Phosphorylation of focal adhesion kinase tyrosine 397 critically mediates gastrin-releasing peptide's morphogenic properties

• Published in: Journal of cellular physiology 2004-04, Vol.199 (1), p.77-88
• Main Authors: Glover, Sarah, Delaney, Melissa, Dematte, Cecile, Kornberg, Lori, Frasco, Melissa, Tran-Son-Tay, Roger, Benya, Richard V.
• Format: Article
• Language: English
• Subjects: Blotting, Western; Bombesin - pharmacology; Cell Adhesion - drug effects; Cell Adhesion - physiology; Cell Line, Transformed; Cell Movement - drug effects; Cell Movement - physiology; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gastrin-Releasing Peptide - pharmacology; Gastrin-Releasing Peptide - physiology; Humans; Immunohistochemistry; Phosphorylation; Promoter Regions, Genetic; Protein-Tyrosine Kinases - drug effects; Protein-Tyrosine Kinases - genetics; Protein-Tyrosine Kinases - metabolism; Receptors, Bombesin - physiology
• ISSN: 0021-9541; 1097-4652
• DOI: 10.1002/jcp.10456

We have proposed that gastrin‐releasing peptide (GRP) and its receptor (GRP‐R) are morphogens that when aberrantly re‐expressed in colon cancer promote tumor cell differentiation and retard metastasis. Because circumstantial evidence suggested that these properties were mediated via focal adhesion kinase (FAK), the purpose of this study was to elucidate the role of GRP‐induced activation of this enzyme on properties fundamental to metastasis including cell attachment, motility, and deformability. To do this, we studied 293 cells, a non‐malignant epithelial cell line that we show expresses GRP and GRPR. To dissect out the role of FAK, 293 cells were modified to inducibly express the dominant negative enzyme FAK‐related non‐kinase (FRNK) under control of a Tet‐On (i.e., doxycycline‐sensitive) promoter. Under serum‐free conditions, GRP acting in an autocrine manner caused FAK to be phosphorylated at Y397; and this could be completely inhibited either by incubating with the specific GRP‐R antagonist D‐Phe6(bombesin) methyl ester, or by upregulating FRNK using doxycycline. To measure cell attachment, we designed a cone‐plate viscometer that recorded the shear stress required to detach cells from their underlying matrix. To assess motility, confluent cells were wounded and behavior assessed by time‐lapse photography. To measure deformability, we recorded the ability of cells to be completely drawn into a micropipette