EVIDENCE THAT INSULIN AND ISOPRENALINE ACTIVATE THE CGMP-INHIBITED LOW-KM CAMP PHOSPHODIESTERASE IN RAT FAT-CELLS BY PHOSPHORYLATION

被引:172
作者
DEGERMAN, E [1 ]
SMITH, CJ [1 ]
TORNQVIST, H [1 ]
VASTA, V [1 ]
BELFRAGE, P [1 ]
MANGANIELLO, VC [1 ]
机构
[1] NHLBI, CELLULAR METAB LAB, BETHESDA, MD 20892 USA
关键词
cAMP-dependent protein kinase; Cilostamide; Immunoprecipitation; Lipolysis;
D O I
10.1073/pnas.87.2.533
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Incubation of intact rat fat cells with maximally effective concentrations of insulin (1 nM, 12 min) or isoprenaline (300 nM, 3 min) increased particulate cGMP- and cilostamide-inhibited, low-Km cAMP phosphodiesterase (cAMP-PDE) activity by about 50% and 100%, respectively. In 32P-labeled cells, these agents induced serine 32P-phosphorylation of a 135-kDa particulate protein and, to a variable and lesser extent, a 44-kDa protein, which were selectively immunoprecipitated by anti-cAMP-PDE, as analyzed by SDS/PAGE and autoradiography. In the absence of hormonal stimulation, little phosphorylation was detected (<10% of that with the hormones). The two phosphoproteins were identified as cAMP-PDE or a closely related molecule (in the case of the 44-kDa species, perhaps a proteolytic fragment) since (i) amounts of 32P in the immunoprecipitated 135-kDa protein paralleled enzyme inactivation, (ii) prior incubation of the anti-cAMP-PDE with the pure rat or bovine enzyme selectively blocked the immunoprecipitation of the phosphoproteins, (iii) 135- and 44-kDa proteins reacted with the anti-cAMP-PDE on Western immunoblots, and (iv) the two phosphoproteins copurified with cAMP-PDE activity through DEAE-Sephacel chromatography and were isolated by highly selective affinity chromatography on cilostamide-agarose. Thus, in fat cells, catecholamine- and insulin-induced activation of the cAMP-PDE may be mediated via phosphorylation by cAMP-dependent protein kinase and an insulin-activated serine protein kinase, respectively.
引用
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页码:533 / 537
页数:5
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