Lawrence J C, Hiken J F, James D E
Department of Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1990 Feb 5;265(4):2324-32.
Phosphorylation of the insulin-regulatable glucose transporter (IRGT) is increased by incubating rat adipocytes with isoproterenol or by incubating microsomal membranes with cAMP-dependent protein kinase. To attempt to locate the sites of phosphorylation, the IRGT (apparent Mr = 46,000) was immunoprecipitated from 32P-labeled adipocytes and cleaved with CNBr or trypsin. Essentially all of the 32P could be recovered in a single CNBr fragment, denoted CB-T (Mr = 8,000), which bound a polyclonal antibody (R820) against a peptide having the sequence of the last 12 amino acids in the COOH terminus of the IRGT. 32P-Labeling of the IRGT was also confined to CB-T when membranes were incubated with [gamma-32P]ATP and cAMP-dependent protein kinase. Isoproterenol increased phosphorylation of CB-T, but insulin was without effect. To resolve phosphorylation sites further, IRGT from 32P-labeled cells was subjected to exhaustive proteolysis with trypsin. Samples were applied to a C-18 column, and 32P-labeled fragments were resolved into three peak fractions by elution with an increasing gradient of acetonitrile. [32P]Phosphoserine was the only phosphoamino acid detected in any of the peaks. Peak III contained approximately 80% of the 32P and was increased by isoproterenol. Almost all of the 32P introduced by cAMP-dependent protein kinase in vitro eluted in Peak III. In all cases, the 32P-labeled species in Peak III were quantitatively immunoprecipitated by R820. Digesting the peptide(s) in Peak III with V8 protease generated a single peak of 32P which eluted at lower acetonitrile than Peak III and contained 32P-labeled species that did not interact with R820. Automated Edman degradation indicated that the serine residue in Peak III phosphorylated by cAMP-dependent protein kinase was the 3rd or 4th residue from the NH2 terminus of the peptide. These findings indicate that phosphorylation of the IRGT is restricted to the presumed intracellular domain at the COOH terminus and that Ser488 is a major site phosphorylated both by cAMP-dependent protein kinase in vitro and in response to isoproterenol in vivo.
通过用异丙肾上腺素孵育大鼠脂肪细胞或用依赖于cAMP的蛋白激酶孵育微粒体膜，可增加胰岛素可调节的葡萄糖转运体（IRGT）的磷酸化。为了尝试定位磷酸化位点，从经32P标记的脂肪细胞中免疫沉淀出IRGT（表观分子量=46,000），并用溴化氰或胰蛋白酶进行切割。基本上所有的32P都可以在一个单一的溴化氰片段中回收，该片段称为CB-T（分子量=8,000），它能结合一种针对IRGT羧基末端最后12个氨基酸序列的肽的多克隆抗体（R820）。当膜与[γ-32P]ATP和依赖于cAMP的蛋白激酶一起孵育时，IRGT的32P标记也局限于CB-T。异丙肾上腺素增加了CB-T的磷酸化，但胰岛素没有作用。为了进一步解析磷酸化位点，将来自32P标记细胞的IRGT用胰蛋白酶进行彻底的蛋白水解。将样品应用于C-18柱，通过用乙腈浓度递增的梯度洗脱，将32P标记的片段分离为三个峰级分。[32P]磷酸丝氨酸是在任何一个峰中检测到的唯一磷酸氨基酸。峰III包含了大约80%的32P，并且被异丙肾上腺素增加。几乎所有在体外由依赖于cAMP的蛋白激酶引入的32P都在峰III中洗脱。在所有情况下，峰III中的32P标记物种都被R820定量免疫沉淀。用V8蛋白酶消化峰III中的肽产生了一个单一的32P峰，其在比峰III更低的乙腈浓度下洗脱，并且包含不与R820相互作用的32P标记物种。自动Edman降解表明，被依赖于cAMP的蛋白激酶磷酸化的峰III中的丝氨酸残基是该肽氨基末端的第3或第4个残基。这些发现表明，IRGT的磷酸化局限于羧基末端假定的细胞内结构域，并且Ser488是在体外被依赖于cAMP的蛋白激酶以及在体内对异丙肾上腺素反应时磷酸化的主要位点。
