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Insulin‐induced formation of macromolecular complexes involved in activation of Phosphodiesterase3B (PDE3B) in 3T3‐L1 adipocytes
Author(s) -
Khan Faiyaz Ahmad,
Nilsson Rebecka,
Tang Yan,
Degerman Eva,
Manganiello Vincent C
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a997-a
Subject(s) - insulin , phosphorylation , insulin receptor , tyrosine phosphorylation , microbiology and biotechnology , biochemistry , protein kinase b , autophosphorylation , chemistry , biology , protein kinase a , insulin resistance , endocrinology
PDE3B is an important regulator of several insulin‐ and cAMP‐dependent signaling pathways. Fractionation of membranes from adipocytes revealed that PDE3B was associated with plasma membrane (PM) and internal membrane (IM) fractions, and that insulin‐induced phosphorylation/activation of PDE3B was greater in IM than PM fractions. Insulin increased tyrosine phosphorylation of IRS‐1, and activated IRS‐1‐associated PI3‐K and PKB in both membrane and cytosolic fractions. Insulin also induced formation of large macromolecular complexes (LMCs), separated during gel filtration (Superose 6) of solubilized membranes, which apparently contain phosphorylated/activated PDE3B and several signaling molecules potentially involved in its activation by insulin, e.g. IRS‐1, PI3‐K, PKB, HSP‐90, and 14‐3‐3. Expression of full length recombinant Flag‐tagged murine (M)PDE3B (M3B) and M3BD604(M3B lacking N‐terminal 604 amino acids) indicated that N‐terminal region of M3B (which contains sites phosphorylated by insulin) was necessary for insulin‐induced activation and recruitment of PDE3B into LMCs. M3BD604 was not activated by insulin, was not recruited into LMCs, and did not interfere with recruitment of endogenous PDE3B. siRNA knockdown of PDE3B indicated that PDE3B was not required for formation of insulin‐induced LMCs. Wortmannin inhibited insulin‐induced LMCs, as well as insulin‐induced phosphorylation/activation of PKB and PDE3B, their co‐immunoprecipitation, and interactions during Superose 6 chromatography. Insulin‐stimulated complex formation of PDE3B and other signaling molecules may be critical for down stream events regulating adipocyte metabolism.