AJP - Renal Physiology, Vol 250, Issue 4 659-F666, Copyright © 1986 by American Physiological Society
Phosphorylation of type II cAMP-dependent protein kinase in renal brush border membranes
M. R. Hammerman
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.
