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Regulatory phosphorylation of the Na+/K+-ATPase from mammalian kidneys and Xenopus oocytes by protein kinases. Characterization of the phosphorylation site for PKC

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Vasilets,  Larisa A.
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;
Institute of Chemical Physics Russian Academy of Science Chernogolovka Moscow region, Russia;

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Fotis,  Heike
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;

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Gärtner,  Eva-Maria
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;

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Citation

Vasilets, L. A., Fotis, H., & Gärtner, E.-M. (1997). Regulatory phosphorylation of the Na+/K+-ATPase from mammalian kidneys and Xenopus oocytes by protein kinases. Characterization of the phosphorylation site for PKC. Annals of the New York Academy of Sciences, 834(1), 585-587. doi:10.1111/j.1749-6632.1997.tb52326.x.


Cite as: http://hdl.handle.net/21.11116/0000-0007-F857-0
Abstract
Protein kinase‐mediated phosphorylation of the Na+/K+‐ATPase has been studied in enzymes purified from pig, dog, sheep, and rat kidneys and in Xenopus oocytes. None of the α subunits from mammalian kidney ATPase is phosphorylated by casein kinase II or Ca2+/calmodulin‐dependent protein kinase. For the purified enzymes, rat protein kinase C (PKC) phosphorylates only the α subunit of the Na+/K+‐ATPase of rat kidney. Selective proteolytic digestion of the rat α1 enzyme under conditions of mild trypsinolysis demonstrates that phosphorylation occurs at the NH2‐terminus before the T2 cleavage site. There are Ser‐16 and Ser‐23 within the cleaved fragment of the rat ATPase that can potentially be phosphorylated by PKC. Only Ser‐16 is present in the other mammalian kidney isoforms that are poorly phosphorylated by PKC. Mutated rat α1 subunits with substitution of Ser‐23 (S23A) or Ser‐16 (S16A) by Ala were coexpressed in Xenopus oocytes together with β subunits, and modulation of transport activity (Table 1) as well as phosphorylation of pumps by PKC in yolk‐free oocyte homogenates (Fig. 1) was investigated. PKC produces incorporation of32P into α subunits of wild‐type pumps and inhibits ouabain‐sensitive 86Rb uptake by 80 ± 3%. By contrast, in the S23A mutant, much weaker phosphorylation and no inhibition of 86Rb uptake by rat PKC were observed. In the S16A mutant, the α subunit still can be phosphorylated by PKC at the Ser‐23 site, but the sensitivity of transport activity to PKC is lost. The data demonstrate that Ser‐23 is the actual site of regulatory phosphorylation for rat brain PKC in rat kidney α1 Na+/K+‐ATPase, but Ser‐16 is also important for PKC‐mediated changes in pump function. Effects of modulation of transport activity of expressed pumps by stimulation of endogenous Xenopus PKC with PMA differ from those of rat brain PKC. Incubation with PMA leads to considerable inhibition of 86Rb uptake in wild‐type pumps as well as in S16A or S23A mutants (Table 1), and all α subunits can be phosphorylated (lanes 2, 3, and 4 in Fig. 1). However, 32P‐incorporation into α subunits of S16A and S23A mutants was less than that for the wild type. This suggests that activation of Xenopus PKC may lead to phosphorylation of sites that are different from those phosphorylated by rat brain PKC.