Phosphorylation of serine 2843 in ryanodine receptor-calcium release channel of skeletal muscle by cAMP-, cGMP- and CaM-dependent protein kinase.
The aim of the present study was to determine the phosphorylation of the purified ryanodine receptor-calcium release channel (RyR) of rabbit skeletal muscle sarcoplasmic reticulum by the cAMP-dependent protein kinase (PK-A), cGMP-dependent protein kinase (PK-G) and Ca(2+)-, CaM-dependent protein kinase (PK-CaM) and the localization of phosphorylation sites. Phosphorylation was highest with PK-A (about 0.9 mol phosphate/mol receptor subunit), between one-half to two-thirds with PK-G and between one-third and more than two-thirds with PK-CaM. Phosphoamino acid analysis revealed solely labeled phosphoserine with PK-A and PK-G and phosphoserine and phosphothreonine with PK-CaM. Reverse-phase high-performance liquid chromatography (HPLC) of cyanogen bromide/trypsin digests of the phosphorylated RyR (purified by gel permeation HPLC) and two-dimensional peptide maps revealed one major phosphopeptide by PK-A and PK-G phosphorylation and several labeled peaks by PK-CaM phosphorylation. Automated Edman sequence analysis of the major phosphopeptide obtained from PK-A and PK-G phosphorylation and one phosphopeptide obtained from PK-CaM phosphorylation yielded the sequence KISQTAQTYDPR (residues 2841-2852) with serine 2843 as phosphorylation site (corresponding to the consensus sequence RKIS), demonstrating that all three protein kinases phosphorylate the same serine residue in the center of the receptor subunit, a region proposed to contain the modulator binding sites of the calcium release channel.
Pharmakologisches Institut der Universität Wien, Vienna, Austria.
J
J
Suko
I
I
Maurer-Fogy
B
B
Plank
O
O
Bertel
W
W
Wyskovsky
M
M
Hohenegger
G
G
Hellmann
D000595:Amino Acid Sequence; D000818:Animals; D015220:Calcium Channels / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000242:Cyclic AMP / Q000494:pharmacology*; D006152:Cyclic GMP / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D011494:Protein Kinases / Q000494:pharmacology*; D011817:Rabbits; D011950:Receptors, Cholinergic / Q000302:isolation & purification / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D012694:Serine / Q000378:metabolism*
D015220:Calcium Channels; D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; D012694:Serine; D000242:Cyclic AMP; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D006152:Cyclic GMP