Phosphorylation of type II cAMP-dependent protein kinase in renal brush border membranes.

We have previously demonstrated cAMP-dependent 32P phosphorylation and dephosphorylation of a 62,000 relative molecular weight (Mr) protein in autoradiograms of sodium dodecyl sulfate polyacrylamide gels originating from canine renal brush border membranes. In the current studies 32P phosphorylation of the 62,000 Mr protein that was independent of cAMP was noted in the presence of Zn2+. Under these conditions, cAMP inhibited the 32P phosphorylation of this protein. Concentration-dependent photoaffinity labeling of a band with Mr 60,000 in autoradiograms of gels resulted from incubation of membranes with cyclic 8-azidoadenosine-3',5'-monophosphate (8-N3-[32P]cAMP) followed by exposure to light. In the presence of Zn2+ and ATP, an apparent shift in the Mr of a portion of the photoaffinity-labeled band to 62,000 was seen. The 62,000 Mr phosphoprotein in detergent-solubilized supernatants of brush border membranes was immunoprecipitated with antibodies directed against the regulatory subunit of type II cAMP-dependent protein kinase. Our observations strongly suggest that the 62,000 Mr protein is the regulatory subunit.