Insulin-induced formation of macromolecular complexes involved in activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and its interaction with PKB
Insulin-induced formation of macromolecular complexes involved in activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and its interaction with PKB
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DOI：
10.1042/bj20060960
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发表时间：
2007-06-01
影响因子：
4.1
通讯作者：
Manganiello, Vincent C.
中科院分区：
文献类型：
--
作者：
Ahmad, Faiyaz;Lindh, Rebecka;Manganiello, Vincent C.
3T3-L1脂肪细胞膜的分级显示，PDE3B(磷酸二酯酶3B)与质膜(PM)和内质网(ER)/高尔基组分有关，胰岛素诱导的PDE3B在内膜的磷酸化/活化程度高于PM组分，并且PDE3B在膜组分之间没有明显的移位。胰岛素还诱导形成大的大分子复合体，通过凝胶过滤(Superose 6柱)分离，其中明显含有磷酸化/激活的PDE3B和可能参与其被胰岛素激活的信号分子，如IRS-1(胰岛素受体底物-1)，IRS-2，PI3K P85[PI3K的P85亚单位(磷酸肌醇3-激酶)]，PKB(蛋白激酶B)，HSP-90(热休克蛋白90)和14-3-3。重组鼠(M)PDE3B和M3B Delta 604(MPDE3B缺失N-末端604个氨基酸)的全长表达表明，MPDE3B的N-末端区域是胰岛素诱导的PDE3B激活和募集所必需的。PDE3B的siRNA(小干扰RNA)敲除表明PDE3B不是胰岛素诱导的复合体形成所必需的。Wortmannin抑制胰岛素诱导的大分子复合体的组装，抑制PKB和PDE3B的磷酸化/活化，以及它们的免疫共沉淀。另一种PI3K抑制剂LY294002和酪氨酸激酶抑制剂Genistein也能抑制胰岛素诱导的PDE3B活化及其与PKB的免疫共沉淀。共聚焦显微镜显示PDE3B和PKB共定位。在Superose 12层析中，重组MPDE3B与重组pPKB(磷酸化/活化的PKB)共沉淀和共洗脱的程度高于去磷酸化/活化的PKB或p-Delta PKB[pPKB缺乏其PH结构域(Pleckstrin同源域)]。截短的重组MPDE3B蛋白和pPKB不能有效地共沉淀，这表明它们相互作用的结构决定因素位于MPDE3B的N末端部分，或受其调控。PDE3B在大分子复合体中的募集可能对胰岛素调节特定的cAMP池和信号通路至关重要，例如脂解作用。
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 (phospho-inosifide 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 M3B Delta 604 (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 coimmunoprecipitation. 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-Delta PKB [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.