Collagen IV regulates Caco-2 migration and ERK activation via alpha1beta1- and alpha2beta1-integrin-dependent Src kinase activation.
Our previous work indicates intestinal epithelial cell ERK activation by collagen IV, a major component of the intestinal epithelial basement membrane, requires focal adhesion kinase (FAK) and suggests FAK and ERK may have important roles in regulating intestinal epithelial cell migration. We therefore sought to identify FAK downstream targets regulating intestinal epithelial cell spreading, migration, and ERK activation on collagen IV and the integrins involved. Both dominant-negative Src and Src inhibitor PP2 strongly inhibited collagen IV ERK activation in Caco-2 intestinal epithelial cells. Collagen IV stimulated Grb2 binding site FAK Y925 phosphorylation, which was inhibited by PP2 and required FAK Y397 autophosphorylation. Additionally, FAK Y925F expression blocked collagen IV ERK activation. alpha(1)beta(1)- Or alpha(2)beta(1)-integrin blockade with alpha(1)- or alpha(2)-integrin subunit antibodies indicated that either integrin can mediate adhesion, cell spreading, and FAK, Src, and ERK activation on collagen IV. Both dominant-negative Src and PP2 inhibited Caco-2 spreading on collagen IV. PP2 inhibited p130(Cas) tyrosine phosphorylation, but dominant-negative p130(Cas) did not inhibit cell spreading. PP2 inhibited Caco-2 migration on collagen IV much more strongly than the mitogen-activated protein kinase kinase inhibitor PD-98059, which completely inhibited collagen IV ERK activation. These results suggest a pathway for collagen IV ERK activation requiring Src phosphorylation of FAK Y925 not previously described for this matrix protein and suggest either alpha(1)beta(1)- or alpha(2)beta(1)-integrins can regulate Caco-2 spreading and ERK activation on collagen IV via Src. Additionally, these results suggest Src regulates Caco-2 migration on collagen IV primarily through ERK-independent pathways.
Sanders
Matthew A
MA
Department of Surgery, Wayne State University, Detroit, MI 48201-1932, USA.
Basson
Marc D
MD
D019138:Antibodies, Blocking / Q000494:pharmacology; D015153:Blotting, Western; D018938:Caco-2 Cells; D002465:Cell Movement / Q000187:drug effects; D024141:Collagen Type IV / Q000502:physiology*; D003470:Culture Media; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D039222:Integrin alpha1beta1 / Q000037:antagonists & inhibitors / Q000502:physiology*; D038982:Integrin alpha2beta1 / Q000037:antagonists & inhibitors / Q000502:physiology*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000502:physiology; D011743:Pyrimidines / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*
C412373:AG 1879; D019138:Antibodies, Blocking; D024141:Collagen Type IV; D003470:Culture Media; D004791:Enzyme Inhibitors; D039222:Integrin alpha1beta1; D038982:Integrin alpha2beta1; D011743:Pyrimidines; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D019061:src-Family Kinases; D020928:Mitogen-Activated Protein Kinases