Expression, Structure/function And Regulation Of Phospho

Heart, Lung, And Blood Institute

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Abstract

Previous studies have indicated that PDE3B appears, or markedly increases, during differentiation of 3T3-L1 adipocytes. Activation of PDE3B in adipocytes, hepatocytes, pancreatic beta cells and PDE3A (presumably) in oocytes is important in effects of insulin on inhibition of lipolysis and glycogenolysis and IGF-1 in inhibition of insulin secretion and stimulation of oocyte maturation.[unreadable] [unreadable] Incubation of 3T3-L1 preadipocytes with isobutylmethylxanthing (IBMX), dexamethasone (Dex), and insulin, alone or in combination, demonstrated that IBMX, which increased CREB phosphorylation, was the predominant regulator of PDE3B expression. Real-time PCR and immunoblotting indicated that, in 3T3-L1 preadipocytes, IBMX-stimulated induction of PDE3B mRNA and protein was markedly inhibited by dominant-negative CREB proteins. By transfecting preadipocytes, differentiating preadipocytes, and HEK293A cells with luciferase reporter vectors containing different fragments of the 5'-flanking region of the PDE3B gene, we identified a distal promoter that contained canonical cis-acting cAMP response elements (CRE), and a proximal, GC-rich promoter region, which contained atypical CRE elements. Mutation of the CRE sequences dramatically reduced distal promoter activity; H89 inhibited IBMX-stimulated CREB phosphorylaiton and proximal and distal promoter activities. Distal promoter activity was stimulated by IBMX and phorbol ester (PMA) in Raw264.7 monocytes, but only by IBMX in 3T3-L1 preadipocytes. Chromatin immunoprecipitation analyses with specific antibodies against CREB, phospho-CREB, and CBP/p300 showed that these proteins associated with both distal and proximal promoters, and that interaction of phospho-CREB, the active form of CREB, with both PDE3B promoter regions was increased in IBMX-treated preadipocytes. These results indicate that CRE elements in distal and proximal promoter regions and activation of CREB proteins play a crucial role in transcriptional regulation of PDE3B expression during preadipocyte differentiation.[unreadable] [unreadable] Fractionation of membranes from 3T3-L1 adipocytes revealed that PDE3B was associated with plasma membrane (PM) and endoplasmic reticulum (ER)/Golgi fractions, and that insulin-induced phosphorylation/activation of PDE3B was greater in internal membranes than PM fractions. Insulin increased tyrosine phosphorylaiton of insulin receptor substrate-1 (IRS-1), and activated IRS-1-associated phosphatidylinositol 3-kinase (P13-K) and protein kinase B (PKB) in both intracellular membrane and cytosolic fractions. Insulin also induced formation of large macromolecular complexes,separated during gel filtration (Superose 6 columns) of solubilized membranes, which apparently contain phosphorylated/activated PDE3B and several signaling molecules potentially involved in its activation by insulin, e.g., IRS-1, P13-K p85, PKB, HSP-90, 14-2-2. Confocal microscopy also indicated colocalization of PDE3B and PKB. Wortmannin inhibited insulin-induced assembly of macromolecular complexes, and insulin-induced phosphorylaiton/activation of PKB and PDE3B, as well as their co-immunoprecipitaiton and interaction during Superose 6 chromatography. Recombinant mouse (M)PDE3B co-immunoprecipitated and co-eluted during Superose 12 chromatography to a greater extent with recombinant p-PKB (phosphorylated/activated PKB) than dephospho PKB or p-deltaPKB (p-PKB lacking its PH domain). Co-immunoprecipitation of truncated recombinant MPDE3B proteins and p-PKB suggested that structural determinants for their interaction seem to reside in, or be regulated by, the N-terminal portion of MPDE3B.[unreadable] [unreadable] Phosphodiestease 3B has previously been shown to be activated by insulin and cAMP increasing hormones in adipocytes and hepatocytes. Hormone-induced phosphorylation of the enzyme has mainly been studied in adipocytes, where insulin and cAMP increasing hormones have been shown to induce activation and serine phhhosphorylation of PDE3B. S273 and S296 have been shown to be targets for hormone-induced effects on adipocyte PDE3B. Also, indications for additional sites have been reported for the adipocyte PDE3B whereas no information with regard to sites phosphorylated in the 135 kDA hepatocyte PDE3B has been presented. We used an adenoviral system to express recombinant flag-tagged PDE3B in primary rat adipocytes (Degerman and Swedish collaborators) and H411E hepatoma cells. The phosphorylation of recombinant PDE3B from primary rat adipocytes and H411E hepatoma cells after treatment with insulin, cAMP increasing agents or the phosphatase inhibitor, calyculin A were analysed by two dimensional (2-D) tryptic phosphopeptide mapping and mass spectrometry. We found that (1) Recombinant PDE3B has similar characteristics to endogenous adipocyte PDE3B regarding intracellular localizaiton, regulation by hormones and regulation of lipolysis in adipocytes. (2) PDE3B is multisite phosphorylated in adipocytes as well as hepatocytes in response to insulin, cAMP increasing agents and calyculin A. (3) The pattern of PDE3B phosphorylation show striking similarities both between different stimuli and between cell types. (4) Several PDE3B phosphorylation sites could be identified: Ser273, Ser296, S421, S424/5, S474 and S536 in adipocyte PDE3B (Degerman, Lund) and S273, S277, S296, S421, S474, S507, S536 in hepatocyte PDE3B (Fales, NHLBI).[unreadable] [unreadable] Since cyclic nucleotide phosphodiesterase 3B (PDE3B) is highly expressed in adipocytes, hepatocytes and pancreatic beta cells, it role in regulating energy homeostasis was studied in Pde3b-/- (KO) mice. In KO adipocytes, adipocyte size decreased, insulin-stimulated phosphorylation of protein kinase B (PKB) and activation of glucose uptake was unchanged, catecholamine-stimulated lipolysis and insulin-stimulated lipogenesis was enhanced, but insulin inhibition of catecholamine-stimulated lipolysis was blocked. Glucose, alone or in combination with glucagon-like peptide-1, increased insulin-secretion to a greater extent in isolated pancreatic KO islets, although pancreatic islet size and morphology and levels of immunoreactive insulin and glucagon were unchanged. The Beta3-adrenergic agonist CL 316,243 (CL)increased lipolysis and serum insulin to a greater extent in KO mice, but there was less reduction in blood glucose CL-treated KO mice. INsulin tolerance tests indicated insulin resistance in KO mice, with the liver appearing as an important site of alterations in insulin-sensitive glucose production, as judged by hyperinsulinemic-euglycemic clamps. In KO mice, liver triglyceride and cAMP content were increased, and liver content/phosphorylation state of several insulin-signaling, gluconeogenic and inflammation/stress-related components were altered. Thus, PDE3B may be important in regulating certain cAMP-signaling pathways, including lipolysis, insulin-induced antilipolysis, and cAMP-mediated insulin-ssecretion. Alteres expression/regulation of PDE3B in different tissues may contribute to dysregulated metabolic states, including systemic insulin resistance.