Phosphorylation in situ of atrial natriuretic peptide prohormone at the cyclic AMP-dependent site.
Previously we have shown that atrial natriuretic peptides (ANP) are phosphorylated at Ser-104 by cyclic AMP-dependent protein kinase (Rittenhouse, J. Moberly, L., O'Donnell, M.E., Owen, N.E., and Marcus, F. (1986) J. Biol. Chem. 261, 7607-7610). In our present study, atrial natriuretic peptide prohormone (pro-ANP) purified from extracts of rat atria that had been incubated in situ with [32P]orthophosphate was found to be phosphorylated. The site of in situ phosphorylation was localized to the ANP region of the prohormone and was further delineated to a chymotryptic peptide having the same retention time by high performance liquid chromatography as the hexapeptide Arg101-Arg102-Ser103-Ser(P)104-Cys105-Phe106+ ++. This hexapeptide was also formed from in vitro phosphorylated pro-ANP and synthetic ANP. It is thus likely that Ser-104 is the site of phosphorylation of pro-ANP both in situ and in vitro. Incubation of atria with intracellular cyclic AMP-elevating agents consistently increased the specific radioactivity of the resulting purified pro-ANP by 5-10-fold. However, the overall extent of phosphorylation was low, suggesting that a subpopulation of pro-ANP molecules are phosphorylated at Ser-104 by a cyclic AMP-mediated pathway.
Rittenhouse
J
J
Department of Biological Chemistry and Structure, Chicago Medical School, Illinois 60064.
Moberly
L
L
Ahmed
H
H
Marcus
F
F
D000595:Amino Acid Sequence; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000302:isolation & purification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000378:metabolism*; D006325:Heart Atria / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011498:Protein Precursors / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013917:Thrombin / Q000378:metabolism
D010446:Peptide Fragments; D011498:Protein Precursors; D009320:Atrial Natriuretic Factor; D000242:Cyclic AMP; D011494:Protein Kinases; D013917:Thrombin