title: A separate pool of cardiac phospholemman that does not regulate or associate with the sodium pump: multimers of phospholemman in ventricular muscle.
abstract: BACKGROUND
  Phospholemman regulates the plasmalemmal sodium pump in excitable tissues.
  
  
  RESULTS
  In cardiac muscle, a subpopulation of phospholemman with a unique phosphorylation signature associates with other phospholemman molecules but not with the pump.
  
  
  CONCLUSIONS
  Phospholemman oligomers exist in cardiac muscle.
  
  
  CONCLUSIONS
  Much like phospholamban regulation of SERCA, phospholemman exists as both a sodium pump inhibiting monomer and an unassociated oligomer. Phospholemman (PLM), the principal quantitative sarcolemmal substrate for protein kinases A and C in the heart, regulates the cardiac sodium pump. Much like phospholamban, which regulates the related ATPase SERCA, PLM is reported to oligomerize. We investigated subpopulations of PLM in adult rat ventricular myocytes based on phosphorylation status. Co-immunoprecipitation identified two pools of PLM: one not associated with the sodium pump phosphorylated at Ser(63) and one associated with the pump, both phosphorylated at Ser(68) and unphosphorylated. Phosphorylation of PLM at Ser(63) following activation of PKC did not abrogate association of PLM with the pump, so its failure to associate with the pump was not due to phosphorylation at this site. All pools of PLM co-localized to cell surface caveolin-enriched microdomains with sodium pump α subunits, despite the lack of caveolin-binding motif in PLM. Mass spectrometry analysis of phosphospecific immunoprecipitation reactions revealed no unique protein interactions for Ser(63)-phosphorylated PLM, and cross-linking reagents also failed to identify any partner proteins for this pool. In lysates from hearts of heterozygous transgenic animals expressing wild type and unphosphorylatable PLM, Ser(63)-phosphorylated PLM co-immunoprecipitated unphosphorylatable PLM, confirming the existence of PLM multimers. Dephosphorylation of the PLM multimer does not change sodium pump activity. Hence like phospholamban, PLM exists as a pump-inhibiting monomer and an unassociated oligomer. The distribution of different PLM phosphorylation states to different pools may be explained by their differential proximity to protein phosphatases rather than a direct effect of phosphorylation on PLM association with the pump.
authors.affiliation: Division of Cardiovascular & Diabetes Medicine, College of Medicine, Dentistry and Nursing, University of Dundee, Dundee DD1 9SY, UK.
authors.forename: Krzysztof J
authors.initials: KJ
authors.lastname: Wypijewski
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Howie
authors.forename: Louise
authors.initials: L
authors.lastname: Reilly
authors.forename: Lindsay B
authors.initials: LB
authors.lastname: Tulloch
authors.forename: Karen L
authors.initials: KL
authors.lastname: Aughton
authors.forename: Linda M
authors.initials: LM
authors.lastname: McLatchie
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Shattock
authors.forename: Sarah C
authors.initials: SC
authors.lastname: Calaghan
authors.forename: William
authors.initials: W
authors.lastname: Fuller
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D021941:Caveolae / Q000378:metabolism; D005404:Fixatives / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D006352:Heart Ventricles / Q000166:cytology* / Q000378:metabolism; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D060066:Protein Interaction Maps; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000378:metabolism; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
chemical_list: D005404:Fixatives; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D010750:Phosphoproteins; D021122:Protein Subunits; C069141:phospholemman; D005557:Formaldehyde; D000254:Sodium-Potassium-Exchanging ATPase
keywords: Caveolae; FXYD Proteins; Heart; Na,K-ATPase; PP2A; Phospholemman; Protein Palmitoylation; Protein Phosphatase; Protein Phosphorylation; Serine-Threonine Protein Phosphatase