LOCATION AND FUNCTION OF 3 SITES PHOSPHORYLATED ON RAT ACETYL-COA CARBOXYLASE BY THE AMP-ACTIVATED PROTEIN-KINASE
LOCATION AND FUNCTION OF 3 SITES PHOSPHORYLATED ON RAT ACETYL-COA CARBOXYLASE BY THE AMP-ACTIVATED PROTEIN-KINASE
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DOI：
10.1111/j.1432-1033.1990.tb15293.x
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发表时间：
1990-01-12
期刊：
影响因子：
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通讯作者：
HARDIE, DG
中科院分区：
文献类型：
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作者：
DAVIES, SP;SIM, ATR;HARDIE, DG
1.我们已经测序了两个胰蛋白酶/胰凝乳蛋白酶肽（TC 3和TC 3a）含有第三个网站磷酸化大鼠乙酰辅酶A羧化酶的AMP激活的蛋白激酶。与根据cDNA序列预测的大鼠乙酰辅酶A羧化酶完整序列的比较[Lopez-Casillas et al.（1988）Proc. Natl. Acad. Sci. USA 85，5784-5788]显示该位点对应于Ser 1215。2.将cDNA序列与先前的氨基酸序列数据进行比较，确定AMP激活的钙蛋白激酶的另外两个位点为Ser 79和Ser 1200。现在可以鉴定出由6种蛋白激酶在体外磷酸化的总共8个丝氨酸残基：其中6个（Ser 23、Ser 25、Ser 29、Ser 77、Ser 79和Ser 95）聚集在氨基末端区域，而2个（Ser 1200和Ser 1215）位于中心区域。3.通过环AMP依赖性蛋白激酶的Ser 77和Ser 1200的先前磷酸化阻止了随后通过AMP活化的蛋白激酶的Ser 79和Ser 1200的磷酸化，但不阻止Ser 1215的磷酸化。在这些条件下，Ser 1215的磷酸化与酶活性的变化无关。4.天然乙酰辅酶A羧化酶的有限胰蛋白酶处理选择性地切割掉含有Ser 79的高度磷酸化的氨基末端区域。5. AMP激活的蛋白激酶在Ser 79和Ser 1200处的磷酸化显著降低了Vmax，并增加了柠檬酸盐的A0.5。通过环AMP依赖性蛋白激酶在Ser 77和Ser 1200处的磷酸化引起柠檬酸盐的A0.5和Vmax的更适度的变化。去磷酸化，或使用胰蛋白酶去除含有Ser 77/70的氨基末端区域，逆转所有这些作用。6.这些结果表明，AMP激活的蛋白激酶对乙酰辅酶A羧化酶活性的影响完全是由Ser 79的磷酸化介导的，而不是Ser 1200和Ser 1215。环AMP依赖性蛋白激酶的较小作用是由Ser 77的磷酸化介导的。
1. We have sequenced two tryptic/chymotryptic peptides (TC3 and TC3a) containing a third site phosphorylated on rat acetyl-CoA carboxylase by the AMP-activated protein kinase. Comparison with the complete sequence of rat acetyl-CoA carboxylase predicted from the cDNA sequence [Lopez-Casillas et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5784-5788] shows that this site corresponds to Ser1215. 2. Comparison of the cDNA sequence with previous amino acid sequence data identifies the other two sites for the AMP-activated perotein kinase as Ser79 and Ser1200. A total of eight serine residues phosphorylated in vitro by six protein kinases can now be identified: six of these (Ser23, Ser25, Ser29, Ser77, Ser79 and Ser95) are clustered in the amino terminal region, while two (Ser1200 and Ser1215) are located in the central region. 3. Prior phosphorylation of Ser77 and Ser1200 by cyclic-AMP-dependent protein kinase prevents subsequent phosphorylation of Ser79 and Ser1200, but not Ser1215, by the AMP-activated protein kinase. Phosphorylation of Ser1215 under these conditions is not associated with a change in enzyme activity. 4. Limited trypsin treatment of native acetyl-CoA carboxylase selectively cleaves off the highly phosphorylated amino-terminal region containing Ser79. 5. Phosphorylation at Ser79 and Ser1200 by the AMP-activated protein kinase dramatically decreases Vmax and increases the A0.5 for citrate. Phosphorylation at Ser77 and Ser1200 by cyclic-AMP-dependent protein kinase causes more modest changes in the A0.5 for citrate and the Vmax. Dephosphorylation, or removal of the amino-terminal region containing Ser77/70 using trypsin, reverses all of these effects. 6. These results suggest that the effects of the AMP-activated protein kinase on acetyl-CoA carboxylase activity are mediated entirely by phosphorylation of Ser79, and not Ser1200 and Ser1215. The smaller effects of cyclic-AMP-dependent protein kinase are mediated by phosphorylation of Ser77.