Submitted on July 8, 2008
Revised on November 10, 2008
Accepted on November 11, 2008
Increased extracellular pressure enhances cancer cell integrin binding affinity through phosphorylation of 
1-integrin at threonine 788/789
David H. Craig1, Christopher P Gayer2, Keri L Schaubert3, Yanzhang Wei4, Jinhua Li5, Yasmina Laouar3, and Marc D Basson2*
1 John D. Dingell VA Medical Center, Wayne State University
2 Wayne State University
3 University of Michigan
4 Clemson University
5 Greenville Hospital System
* To whom correspondence should be addressed. E-mail: marc.basson{at}va.gov.
Increased extracellular pressure stimulates 
1-integrin-dependent cancer cell adhesion. We asked whether pressure-induced adhesion is mediated by changes in 1-integrin binding affinity or avidity and whether these changes are phosphorylation-dependent. We evaluated integrin affinity and clustering in human SW620 colon cancer cells by measuring differences in binding between soluble Arg-Gly-Asp (RGD)-Fc ligands and RGD-Fc-F(ab)2 multimeric complexes under ambient and 15 mmHg increased pressure. Phosphorylation of 1-integrin S785 and T788/9 residues in SW620 and primary malignant colonocytes were assessed in parallel. We further employed GD25 1-integrin null murine fibroblasts stably transfected with either wild-type 1A-integrin, S785A, TT788/9AA or T788D mutants to investigate the role of 1-integrin site-specific phosphorylation. SW620 binding of RGD-Fc-F(ab)2 multimeric complexes, but not soluble RGD-Fc ligands was sensitive to integrin clustering. RGD-Fc ligand binding was significantly increased under elevated pressure, suggesting pressure modulates 1-integrin affinity. Pressure stimulated both 1-integrin S785 and T788/9 phosphorylation. GD25 1A-integrin wild-type and S785A cells displayed an increase in adhesion to fibronectin under elevated pressure, an effect absent in 1-integrin null and TT788/9AA cells. T788D substitution significantly elevated basal cell adhesion, but displayed no further increase under pressure. These results suggest pressure-induced cell adhesion is mediated by 1-integrin T788/9 phosphorylation-dependent changes in integrin binding affinity.
