In vivo phosphorylation of the Na,K-ATPase alpha subunit in sciatic nerves of control and diabetic rats: effects of protein kinase modulators.
The phosphorylation state of the Na,K-ATPase alpha subunit has been examined in 32P-labeled sciatic nerves of control and streptozotocin-treated diabetic rats. Intact nerves were challenged with protein kinase (PK) modulators and alpha-subunit 32P labeling was analyzed after immunoprecipitation. In control nerves, the PKC activator phorbol 12-myristate 13-acetate (PMA) had little effect on alpha-subunit 32P labeling. In contrast, staurosporine, a PKC inhibitor, and extracellular calcium omission decreased it. In Ca(2+)-free conditions, PMA restored the labeling to basal levels. The cAMP-raising agent forskolin reduced the 32P labeling of the alpha subunit. The results suggest that nerve Na,K-ATPase is tonically phosphorylated by PKC in a Ca(2+)-dependent manner and that PKA modulates the phosphorylation process. In nerves of diabetic rats, PMA increased 32P labeling of the alpha subunit. In contrast to staurosporine or extracellular calcium omission, the decreased state of phosphorylation seen with forskolin was no longer significant in diabetic nerves. No change in the level of alpha-subunit isoforms (alpha 1 or alpha 2) was detected by Western blot analysis in such nerves. In conclusion, the altered effect of PK activators on Na,K-ATPase phosphorylation state is consistent with the view that a defect in PKC activation exists in diabetic nerves.
Borghini
I
I
Département de Médecine, Centre Médical Universitaire, CH-1211 Geneva, Switzerland.
Geering
K
K
Gjinovci
A
A
Wollheim
C B
CB
Pralong
W F
WF
D000470:Alkaloids / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D003921:Diabetes Mellitus, Experimental / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012016:Reference Values; D012584:Sciatic Nerve / Q000201:enzymology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification / Q000378:metabolism*; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D000470:Alkaloids; D007527:Isoenzymes; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010750:Phosphoproteins; D005576:Colforsin; D011493:Protein Kinase C; D000254:Sodium-Potassium-Exchanging ATPase; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium