Phosphorylation of the β1 integrin cytoplasmic domain:: Toward an understanding of function and mechanism

As F9 stem cells differentiate into parietal endoderm they form focal adhesion 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 residue, mimicking different phosphorylation states, can alter the subcellular localization of the subunit in differentiating F9 cells. We now extend these observations in an attempt to substantiate the function of beta 1 phosphorylation 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 parietal endoderm, including PP2A, Using a crosslinking approach, where antibodies 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 (ILK) co-immunoprecipitates with beta 1 in parietal endoderm and localizes to focal adhesion sites. Okadaic acid treatment significantly decreases the level of ILK associated with beta 1. A possible role for regulated beta 1 phosphorylation in cell migration is discussed. (C) 2000 Academic Press.