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 (32)P-labeled within 5 s by incubation with [gamma-(32)P]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-(32)P]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.
Kurihara
K
K
Department of Oral Physiology, School of Dentistry, Meikai University, Sakado, Saitama 350-0283, Japan. kkinji@dent.meikai.ac.jp
Nakanishi
N
N
Ueha
T
T
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D015536:Down-Regulation; D020035:Holoenzymes / Q000378:metabolism; D007425:Intracellular Membranes / Q000201:enzymology*; D007527:Isoenzymes / Q000378:metabolism; D008297:Male; D010306:Parotid Gland / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; C415703:Akap5 protein, rat; D002352:Carrier Proteins; D020035:Holoenzymes; D007527:Isoenzymes; D012333:RNA, Messenger; D017868:Cyclic AMP-Dependent Protein Kinases; D000254:Sodium-Potassium-Exchanging ATPase