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Identification of a Cytoskeleton-bound Form of Phospholemman with Unique C-Terminal Immunoreactivity

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Abstract

Phospholemman (PLM) is a 72-amino acid transmembrane protein thought to function in Na,K-ATPase regulation or assembly, similar to other members of the FXYD family of proteins. Unique to PLM among these regulatory proteins are sites for C-terminal phosphorylation by PKA and PKC, although a role for phosphorylation in PLM function remains unclear. To study PLM phosphorylation, we used PLM phosphopeptides to generate antibodies to specifically detect phosphorylated PLM. Peptide affinity chromatography isolated two populations of antibodies: one reacting with standard PLM, a collection of closely-spaced 15-kDa protein bands by SDS-PAGE. About 20% of PLM antibodies reacted specifically with a single distinct form of PLM. Levels of this second immunological form (PLM-b) were increased with overexpression of PLM cDNA, and also reacted with a monoclonal antibody against the PLM N-terminus. In complete contrast to standard PLM, however, PLM-b was quantitatively insoluble in nonionic detergents and was released from tight binding by colchicine. Antibodies to PLM-b were present in two different antisera raised to the phosphorylated C-terminal peptide (residues 57–70), but not in antiserum raised to the non-phosphorylated C-terminal peptide. Despite an apparent relationship between PLM-b and phosphorylated PLM, PLM-b levels were not affected by treatment of heart cells with isoproterenol. PLM-b appears to represent a cytoskeleton-attached detergent-insoluble form of PLM with distinctive C-terminal immunoreactivity that might have implications for PLM structure and function.

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Notes

  1. Abbreviations: Ad.PLM, recombinant adenovirus encoding canine PLM; DMEM, Dulbeccos’ modified Eagle’s medium; ECL, enhanced chemilumenescence; HEK, human embryonic kidney; HRP, horseradish peroxidase; KLH, keyhole limpet hemocyanin; PAC, pulmonary arterial cells; PLM, phospholemman; PLM-b, tightly bound form of PLM; P-S, PLM 57–70 containing phospho-Ser68; P-T, PLM 57–70 containing phospho-Thr69; PVDF, polyvinyl difluoride.

  2. S. Cala and L. Jones, unpublished observations.

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Acknowledgments

We thank Dr. Larry R. Jones and Dr. Joseph Y. Cheung for providing us with anti-PLM antibodies. This work was funded by the American Health Assistance Foundation, National Heart Foundation Grant H1999-020.

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  1. Program in Molecular and Cellular Cardiology, Wayne State University School of Medicine, 421 East Canfield Avenue, Detroit, MI, 48201, USA

    C.E. Kelly, M.L. Ram, S.A. Francis, T.D. Houle & S.E. Cala

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  1. C.E. Kelly
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  2. M.L. Ram
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  3. S.A. Francis
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  4. T.D. Houle
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  5. S.E. Cala
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Correspondence to S.E. Cala.

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Kelly, C., Ram, M., Francis, S. et al. Identification of a Cytoskeleton-bound Form of Phospholemman with Unique C-Terminal Immunoreactivity. J Membrane Biol 202, 127–135 (2004). https://doi.org/10.1007/s00232-004-0724-5

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  • DOI: https://doi.org/10.1007/s00232-004-0724-5

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