Role of protein kinase C in phospholemman mediated regulation of α₂β₁ isozyme of Na⁺/K⁺-ATPase in caveolae of pulmonary artery smooth muscle cells.
We have recently reported that α(2)β(1) and α(1)β(1) isozymes of Na(+)/K(+)-ATPase (NKA) are localized in the caveolae whereas only the α(1)β(1) isozyme of NKA is localized in the non-caveolae fraction of pulmonary artery smooth muscle cell membrane. It is well known that different isoforms of NKA are regulated differentially by PKA and PKC, but the mechanism is not known in the caveolae of pulmonary artery smooth muscle cells. Herein, we examined whether this regulation occurs through phospholemman (PLM) in the caveolae. Our results suggest that PKC mediated phosphorylation of PLM occurs only when it is associated with the α(2) isoform of NKA, whereas phosphorylation of PLM by PKA occurs when it is associated with the α(1) isoform of NKA. To investigate the mechanism of regulation of α(2) isoform of NKA by PKC-mediated phosphorylation of PLM, we have purified PLM from the caveolae and reconstituted into the liposomes. Our result revealed that (i) in the reconstituted liposomes phosphorylated PLM (PKC mediated) stimulate NKA activity, which appears to be due to an increase in the turnover number of the enzyme; (ii) phosphorylated PLM did not change the affinity of the pump for Na(+); and (iii) even after phosphorylation by PKC, PLM still remains associated with the α(2) isoform of NKA.
Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India.
Kuntal
K
Dey
Soumitra
S
Roy
Biswarup
B
Ghosh
Sajal
S
Chakraborti
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D021941:Caveolae / Q000201:enzymology* / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000201:enzymology* / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology*; D055550:Protein Stability; D011651:Pulmonary Artery / Q000166:cytology*; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification / Q000378:metabolism*
D007527:Isoenzymes; D008081:Liposomes; D008565:Membrane Proteins; D010750:Phosphoproteins; C069141:phospholemman; D012964:Sodium; D011493:Protein Kinase C; D000254:Sodium-Potassium-Exchanging ATPase