Phosphorylation of the beta1 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 beta1 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 beta1 phosphorylation and determine how the phosphorylation levels are regulated. We show that treatment of parietal endoderm with okadaic acid induces an increase in beta1 phosphorylation and selective loss of beta1 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 beta1. Immunocytochemistry shows PP2A colocalizing to focal adhesion sites with beta1. In addition integrin-linked kinase (ILK) co-immunoprecipitates with beta1 in parietal endoderm and localizes to focal adhesion sites. Okadaic acid treatment significantly decreases the level of ILK associated with beta1. A possible role for regulated beta1 phosphorylation in cell migration is discussed.
Mulrooney
J
J
Department of Biology, Wesleyan University, Middletown, Connecticut 06459-0170, USA.
Foley
K
K
Vineberg
S
S
Barreuther
M
M
Grabel
L
L
D000818:Animals; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D039121:Integrin alpha6beta1; D019012:Integrin beta1 / Q000378:metabolism*; D016023:Integrins / Q000378:metabolism; D051379:Mice; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D017469:Receptors, Fibronectin / Q000378:metabolism; D014407:Tumor Cells, Cultured; D000099200:Scaffold Protein ILK
D039121:Integrin alpha6beta1; D019012:Integrin beta1; D016023:Integrins; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases; D017346:Protein Serine-Threonine Kinases; D017469:Receptors, Fibronectin; D000099200:Scaffold Protein ILK