Purification and complete sequence determination of the major plasma membrane substrate for cAMP-dependent protein kinase and protein kinase C in myocardium

Cathy J. Palmer, Bruce T. Scott, Larry Jones

Research output: Contribution to journalArticle

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Abstract

A protein of apparent Mr = 15,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is the major plasma membrane substrate for cAMP-dependent protein kinase (PK-A) and protein kinase C (PK-C) in several different tissues. In the work described here, we purified, cloned, and sequenced the canine cardiac sarcolemmal "15-kDa protein." The amino terminus of the purified protein was not blocked, allowing determination of 50 consecutive residues by standard Edman degradation. Overlapping proteolytic phospho-peptides yielded 22 additional residues at the carboxyl terminus. Dideoxy sequencing of the full-length cDNA confirmed that the 15-kDa protein contains 72 amino acids, plus a 20-residue signal sequence. The mature protein has a calculated Mr = 8409. There is one hydrophobic membrane-spanning segment composed of residues 18-37. The acidic amino-terminal end (residues 1-17) of the protein is oriented extracellularly, whereas the basic carboxyl-terminal end (residues 38-72) projects into the cytoplasm. The positively charged carboxyl terminus contains the phosphorylation sites for PK-A and PK-C. In the transmembrane region, the 15-kDa protein exhibits 52% amino acid identity with the "γ" subunit of Na,K-ATPase. High stringency Northern blot analysis revealed that 15-kDa mRNA is present in heart, skeletal muscle, smooth muscle, and liver but absent from brain and kidney. We propose the name "phospholemman" for the 15-kDa protein, which denotes the protein's location within the plasma membrane and its characteristic multisite phosphorylation.

Original languageEnglish
Pages (from-to)11126-11130
Number of pages5
JournalJournal of Biological Chemistry
Volume266
Issue number17
StatePublished - 1991

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Cell membranes
Cyclic AMP-Dependent Protein Kinases
Protein Kinase C
Purification
Sequence Analysis
Myocardium
Cell Membrane
Substrates
Proteins
Phosphorylation
Muscle
Amino Acids
Protein Sorting Signals
Electrophoresis
Sodium Dodecyl Sulfate
Northern Blotting
Liver
Protein Kinases
Names
Smooth Muscle

ASJC Scopus subject areas

  • Biochemistry

Cite this

Purification and complete sequence determination of the major plasma membrane substrate for cAMP-dependent protein kinase and protein kinase C in myocardium. / Palmer, Cathy J.; Scott, Bruce T.; Jones, Larry.

In: Journal of Biological Chemistry, Vol. 266, No. 17, 1991, p. 11126-11130.

Research output: Contribution to journalArticle

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