Identification of the Site in the cGMP-inhibited Phosphodiesterase Phosphorylated in Adipocytes in Response to Insulin and Isoproterenol

Abstract

Stimulation of rat adipocytes with insulin and isoproterenol results in serine phosphorylation and activation of the adipocyte cGMP-inhibited 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. Natl. Acad. Sci. U. S. A. 87, 533-537). Here we demonstrate, by two-dimensional phosphopeptide mapping, that the major phosphopeptide generated by trypsin, or trypsin followed by Asp-N protease digestion of [ Insulin controls cell metabolism and proliferation through several multicomponent diverging pathways, which are only partially understood (1-3). One important and well known 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, 1 referred to as the PDE 3 gene family; Ref. 4), which leads to a reduction in cAMP, a decrease in cAMPdependent protein kinase (protein kinase A) 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 (5-11).Insulin and cAMP-increasing agents induce serine phosphorylation and activation of cGI PDE in rat adipocytes (12, 13). An insulin-stimulated cGI PDE serine protein kinase activity in rat adipocytes has previously been partially characterized (14,15). In the presence of both isoproterenol and insulin, the phosphorylation/activation of cGI PDE is more than additive (13, 16), suggesting cross-talk between the two signal transduction pathways.To further dissect the components of the antilipolytic signaling pathway controlling the cGI PDE, we used two-dimensional tryptic phosphopeptide mapping and other methods to analyze the site(s) phosphorylated in this enzyme in 32 P-labeled rat adipocytes incubated with insulin, isoproterenol, or both. Since the amount of labeled 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 cGI PDE phosphorylated in adipocytes, or from recombinant cGI PDE expressed in NIH 3006 human fibroblasts 2 (rcGI PDE) phosphorylated by protein kinase A, with peptides synthesized on the basis of the deduced sequence of rat adipocyte cGI PDE (17) and phosphorylated by protein kinase A. Peptides chosen for these studies contained consensus sequences for phosphorylation by protein kinase A (18), since preliminary results indicated that the site(s) phosphorylated during incubation of adipocytes with insulin or isoproterenol was located in the same tryptic phosphopeptide.EXPERIMENTAL PROCEDURES

Mechanisms of inhibition of lipolysis by insulin, vanadate and peroxovanadate in rat adipocytes

“…PDE3B has previously been identified as an important component in the anti-lipolytic pathway of insulin [12]. Phosphorylation of PDE3B (serine-302) has been shown to be associated with PDE3B activation [19]. Incubation of $#P-labelled adipocytes with 1 nM insulin, 5 mM vanadate, 0.01 mM pV or 0.1 mM pV resulted in phosphorylation of PDE3B (Figure 6).…”

“…The piece of membrane containing phosphorylated IRS-1 was excised and the phosphoprotein was either subjected to phosphoamino acid analysis or digested with trypsin. Tryptic phosphopeptides were subjected to two-dimensional phosphopeptide mapping with the Hunter thin-layer electrophoresis (TLE) apparatus (HTLE-7000 ; C.B.S Scientific Co.) in the first dimension and TLC in the second dimension as described previously [19]. Phosphoamino acid analysis was performed as described by Boyle et al [20].…”

Phosphorylation and activation of phosphodiesterase type 3B (PDE3B) in adipocytes in response to serine/threonine phosphatase inhibitors: deactivation of PDE3B in vitro by protein phosphatase type 2A

“…Two-dimensional tryptic phosphopeptide mapping of PDE3B was performed essentially as described previously [41] with some modification [9]. Immuno-isolated [$#P]PDE3B was subjected to SDS\PAGE and Western blotting.…”

“…The PDE3 family has two members, PDE3A and PDE3B, that are expressed in a tissue-specific manner [6]. PDE3B is activated and phosphorylated in adipocytes [7][8][9] and hepatocytes [10] in response to insulin and cAMP-increasing hormones and in FDCP2 myeloid cells in response to insulin-like growth factor I (IGF-I ; F. Ahmad, L. N. Cong, L. Stenson Holst, L.-M. Wang, J. Pierce, T. Rahn Landstro$ m, M. Quon, E. Degerman and V. Manganiello, unpublished work).…”

“…The cAMPmediated activation of PDE3B, presumably catalysed by protein kinase A, has been suggested to be important in feedback regulation of cAMP [18]. Hormone-induced regulation of PDE3B has been studied extensively in primary rat adipocytes where phosphorylation of serine 302 [6][7][8][9] and activation of PDE3B is thought to be the major mechanism whereby insulin antagonizes protein kinase Amediated phosphorylation and activation of hormone-sensitive lipase and lipolysis [16,17,19,20]. Although phosphatidylinositol 3-kinase is most probably involved as an upstream regulator of PDE3B in the insulin-signalling pathway [21], the kinase that phosphorylates and activates PDE3B directly has not been identified.…”