Identification of the phosphorylation site in vitro for cAMP-dependent protein kinase on the rat adipocyte cGMP-inhibited cAMP phosphodiesterase.
Rat adipocyte cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) appears to be dually regulated in intact cells by serine phosphorylations induced by isoprenaline and insulin, respectively (Degerman, E., Smith, C. J., Tornqvist, H., Vasta, V., Belfrage, P., and Manganiello, V. C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 533-537; Smith, C. J., Vasta, V., Degerman, E., Belfrage, P., and Manganiello, V. C. (1991) J. Biol. Chem. 266, 13385-13390). Since cAMP-dependent protein kinase (cAMP-PK) catalyzes the beta-adrenergic effects, the site in the isolated cGI-PDE phosphorylated by this kinase was explored. A peptide, LRRSSGASGLLTSEHHSR (P18), corresponding to the amino acid sequence Leu423-Arg440 in the putative regulatory domain of the rat adipocyte cGI-PDE was synthesized. It contains a consensus substrate sequence -RRXS- for cAMP-PK within two tryptic cleavage sites and was readily phosphorylated by cAMP-PK. Two phosphopeptides, identified as RS-[32P]SGASGLLTSEHHSR and S-[32P]SGASGLLTSEHHSR, were obtained after stoichiometric phosphorylation and trypsinization of the peptide. These two peptides and the two main tryptic phosphopeptides obtained from immunoisolated [32P]cGI-PDE phosphorylated with cAMP-PK in a solubilized crude adipocyte membrane fraction were immuno-precipitated by an affinity-purified polyclonal antibody raised against P18 and exhibited the same chromatographic and electrophoretic profiles in three different separation systems. Similar radiosequencing profiles indicated that the second most N-terminal serine, corresponding to Ser-427 in the intact cGI-PDE, was phosphorylated by cAMP-PK in both P18 and authentic cGI-PDE. It is concluded that serine 427 is the target for cAMP-PK phosphorylation of the rat adipocyte cGI-PDE in vitro.
Rascón
A
A
Department of Medical and Physiological Chemistry, University of Lund, Sweden.
Degerman
E
E
Taira
M
M
Meacci
E
E
Smith
C J
CJ
Manganiello
V
V
Belfrage
P
P
Tornqvist
H
H
D017667:Adipocytes / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002462:Cell Membrane / Q000201:enzymology; D016384:Consensus Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004822:Epididymis; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity; D014357:Trypsin
D000906:Antibodies; D010446:Peptide Fragments; D010455:Peptides; D010748:Phosphopeptides; D017868:Cyclic AMP-Dependent Protein Kinases; D014357:Trypsin