Protein kinase C phosphorylation of purified Na,K-ATPase: C-terminal phosphorylation sites at the alpha- and gamma-subunits close to the inner face of the plasma membrane.
The alpha-subunit of the Na,K-ATPase is phosphorylated at specific sites by protein kinases A and C. Phosphorylation by protein kinase C (PKC) is restricted to the N terminus and takes place to a low stoichiometry, except in rat. Here we show that the alpha-subunit of shark Na,K-ATPase can be phosphorylated by PKC at C-terminal sites to stoichiometric levels in the presence of detergents. Two novel phosphorylation sites are possible candidates for this PKC phosphorylation: Thr-938 in the M8/M9 loop located very close to the PKA site, and Ser-774, in the proximal part of the M5/M6 hairpin. Both sites are highly conserved in all known alpha-subunits, indicating a physiological role. A similar pattern of detergent-mediated phosphorylation by PKC was found in pig kidney Na,K-ATPase alpha-subunit. Interestingly, the kidney-specific gamma-subunit was phosphorylated by PKC in the presence of detergent. The close proximity of the novel PKC sites to the membrane suggests that targeting proteins to tether PKC into the membrane phase is important in controlling the in vivo phosphorylation of this novel class of membrane-adjacent PKC sites. It is suggested that in purified preparations where functional targeting may be impaired detergents are needed to expose the sites.
Mahmmoud
Yasser A
YA
Department of Biophysics, University of Aarhus, Aarhus, DK-8000 Denmark.
Cornelius
Flemming
F
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D007668:Kidney / Q000201:enzymology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013552:Swine; D013912:Threonine / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry
D003902:Detergents; D013912:Threonine; D012694:Serine; D011493:Protein Kinase C; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase