O-5: Identification of the site in the cGMP-inhibited phosphodiesterase phosphorylated in adipocytes in response to insulin and isoproterenol

Abstract

Insulin controls cell metabolism and proliferation through several diverging pathways which are only partially understood. One important metabolic effect is to antagonize hormone-activated lipolysis in adipose tissue, brought about mainly via insulin-mediated activation of a membrane-associated cGMP-inhibited phosphodiesterase [cGI PDE] which leads to a reduction in cAMP, a decrease in cAMP dependent protein kinase (A-kinase) activity and a lowering of the phosphorylation and thereby the activity of the hormone-sensitive lipase, the rate limiting enzyme in the regulation of lipolysis (I).Insulin as well as cAMP-increasing agents induce serine phosphorylation and activation of cGI PDE in rat adipocytes (2,3). In the presence of both isoproterenol and insulin the phosphorylation/activation of cGI PDE is more than additive (3), suggesting crosstalk between the two signal transduction pathways (Fig.1). NSULIN ECEPTO IRS1I2 P13 CGPDE -T AMP .4ATP (A KINASE) 4 LIPOLYSIS beta adrenergic eecepîoe Fig. I. Working hypothesis for the antilipolytic action of insulin.To further dissect the antilipolytic signalling pathway controlling the cGl PDE we used two-dimensional tryptic phosphopeptide mapping and other methods to analyze the site(s) phosphorylated in cGI PDE in 32P-labelled rat aidpocytes incubated with insulin, isoproterenol or both. Since the amount of labelled phosphopeptides that could be obtained from experiments with intact cells was far less than that required for amino acid sequencing, the phosphorylation site(s) was identified by comparison of properties of phosphopeptides from cGT PDE phosphorylated in adipocytes. or from recombinant cGl PDE expressed in NIE-1 3006 murine fibroblasts phosphorylated by A-kinase, with peptides synthesized on the basis of the deduced sequence of rat adipocyte cGI PDE and phosphorylated by A-kinase. Peptides chosen for these studies contained consensus sequences for phosphorylation by A-kinase since preliminary results indicated that the site(s) phosphorylated during incubation of adipocytes with insulin or isoproterenol was located in the same tryptic phosphopeptide.Here we demonstrate, by two-dimensional phosphopeptide mapping, that the major phosphopeptide generated by trypsin, or trypsin followed by Asp-N protease digestion of 3P-cGl PDE phosphorylated in adipocytes in response to isoproterenol and/or insulin, in each case co-migrates with lo