Characterization of protein serine/threonine phosphatases in rat pancreas and development of an endogenous substrate-specific phosphatase assay.
Protein phosphatases have recently been recognized to represent an important, independently regulated portion of cellular signaling cascades. Although reversible phosphorylation of multiple pancreatic proteins has been described, suggesting a role for these enzymes, little is known about the characteristics of protein phosphatases in this organ. In this work, we have characterized and quantified the serine/threonine phosphatases present in pancreatic cytosol and plasma membranes. Using a sensitive and specific in vitro assay with standard substrates (phosphorylase a and phosphocasein), the predominant enzymes represented protein phosphatase 2A in cytosol and protein phosphatase 1 in plasma membranes, with both compartments having substantial amounts of both of these enzymes. Both compartments also had protein phosphatase 2B activity, whereas protein phosphatase 2C was only measurable in the plasma membrane fraction. Further, a novel assay was developed and validated in which the action of an endogenous protein phosphatase on a specific cellular phosphoprotein was studied. For this, we utilized as substrate the cholecystokinin receptor which had been phosphorylated in response to agonist stimulation of the intact acinar cell. This type of assay will be key for the analysis of the mediation and regulation of dephosphorylation events which actually occur in the cell.
Lutz
M P
MP
Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, Minnesota 55905.
Pinon
D I
DI
Miller
L J
LJ
D000818:Animals; D001345:Autoradiography; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D007124:Immunoenzyme Techniques; D008297:Male; D010179:Pancreas / Q000201:enzymology* / Q000648:ultrastructure; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D051381:Rats; D017207:Rats, Sprague-Dawley; D011949:Receptors, Cholecystokinin / Q000378:metabolism*; D013379:Substrate Specificity
D011949:Receptors, Cholecystokinin; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2