IDENTIFICATION OF THE SITE IN THE CGMP-INHIBITED PHOSPHODIESTERASE PHOSPHORYLATED IN ADIPOCYTES IN RESPONSE TO INSULIN AND ISOPROTERENOL

Stimulation of rat adipocytes with insulin and isoproterenol results i n serine phosphorylation and activation of the adipocyte cGMP-inhibite d phosphodiesterase (cGI PDE), events believed to be important in the antilipolytic action of insulin (Degerman, E., Smith, C. J., Tornqvist , H., Vasta, V., Manganiello, V. C., and Belfrage, P. (1990) Proc. Nat l. Acad. Sci. U. S. A. 87, 533-537). Here we demonstrate, by two-dimen sional phosphopeptide mapping, that the major phosphopeptide generated by trypsin, or trypsin followed by Asp-N protease digestion of [P-32] cGI PDE phosphorylated in adipocytes in response to isoproterenol and/ or insulin, in each case co-migrates with the phosphopeptide released by the same treatment of M(297)FRRPS((P) under bar)LPCISREQ(310). This peptide was synthesized based on the deduced sequence of the cloned r at adipocyte cGI PDE and phosphorylated by cAMP-dependent protein kina se (protein kinase A). Radiosequencing of authentic and synthetic tryp tic P-32-peptides showed that a single site in cGI PDE (Ser(302)) was phosphorylated in adipocytes incubated with isoproterenol and/or insul in. The more than additive phosphorylation and activation of cGI PDE i n response to the two hormones found in this report and previously (Sm ith, C. J., Vasta, V., Degerman, E., Belfrage, P., and Manganiello, V. C. (1991) J. Biol. Chem. 266, 13385-13390) is proposed to reflect cro ss-talk between their respective signal transduction pathways at the l evel of the cGI PDE serine protein kinase or upstream regulatory compo nent(s).