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Updated: Aug 18, 2026
Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis
Published on: April 1, 2022
Identification of a cytoskeleton-bound form of phospholemman with unique C-terminal immunoreactivity
C E Kelly1, M L Ram, S A Francis
1Program in Molecular and Cellular Cardiology, Wayne State University School of Medicine, 421 East Canfield Avenue, Detroit, MI 48201, USA.
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.
Insights
Phospholemman (PLM) is a transmembrane protein involved in Na,K-ATPase regulation. A distinct PLM-b form, insoluble in detergents, was identified, suggesting a role in protein structure and function.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Phospholemman (PLM) is a transmembrane protein within the FXYD family, implicated in regulating Na,K-ATPase.
- PLM possesses unique C-terminal phosphorylation sites (PKA, PKC), but the functional significance remains elusive.
Purpose of the Study:
- To investigate the phosphorylation of Phospholemman (PLM).
- To develop specific antibodies for detecting phosphorylated PLM and characterize distinct PLM forms.
Main Methods:
- Generation of antibodies using PLM phosphopeptides.
- Peptide affinity chromatography to isolate antibody populations.
- SDS-PAGE and Western blotting to analyze PLM forms and immunoreactivity.
- Investigating PLM-b solubility and release from binding using colchicine.
Main Results:
- Two antibody populations were isolated: one recognizing standard PLM, and another specific to a distinct form, PLM-b.
- PLM-b, a detergent-insoluble form, showed unique C-terminal immunoreactivity and was released by colchicine.
- PLM-b levels increased with PLM cDNA overexpression and reacted with N-terminal antibodies, but were unaffected by isoproterenol treatment.
Conclusions:
- PLM-b represents a cytoskeleton-associated, detergent-insoluble form of PLM.
- PLM-b's distinct C-terminal immunoreactivity suggests implications for PLM structure and function.
- Further research is needed to elucidate the precise role of PLM phosphorylation in protein function.
