In vitro phosphorylation of phosphatidylethanolamine N-methyltransferase by cAMP-dependent protein kinase: lack of in vivo phosphorylation in response to N6-2'-O-dibutryladenosine 3',5'-cyclic monophosphate.
Phosphorylation of rat liver phosphatidylethanolamine (PE) N-methyltransferase by cAMP-dependent protein kinase was investigated. The 18 kDa methyltransferase was found to be phosphorylated in vitro by cAMP-dependent protein kinase on a serine residue. The stoichiometry of phosphate incorporation reached a maximum of 0.25 mol phosphate/mol methyltransferase at 30 min. Resolution of the phosphorylated methyltransferase by two-dimensional gel electrophoresis showed that two isoproteins were substrates. Phosphorylation of the purified PE N-methyltransferase for up to 1 h had no effect on the methylation of PE, PMME or PDME. To test for in vivo phosphorylation, isolated rate hepatocytes were exposed to 0.5 mM N6-2'-O-dibutryladenosine 3':5'-cyclic monophosphate (DiB-cAMP) and the phosphorylation state of microsomal proteins evaluated by two-dimensional gel electrophoresis, nitrocellulose blotting and autoradiography. The same nitrocellulose blots were probed with a rabbit anti-PE N-methyltransferase antibody, immunochemically stained and aligned with the autoradiogram. No phosphorylated proteins co-migrated with the methyltransferase under non-phosphorylating conditions, or when hepatocytes were exposed to the cAMP analogue for up to 2 h. Oddly, DiB-cAMP increased both PE- and PMME-dependent activity in isolated microsomes, but decreased PE to PC conversion measured in intact hepatocytes. The results indicated that PE N-methyltransferase is poorly phosphorylated by cAMP-dependent protein kinase in vitro, and is not phosphorylated in intact hepatocytes treated with a cAMP analogue.
Ridgway
N D
ND
Lipid and Lipoprotein Research Group, University of Alberta, Edmonton, Canada.
Vance
D E
DE
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology*; D000242:Cyclic AMP / Q000494:pharmacology*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008099:Liver / Q000201:enzymology; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D050918:Phosphatidylethanolamine N-Methyltransferase; D010714:Phosphatidylethanolamines / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats
D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D047428:Protein Kinase Inhibitors; C032000:phosphatidyl-N-monomethylaminoethanol; C026508:phosphatidyldimethylethanolamine; D003994:Bucladesine; D000242:Cyclic AMP; D008780:Methyltransferases; D050918:Phosphatidylethanolamine N-Methyltransferase; D011494:Protein Kinases