Haystead T A, Campbell D G, Hardie D G
Biochemistry Department, Dundee University, Scotland.
Eur J Biochem. 1988 Aug 1;175(2):347-54. doi: 10.1111/j.1432-1033.1988.tb14203.x.
We have examined the sites phosphorylated on acetyl-CoA carboxylase in response to insulin in isolated adipocytes. Two tryptic peptides derived from the enzyme become more radioactive after treatment of 32P-labelled cells with insulin. One of these (T4a) accounts for a large part of the total increase in phosphate observed after insulin treatment, and comigrates with the peptide containing the sites phosphorylated in vitro by casein kinase-2. The other may correspond to the 'I' site peptide originally described by Brownsey and Denton in 1982: labelling of this peptide is stimulated at least threefold by insulin treatment, but it is a minor phosphopeptide and, even after insulin treatment, accounts for only about 2.5% of the enzyme-bound phosphate (equivalent to less than 0.1 mol phosphate/mol 240-kDa subunit). Two other major tryptic phosphopeptides (T1 and T4b) labelled in adipocytes do not change significantly in response to insulin, and comigrate with peptides containing sites phosphorylated in vitro by cyclic-AMP-dependent protein kinase and calmodulin-dependent multiprotein kinase respectively. We have sequenced peptides T4a and T4b from acetyl-CoA carboxylase derived from control and insulin-treated adipocytes, and also after phosphorylation in vitro with casein kinase-2 and the calmodulin-dependent multiprotein kinase. The results show that T4a and T4b are forms of the same peptide containing phosphate groups on different serine residues: Phe-Ile-Ile-Gly-Ser4-Val-Ser5-Gln-Asp-Asn-Ser6-Glu-Asp -Glu-Ile-Ser-Asn-Leu-. Site 5 was phosphorylated by the calmodulin-dependent protein kinase and site 6 by casein kinase-2. Migration in the T4a position was exclusively associated with phosphorylation in site 6, irrespective of the presence of phosphate in sites 4 and 5. Sites 5 and 6 were partially phosphorylated in control adipocytes, and there were also small amounts of phosphate in site 4. On stimulation with insulin, phosphorylation appeared to occur primarily at site 6, thus accounting for the increase in 32P-labelling of T4a. We were unable to isolate sufficient quantities of the other insulin-sensitive peptide to determine its sequence. Our results are consistent with the idea that insulin activates either casein kinase-2, or a protein kinase which has the same specificity as casein kinase-2. The function of this modification is not clear, since phosphorylation by casein kinase-2 has no direct effect on acetyl-CoA carboxylase activity.
我们研究了分离的脂肪细胞中乙酰辅酶A羧化酶上响应胰岛素而发生磷酸化的位点。在用胰岛素处理32P标记的细胞后，该酶产生的两种胰蛋白酶肽变得更具放射性。其中一种（T4a）占胰岛素处理后观察到的总磷酸盐增加量的很大一部分，并且与含有酪蛋白激酶-2体外磷酸化位点的肽一起迁移。另一种可能对应于布朗西和丹顿于1982年最初描述的“I”位点肽：胰岛素处理可使该肽的标记至少增加三倍，但它是一种次要的磷酸肽，即使在胰岛素处理后，也仅占酶结合磷酸盐的约2.5%（相当于每摩尔240 kDa亚基中磷酸盐含量少于0.1摩尔）。脂肪细胞中标记的另外两种主要胰蛋白酶磷酸肽（T1和T4b）对胰岛素的反应没有明显变化，并且分别与含有环磷酸腺苷依赖性蛋白激酶和钙调蛋白依赖性多蛋白激酶体外磷酸化位点的肽一起迁移。我们对来自对照和胰岛素处理的脂肪细胞的乙酰辅酶A羧化酶的肽T4a和T4b进行了测序，并且在酪蛋白激酶-2和钙调蛋白依赖性多蛋白激酶体外磷酸化后也进行了测序。结果表明，T4a和T4b是同一肽的不同形式，在不同的丝氨酸残基上含有磷酸基团：苯丙氨酸-异亮氨酸-异亮氨酸-甘氨酸-丝氨酸4-缬氨酸-丝氨酸5-谷氨酰胺-天冬氨酸-天冬酰胺-丝氨酸6-谷氨酸-天冬氨酸-谷氨酸-异亮氨酸-丝氨酸-天冬酰胺-亮氨酸-。位点5由钙调蛋白依赖性蛋白激酶磷酸化，位点6由酪蛋白激酶-2磷酸化。在T4a位置的迁移仅与位点6的磷酸化相关，而与位点4和5中是否存在磷酸盐无关。位点5和6在对照脂肪细胞中部分磷酸化，位点4中也有少量磷酸盐。在用胰岛素刺激时，磷酸化似乎主要发生在位点6，从而导致T4a的32P标记增加。我们无法分离出足够量的其他胰岛素敏感肽来确定其序列。我们的结果与胰岛素激活酪蛋白激酶-2或具有与酪蛋白激酶-2相同特异性蛋白激酶的观点一致。这种修饰的功能尚不清楚，因为酪蛋白激酶-2的磷酸化对乙酰辅酶A羧化酶活性没有直接影响。
