Regulation of Na+-K+-ATPase by cAMP-dependent protein kinase anchored on membrane via its anchoring protein

Na+-K+ ATPase alpha-subunits in basolateral membrane vesicles (BLMVs) purified from rat parotid glands were P-32-labeled within 5 s by incubation with [gamma-P-32] ATP at 37 degrees C in the presence of cAMP, but no labeling occurred without cAMP. Phosphorylation of Na+-K+-ATPase was associated with a decrease in its activity. This alpha-subunit phosphorylation disappeared when BLMVs were briefly incubated with cAMP and subsequent washing before the incubation with [gamma-P-32]ATP, indicating that catalytic subunit of protein kinase A (PKA) associated to BLMVs via binding with its RII regulatory subunit anchored on the membrane. In the absence of cAMP, a PKA catalytic subunit readily reassociated with the membrane-bound RII subunit. HT-31 peptide inhibited the Na+-K+-ATPase phosphorylation by membrane- bound endogenous PKA, indicating an involvement of A-kinase anchoring protein (AKAP). AKAP-150 protein in BLMVs was shown by immunoblotting and an RII overlay assay and was coimmunoprecipitated by anti-RII antibody. These results show that Na+-K+-ATPase of rat parotid gland acinar cells is regulated in vivo by membrane- anchored PKA via AKAP rather than by free cytosolic PKA.