Role of phospholemman phosphorylation sites in mediating kinase-dependent regulation of the Na+-K+-ATPase.
Phospholemman (PLM) is a major target for phosphorylation mediated by both PKA (at Ser68) and PKC (at both Ser63 and Ser68) in the heart. In intact cardiac myocytes, PLM associates with and inhibits Na(+)-K(+)-ATPase (NKA), mainly by reducing its affinity for internal Na(+). The inhibition is relieved upon PLM phosphorylation by PKA or PKC. The aim here was to distinguish the role of the Ser63 and Ser68 PLM phosphorylation sites in mediating kinase-induced modulation of NKA function. We expressed wild-type (WT) PLM and S63A, S68A, and AA (Ser63 and Ser68 to alanine double mutant) PLM mutants in HeLa cells that stably express rat NKA-α(1) and we measured the effect of PKA and PKC activation on NKA-mediated intracellular Na(+) concentration decline. PLM expression (WT or mutant) significantly decreased the apparent NKA affinity for internal Na(+) and had no significant effect on the maximum pump rate (V(max)). PKA activation with forskolin (20 μM) restored NKA Na(+) affinity in cells expressing WT but not AA PLM and did not affect V(max) in either case. Similarly, PKC activation with 300 nM phorbol 12,13-dibutyrate increased NKA Na(+) affinity in cells expressing WT, S63A, and S68A PLM and had no effect in cells expressing AA PLM. Neither forskolin nor phorbol 12,13-dibutyrate affected NKA function in the absence of PLM. We conclude that PLM phosphorylation at either Ser63 or Ser68 is both necessary and sufficient for completely relieving the PLM-induced NKA inhibition.
Han
Fei
F
Department of Pathology, Northwestern University, Feinberg School of Medicine, Chicago, Illinois, USA.
Bossuyt
Julie
J
Martin
Jody L
JL
Despa
Sanda
S
Bers
Donald M
DM
D000818:Animals; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D012964:Sodium / Q000032:analysis / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D008565:Membrane Proteins; D010750:Phosphoproteins; C069141:phospholemman; D005576:Colforsin; D015240:Phorbol 12,13-Dibutyrate; D012964:Sodium; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; C499075:Atp1a1 protein, rat; D000254:Sodium-Potassium-Exchanging ATPase