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

被引:44
作者
Ahmad, Faiyaz
Lindh, Rebecka
Tang, Yan
Weston, Marie
Degerman, Eva
Manganiello, Vincent C.
机构
[1] NHLBI, Pulm Crit Care Med Branch, NIH, Bethesda, MD 20892 USA
[2] Lund Univ, Dept Expt Med Sci, S-22184 Lund, Sweden
关键词
confocal microscopy; gel filtration; insulin; macromolecular complex; phosphodiesterase 3B (PDE3B); phosphorylation;
D O I
10.1042/BJ20060960
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:257 / 268
页数:12
相关论文
共 37 条
[1]  
Ahmad F, 1999, J IMMUNOL, V162, P4864
[2]   Cyclic nucleotide phosphodiesterase 3B is a downstream target of protein kinase B and may be involved in regulation of effects of protein kinase B on thymidine incorporation in FDCP2 cells [J].
Ahmad, F ;
Cong, LN ;
Holst, LS ;
Wang, LM ;
Landstrom, TR ;
Pierce, JH ;
Quon, MJ ;
Degerman, E ;
Manganiello, VC .
JOURNAL OF IMMUNOLOGY, 2000, 164 (09) :4678-4688
[3]  
Ahmad Faiyaz, 2005, V307, P93
[4]   Insulin signaling and the regulation of glucose transport [J].
Chang, L ;
Chiang, SH ;
Saltiel, AR .
MOLECULAR MEDICINE, 2004, 10 (7-12) :65-71
[5]   The 47 kDa Akt substrate associates with phosphodiesterase 3B and regulates its level in adipocytes [J].
Chavez, JA ;
Gridley, S ;
Sano, H ;
Lane, WS ;
Lienhard, GE .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2006, 342 (04) :1218-1222
[6]   Alterations in regulation of energy homeostasis in cyclic nucleotide phosphodiesterase 3B-null mice [J].
Choi, Young Hun ;
Park, Sunhee ;
Hockman, Steven ;
Zmuda-Trzebiatowska, Emilia ;
Svennelid, Fredrik ;
Haluzik, Martin ;
Gavrilova, Oksana ;
Ahmad, Faiyaz ;
Pepin, Laurent ;
Napolitano, Maria ;
Taira, Masato ;
Sundler, Frank ;
Holst, Lena Stenson ;
Degerman, Eva ;
Manganiello, Vincent C. .
JOURNAL OF CLINICAL INVESTIGATION, 2006, 116 (12) :3240-3251
[7]   Role of caveolin-1 in the modulation of lipolysis and lipid droplet formation [J].
Cohen, AW ;
Razani, B ;
Schubert, W ;
Williams, TM ;
Wang, XB ;
Iyengar, P ;
Brasaemle, DL ;
Scherer, PE ;
Lisanti, MP .
DIABETES, 2004, 53 (05) :1261-1270
[8]   Leptin-mediated activation of human platelets: involvement of a leptin receptor and phosphodiesterase 3A-containing cellular signaling complex [J].
Elbatarny, HS ;
Maurice, DH .
AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM, 2005, 289 (04) :E695-E702
[9]   Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase [J].
Feng, JH ;
Park, J ;
Cron, P ;
Hess, D ;
Hemmings, BA .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2004, 279 (39) :41189-41196
[10]  
Francis SH, 2001, PROG NUCLEIC ACID RE, V65, P1