Abstract
The atrial natriuretic peptide (ANP) stimulates cGMP production and protein phosphorylation in a particulate fraction of cultured rat aortic smooth muscle cells. Three proteins of 225, 132, and 11 kDa were specifically phosphorylated in response to ANP treatment, addition of cGMP (5 nM), or addition of purified cGMP-dependent protein kinase. The cAMP-dependent protein kinase inhibitor had no effect on the cGMP-stimulated phosphorylation of the three proteins but inhibited cAMP-dependent phosphorylation of a 17-kDa protein. These results demonstrate that the particulate cGMP-dependent protein kinase mediates the phosphorylation of the 225-, 132-, and 11-kDa proteins. The 11-kDa protein is phospholamban based on the characteristic shift in apparent Mr from 11,000 to 27,000 on heating at 37 degrees C rather than boiling prior to electrophoresis. ANP (1 microM) increased the cGMP concentration approximately 4-fold in the particulate fractions, from 4.3 to 17.7 nM, as well as the phosphorylation of the 225-, 132-, and 11-kDa proteins. In contrast, the biologically inactive form of ANP, carboxymethylated ANP (1 microM), did not stimulate phosphorylation of any proteins nor did the unrelated peptide hormone, angiotensin II (1 microM). These results demonstrate the presence of the cGMP-mediated ANP signal transduction pathway in a particulate fraction of smooth muscle cells and the specific phosphorylation of three proteins including phospholamban, which may be involved in ANP-dependent relaxation of smooth muscle.
Highlights
The apparentM,of the proteins phosphorylated in phosphorylated in response to thecyclic nucleotides is indicated with response to cGMPis indicated with arrows
Protein Kinases and Substrates-The presence of endogenous proteinkinases andsubstratesin cultured vascular smooth muscle cell particulate fractions was investigated by adding a variety of exogenous regulators in the presence of [-y-"P]ATP and separating the resultant proteins on SDSpolyacrylamide gels
Four protein kinase C substrates are phosphorylated with molecular weights 32,000, 31,000, 21,000, and 19,000, in the presence of calcium/phorbol 1-myristate 8-acetatelphosphatidylserine. Both cGMP andcAMP stimulated the phosphorylation of proteins of molecular weights 225,000,132,000, and 11,000, andin addition,cAMPstimulated the phosphorylation of proteins of molecular weights 17,000, 26,000, 43,000, and 151,000
Summary
The apparentM,of the proteins phosphorylated in phosphorylated in response to thecyclic nucleotides is indicated with response to cGMPis indicated with arrows. The position of standard molecular weight marker proteins molecular weight marker proteins is shown on the left. This experi- is shown on the left. This experiment is representative of three ment is representative of four experiments with the same pattern of separate experiments with the same pattern of protein phosphorylaprotein phosphorylation. Modification reaction was monitored by high performance liquid chromatography
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