3β underwent inactivation and later was observed to increase to the original level within 24 h. Direct phosphorylation of S1, requiring pre-phosphorylation of Ser-726 in the +4 position, was demonstrated using purified GSK3 and a synthetic peptide containing FAK residues 714–730. An inhibitory role for S1 phosphorylation in FAK signalling was indicated by findings that both alanine substitution for S1 and dephosphorylation of S1 by PP1 (serine/threonine protein phosphatase type-1) resulted in an increase in FAK kinase activity; likewise, this role was also shown by cell treatment with the GSK3 inhibitor LiCl. The inhibitory role was confirmed by the finding that cells expressing FAK with alanine substitution for S1 displayed improved cell spreading and faster migration in wound-healing and trans-well assays. Finally, the finding that S1 phosphorylation increased in cells treated with the PP1 inhibitor okadaic acid indicated targeting of this site by PP1. These results indicate an additional mechanism for regulation of FAK activity during cell spreading and migration, involving Ser-722 phosphorylation modulated through the competing actions of GSK3β and PP1.
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Research Article|
October 10 2005
Regulation of FAK Ser-722 phosphorylation and kinase activity by GSK3 and PP1 during cell spreading and migration
Mariarita Bianchi;
Mariarita Bianchi
*Department of Experimental Pathology, University of Pisa, 56126 Pisa, Italy
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Stefania De Lucchini;
Stefania De Lucchini
†Cell and Developmental Biology Laboratories, Department of Physiology and Biochemistry, University of Pisa, 56010 Pisa, Italy
‡Scuola Normale Superiore of Pisa, 56126 Pisa, Italy
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Oriano Marin;
Oriano Marin
§Department of Biological Chemistry, University of Padova, 35121 Padova, Italy
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David L. Turner;
David L. Turner
∥Department of Biological Chemistry and Mental Health Research Institute, University of Michigan, Ann Arbor, MI 48109, U.S.A.
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Steven K. Hanks;
Steven K. Hanks
¶Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, U.S.A.
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Emma Villa-Moruzzi
Emma Villa-Moruzzi
1
*Department of Experimental Pathology, University of Pisa, 56126 Pisa, Italy
1To whom correspondence should be addressed (email villa@biomed.unipi.it).
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Publisher: Portland Press Ltd
Received:
February 14 2005
Revision Received:
June 20 2005
Accepted:
June 24 2005
Accepted Manuscript online:
June 24 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 391 (2): 359–370.
Article history
Received:
February 14 2005
Revision Received:
June 20 2005
Accepted:
June 24 2005
Accepted Manuscript online:
June 24 2005
Citation
Mariarita Bianchi, Stefania De Lucchini, Oriano Marin, David L. Turner, Steven K. Hanks, Emma Villa-Moruzzi; Regulation of FAK Ser-722 phosphorylation and kinase activity by GSK3 and PP1 during cell spreading and migration. Biochem J 15 October 2005; 391 (2): 359–370. doi: https://doi.org/10.1042/BJ20050282
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