Deletion analysis of protein kinase Calpha reveals a novel regulatory segment.
Deletion analysis of protein kinase Calpha reveals a novel regulatory segment.
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蛋白激酶 Calpha 的缺失分析揭示了一个新的调控片段。
DOI：
10.1093/oxfordjournals.jbchem.a022176
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发表时间：
1998
影响因子：
2.7
通讯作者：
Riedel,H
中科院分区：
文献类型：
--
作者：
Rotenberg,SA;Zhu,J;Hansen,H;Li,XD;Sun,XG;Michels,CA;Riedel,H
利用药理学和遗传学相结合的方法，我们已经确定C2区的260-280是蛋白激酶Ca(PKCA)催化功能的关键因子。在酿酒酵母中表达了N末端的渐进式截断和选定的内部缺失突变体，并测试了其对去奎宁的敏感性改变，去奎宁是一种PKC抑制剂，其靶点先前被映射到催化结构域。代表N-端多达158个氨基酸残基(ND84和ND158)的PKC突变体显示，50ƒ的去奎宁对激酶活性的抑制率为60%-63%，略高于野生型PKCƒ酶(45%的抑制率)。N端缺失AS 1-2 6 2的突变体ND2 6 2对50ƒ的抑制率可达72%，而缺少16个氨基酸的突变体ND2 78的抑制率仅为总活力的9%。这一结果表明，C2区的一个C-末端片段(AA 263-278)影响低微摩尔浓度下地奎宁的抑制作用。一个内部缺失突变体(D260-280)保留了PKCƒ的整个一级结构，但AS260-280除外，同样地，用50ƒ的Degaldium抑制也只有4%。在没有去奎宁的情况下，尽管存在几乎完整的调节域，该突变株表现出结构性活性(无论是在体外还是在与酿酒酵母的表型试验中)，即使是强大的激活剂TPA也不能进一步刺激该活性。综上所述，我们的发现表明，在PKCƒ的天然结构中，AS260-280所描述的片段调节PKCƒ的活性，并影响PKCƒ对去奎宁的敏感性。
Using a combined pharmacological and genetic approach, we have identified as 260-280 in the C2 region as a critical factor in the catalytic function of protein kinase Ca (PKCa). Progressive truncations from the N-terminus as well as selected internal deletion mutants were expressed in Saccharomyces cerevisiae and tested for altered sensitivity to dequali nium, a PKC inhibitor whose target site was previously mapped to the catalytic domain. PKC mutants representing truncations of up to 158 amino acid residues(aa) from the N-terminus(ND84 and ND158) displayed 60-63% inhibition of kinase activity by 50 ƒÊM dequalinium, somewhat more sensitive than the wild-type PKCƒ¿ enzyme(45% inhibition). Mutant ND262, lacking N-terminal as 1-262, was inhibited by almost 72% with 50 ƒÊM dequalinium, but mutant ND278, which lacked an additional 16 aa, was inhibited by only 9% of total activity. This result suggests that a C-terminal segment of the C2 region(aa 263-278) influences inhibition by dequalinium at low micromolar concentrations. An internal deletion mutant(D260-280) which retains the entire primary structure of PKCƒ¿ except for as 260-280, was similarly inhibited by only 4% with 50 ƒÊM dequalinium. In the absence of dequalinium and despite the presence of a nearly complete regulatory domain, this mutant exhibited constitutive activity(both in vitro and in a phenotypic assay with S. cerevisiae) that could not be further stimulated even by the potent activator TPA. Taken together, our findings suggest that, in the native structure of PKC ƒ¿, the segment described by as 260-280 regulates PKCƒ¿ activity and influences the sensitivity of PKCƒ¿ to dequalinium.