Insulin stimulates the tyrosyl phosphorylation and activation of the 52 kDa peripheral plasma-membrane cyclic AMP phosphodiesterase in intact hepatocytes.
The 52 kDa subunit of the peripheral-plasma-membrane insulin-stimulated high-affinity cyclic AMP phosphodiesterase can be specifically detected by the antibody PM1 by Western-blotting procedures and also can be immunoprecipitated from a hepatocyte extract. PM1-mediated immunoprecipitation from hepatocyte extracts showed that insulin treatment of intact 32P-labelled hepatocytes caused the rapid phosphorylation of the peripheral-plasma-membrane cyclic AMP phosphodiesterase. Phosphoamino acid analysis and the use of a phosphotyrosine-specific antibody indicated that phosphorylation occurred on tyrosyl residue(s) of this phosphodiesterase. Prior treatment of hepatocytes with glucagon (10 nM) completely blocked the insulin-mediated tyrosyl phosphorylation of this 52 kDa protein, as detected with both the PM1 and the anti-phosphotyrosine antibodies. Treatment of hepatocytes with glucagon alone did not increase the phosphorylation state of the peripheral-plasma-membrane cyclic AMP phosphodiesterase. The specific anti-phosphotyrosine antibody also detected the insulin-stimulated phosphorylation of proteins of 180 kDa, 95 kDa and 39 kDa. Prior treatment of hepatocytes with glucagon decreased the ability of insulin to phosphorylate the 180 kDa and 39 kDa species, but not the 95 kDa species.
Pyne
N J
NJ
Department of Biochemistry, University of Glasgow, Scotland, U.K.
Cushley
W
W
Nimmo
H G
HG
Houslay
M D
MD
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D004789:Enzyme Activation; D007328:Insulin / Q000494:pharmacology*; D008099:Liver / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D010766:Phosphorylation; D019000:Phosphotyrosine; D051381:Rats; D011919:Rats, Inbred Strains; D014443:Tyrosine / Q000378:metabolism
D007328:Insulin; D019000:Phosphotyrosine; D014443:Tyrosine; D015105:3',5'-Cyclic-AMP Phosphodiesterases