Integrin Cross-talk in Endothelial Cells Is Regulated by Protein Kinase A and Protein Phosphatase 1

In endothelial cells (ECs) beta 1 integrin function-blocking antibodies inhibit alpha v beta 3 integrin-mediated adhesion to a recombinant alpha 4-laminin fragment (r alpha 4LN fragment). beta 1 integrin sequestration of talin is not the mechanism by which beta 1 integrin modulates alpha v beta 3 integrin ligand binding. Rather, treatment of the ECs with beta 1 integrin function-blocking antibodies enhances cAMP-dependent protein kinase (PKA) activity and increases beta 3 integrin serine phosphorylation. The PKA inhibitor H-89 abrogates the effect of beta 1 integrin function-blocking antibodies on beta 3 integrin serine phosphorylation and EC-r alpha 4LN fragment binding. beta 3 integrin contains a serine residue at position 752. To confirm the importance of this residue in alpha v beta 3 integrin-r alpha 4LN fragment binding, we mutated it to alanine (beta 3S752A) or aspartic acid (beta 3S752D). Chinese hamster ovary (CHO) cells expressing wild type or beta 3S752A integrin attach robustly to ligand. CHO cells expressing beta 3S752D integrin do not. Because the beta 3 cytoplasmic tail lacks a PKA consensus site, it is unlikely that PKA acts directly on beta 3 integrin. Instead, we have tested an hypothesis that PKA regulates beta 3 integrin serine phosphorylation indirectly through phosphorylation of inhibitor-1, which, when phosphorylated, inhibits protein phosphatase 1 (PP1). Treatment of ECs with beta 1 integrin function-blocking antibodies significantly increases phosphorylation of inhibitor-1. Furthermore, blocking PP1 activity pharmacologically inhibits alpha v beta 3-mediated cell adhesion to the r alpha 4LN fragment when both PKA and beta 1 integrin function are inhibited. Concomitantly, there is an increase in serine phosphorylation of the beta 3 integrin cytoplasmic tail. These results indicate a novel mechanism by which beta 1 integrin negatively modulates alpha v beta 3 integrin-ligand binding via activation of PKA and inhibition of PP1 activity.