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Insulin-induced formation of macromolecular complexes involved in activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and its interaction with PKB.

ABSTRACT: Fractionation of 3T3-L1 adipocyte membranes revealed that PDE3B (phosphodiesterase 3B) was associated with PM (plasma membrane) and ER (endoplasmic reticulum)/Golgi fractions, that insulin-induced phosphorylation/activation of PDE3B was greater in internal membranes than PM fractions, and that there was no significant translocation of PDE3B between membrane 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 signalling molecules potentially involved in its activation by insulin, e.g. IRS-1 (insulin receptor substrate-1), IRS-2, PI3K p85 [p85-subunit of PI3K (phosphoinositide 3-kinase)], PKB (protein kinase B), HSP-90 (heat-shock protein 90) and 14-3-3. Expression of full-length recombinant FLAG-tagged murine (M) PDE3B and M3BDelta604 (MPDE3B lacking N-terminal 604 amino acids) indicated that the N-terminal region of MPDE3B was necessary for insulin-induced activation and recruitment of PDE3B. siRNA (small interfering RNA) knock-down of PDE3B indicated that PDE3B was not required for formation of insulin-induced complexes. Wortmannin inhibited insulin-induced assembly of macromolecular complexes, as well as phosphorylation/activation of PKB and PDE3B, and their co-immunoprecipitation. Another PI3K inhibitor, LY294002, and the tyrosine kinase inhibitor, Genistein, also inhibited insulin-induced activation of PDE3B and its co-immunoprecipitation with PKB. Confocal microscopy indicated co-localization of PDE3B and PKB. Recombinant MPDE3B co-immunoprecipitated, and co-eluted during Superose 12 chromatography, to a greater extent with recombinant pPKB (phosphorylated/activated PKB) than dephospho-PKB or p-DeltaPKB [pPKB lacking its PH domain (pleckstrin homology domain)]. Truncated recombinant MPDE3B proteins and pPKB did not efficiently co-immunoprecipitate, suggesting that structural determinants for their interaction reside in, or are regulated by, the N-terminal portion of MPDE3B. Recruitment of PDE3B in macromolecular complexes may be critical for regulation of specific cAMP pools and signalling pathways by insulin, e.g. lipolysis.

SUBMITTER: Ahmad F

PROVIDER: S-EPMC1868803 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Insulin-induced formation of macromolecular complexes involved in activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and its interaction with PKB.

Ahmad Faiyaz F Lindh Rebecka R Tang Yan Y Weston Marie M Degerman Eva E Manganiello Vincent C VC

The Biochemical journal 20070601 2

Fractionation of 3T3-L1 adipocyte membranes revealed that PDE3B (phosphodiesterase 3B) was associated with PM (plasma membrane) and ER (endoplasmic reticulum)/Golgi fractions, that insulin-induced phosphorylation/activation of PDE3B was greater in internal membranes than PM fractions, and that there was no significant translocation of PDE3B between membrane fractions. Insulin also induced formation of large macromolecular complexes, separated during gel filtration (Superose 6 columns) of solubil ...[more]

PMID: 17324123

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Project description:Phosphodiesterase type 3B (PDE3B) has been shown to be activated and phosphorylated in response to insulin and hormones that increase cAMP. In order to study serine/threonine protein phosphatases involved in the regulation of rat adipocyte PDE3B, we investigated the phosphorylation and activation of PDE3B in vivo in response to phosphatase inhibitors and the dephosphorylation and deactivation of PDE3B in vitro by phosphatases purified from rat adipocyte homogenates. Okadaic acid and calyculin A induced dose- and time-dependent activation of PDE3B. Maximal effects were obtained after 30 min using 1 microM okadaic acid (1.8-fold activation) and 300 nM calyculin A (4-fold activation), respectively. Tautomycin and cyclosporin A did not induce activation of PDE3B. Incubation of adipocytes with 300 nM calyculin A inhibited protein phosphatase (PP) 1 and PP2A completely. Okadaic acid (1 microM) reduced PP2A activity by approx. 50% but did not affect PP1 activity, and 1 microM tautomycin reduced PP1 activity by approx. 60% but PP2A activity by only 11%. This indicates an important role for PP2A in the regulation of PDE3B. Furthermore, rat adipocyte PDE3B phosphatase activity co-purified with PP2A but not with PP1 during MonoQ chromatography. As compared with insulin, okadaic acid and calyculin A induced phosphorylation of PDE3B by 2.8- and 14-fold respectively, whereas tautomycin and cyclosporin A had no effect. Both calyculin A and okadaic acid induced phosphorylation on serine 302, the site known to be phosphorylated on PDE3B in response to insulin and isoproterenol (isoprenaline), as well as on sites not identified previously. In summary, PP2A seems to be involved in the regulation of PDE3B in vivo and can act as a PDE3B phosphatase in vitro. In comparison with insulin, calyculin A induced a dramatic activation of PDE3B and both calyculin A and okadaic acid induced phosphorylation on additional sites, which could have a role in signalling pathways not yet identified.

Dataset Information

Insulin-induced formation of macromolecular complexes involved in activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and its interaction with PKB.

Publications

Insulin-induced formation of macromolecular complexes involved in activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and its interaction with PKB.

Similar Datasets

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