In vivo Phosphorylation of the Na,K-ATPase α Subunit in Sciatic Nerves of Control and Diabetic Rats: Effects of Protein Kinase Modulators

The phosphorylation state of the Na,K-ATPase α subunit has been examined in32P-labeled sciatic nerves of control and streptozotocin-treated diabetic rats. Intact nerves were challenged with protein kinase (PK) modulators and α-subunit32P labeling was analyzed after immunoprecipitation. In control nerves, the PKC activator phorbol 12-myristate 13-acetate (PMA) had little effect on α-subunit32P labeling. In contrast, staurosporine, a PKC inhibitor, and extracellular calcium omission decreased it. In Ca2+-free conditions, PMA restored the labeling to basal levels. The cAMP-raising agent forskolin reduced the32P labeling of the α subunit. The results suggest that nerve Na,K-ATPase is tonically phosphorylated by PKC in a Ca2+-dependent manner and that PKA modulates the phosphorylation process. In nerves of diabetic rats, PMA increased32P labeling of the α 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 α-subunit isoforms (α1 or α2) 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.