Phosphorylation of the inositol 1,4,5-trisphosphate receptor by cyclic GMP-dependent protein kinase.
Cyclic GMP (cGMP) inhibits intracellular calcium ([Ca2+]i) mobilization in vascular smooth muscle cells by a mechanism that is not well understood. Because several studies suggest that cGMP inhibits inositol 1,4,5-trisphosphate (IP3) action, we examined the effects of cGMP-dependent protein kinase on IP3 receptor phosphorylation. The purified IP3 receptor was phosphorylated using either the cGMP- or cAMP-dependent protein kinase in vitro. Phosphorylation was time-dependent and stoichiometric using both kinases. Two-dimensional phosphopeptide mapping, high performance liquid chromatography analysis, and amino acid analysis showed that identical sites were phosphorylated using either kinase, and identified serine 1755 as the site of phosphorylation. The synthetic peptide corresponding to serine 1755 (GRRESLTSFG) was phosphorylated with aKm in the range of 30-40 microM by both kinases. The kinetic analysis revealed that this peptide substrate is the best substrate described for cGMP kinase to date. Vascular smooth muscle cells prelabeled with [32P]orthophosphate and treated with atrial natriuretic peptide or sodium nitroprusside to elevate cGMP also resulted in increased labeling of the IP3 receptor. Phosphorylation of IP3 receptor by cGMP kinase may regulate the function of IP3 receptor in vascular smooth muscle cells and contribute to the effect of cGMP to regulate intracellular calcium levels.
Komalavilas
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P
Department of Pathology, University of Alabama, Birmingham 35294-0019.
Lincoln
T M
TM
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta; D015220:Calcium Channels / Q000378:metabolism*; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007700:Kinetics; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D009124:Muscle Proteins; D010750:Phosphoproteins; D018160:Receptors, Cytoplasmic and Nuclear; D015544:Inositol 1,4,5-Trisphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases