Purification and complete sequence determination of the major plasma membrane substrate for cAMP-dependent protein kinase and protein kinase C in myocardium.
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 phosphopeptides 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 "gamma" 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.
Palmer
C J
CJ
Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis 46202.
Scott
B T
BT
Jones
L R
LR
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002135:Calcium-Binding Proteins / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009206:Myocardium / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012508:Sarcolemma / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D004247:DNA; D008565:Membrane Proteins; D010750:Phosphoproteins; D011493:Protein Kinase C; D011494:Protein Kinases; D012313:RNA; D000099211:Phospholamban; C069141:phospholemman