Phosphorylation of the beta 1 integrin cytoplasmic domain: Toward an understanding of function and mechanism

As F9 stem cells differentiate into parietal endoderm they form focal adhes ion sites. There is a concomitant decrease in the level of phosphorylation of S785 in the cytoplasmic domain of the beta 1 integrin subunit, Previous transfection studies demonstrate that site-specific mutations at this resid ue, mimicking different phosphorylation states, can alter the subcellular l ocalization of the subunit in differentiating F9 cells. We now extend these observations in an attempt to substantiate the function of beta 1 phosphor ylation and determine how the phosphorylation levels are regulated, We show that treatment of parietal endoderm with okadaic acid induces an increase in beta 1 phosphorylation and selective loss of beta 1 from focal adhesion sites. Using a PCR approach, we identify two phosphatases expressed in pari etal endoderm, including PP2A, Using a crosslinking approach, where antibod ies are added to live cells, we show that the catalytic subunit of PP2A co- immunoprecipitates with beta 1. Immunocytochemistry shows PP2A colocalizing to focal adhesion sites with beta 1. In addition integrin-linked kinase (I LK) co-immunoprecipitates with beta 1 in parietal endoderm and localizes to focal adhesion sites. Okadaic acid treatment significantly decreases the l evel of ILK associated with beta 1. A possible role for regulated beta 1 ph osphorylation in cell migration is discussed. (C) 2000 Academic Press.