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

Faiyaz Ahmad, Rebecka Lindh, Yan Tang, Marie Weston, Eva Degerman, Vincent C. Manganiello

Research output: Contribution to journalArticle

42 Citations (Scopus)

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 M3BΔ604 (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-ΔPKB [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.

Original languageEnglish (US)
Pages (from-to)257-268
Number of pages12
JournalBiochemical Journal
Volume404
Issue number2
DOIs
StatePublished - Jun 1 2007
Externally publishedYes

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Type 3 Cyclic Nucleotide Phosphodiesterases
Macromolecular Substances
Proto-Oncogene Proteins c-akt
Cyclic Nucleotides
Chemical activation
Insulin
1-Phosphatidylinositol 4-Kinase
Phosphatidylinositols
Phosphorylation
Membranes
Cell membranes
Immunoprecipitation
Phosphotransferases
Cell Membrane
14-3-3 Proteins
HSP90 Heat-Shock Proteins
Insulin Receptor Substrate Proteins
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
Genistein
Confocal microscopy

Keywords

  • Confocal microscopy
  • Gel filtration
  • Insulin
  • Macromolecular complex
  • Phosphodiesterase 3B (PDE3B)
  • Phosphorylation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Insulin-induced formation of macromolecular complexes involved in activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and its interaction with PKB. / Ahmad, Faiyaz; Lindh, Rebecka; Tang, Yan; Weston, Marie; Degerman, Eva; Manganiello, Vincent C.

In: Biochemical Journal, Vol. 404, No. 2, 01.06.2007, p. 257-268.

Research output: Contribution to journalArticle

Ahmad, Faiyaz ; Lindh, Rebecka ; Tang, Yan ; Weston, Marie ; Degerman, Eva ; Manganiello, Vincent C. / Insulin-induced formation of macromolecular complexes involved in activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and its interaction with PKB. In: Biochemical Journal. 2007 ; Vol. 404, No. 2. pp. 257-268.
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KW - Confocal microscopy

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KW - Phosphodiesterase 3B (PDE3B)

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