N-terminal mutations modulate yeast SNF1 protein kinase function.
N-terminal mutations modulate yeast SNF1 protein kinase function.
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发表时间：
1992-11
期刊：
影响因子：
3.3
通讯作者：
F. Estruch;M. Treitel;X. Yang;M. Carlson
中科院分区：
文献类型：
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作者：
F. Estruch;M. Treitel;X. Yang;M. Carlson
SNF1 蛋白激酶是响应葡萄糖剥夺而表达葡萄糖抑制基因所必需的。 SNF4 蛋白与 SNF1 物理相关，并对激酶活性产生积极影响。我们在此报告了显性突变 SNF1-G53R 的特征，该突变被分离为 SNF4 需求的抑制因子。突变的 SNF1-G53R 蛋白仍然对 SNF4 有反应，但在免疫复合物测定中激酶活性大大提高；相反，在蛋白质印迹测定中，该活性是野生型的。激酶结构域 N 端区域（密码子 5-52）的缺失会降低体外激酶活性，但突变型 SNF1-delta N 激酶仍然依赖于 SNF4。 N 末端不是葡萄糖调节反应所必需的。在凝胶过滤色谱中，SNF1、SNF1-G53R 和 SNF1-delta N 蛋白显示出不同的洗脱曲线，与高分子量复合物的差异形成一致。综上所述，结果表明 N 末端对 SNF1 激酶的功能产生积极影响，并且可能参与与 SNF4 以外的积极效应子的相互作用。我们还表明，子结构域 VIII 中保守的苏氨酸残基 210（其他激酶中的磷酸化位点）对于 SNF1 活性至关重要。最后，我们提供的证据表明，当 C 末端被删除时，SNF1 激酶结构域的过度表达对细胞有害。
The SNF1 protein kinase is required for expression of glucose-repressed genes in response to glucose deprivation. The SNF4 protein is physically associated with SNF1 and positively affects the kinase activity. We report here the characterization of a dominant mutation, SNF1-G53R, that was isolated as a suppressor of the requirement for SNF4. The mutant SNF1-G53R protein is still responsive to SNF4 but has greatly elevated kinase activity in immune complex assays; in contrast, the activity is wild type in a protein blot assay. Deletion of the region N-terminal to the kinase domain (codons 5-52) reduces kinase activity in vitro, but the mutant SNF1-delta N kinase is still dependent on SNF4. The N terminus is not required for the regulatory response to glucose. In gel filtration chromatography, the SNF1, SNF1-G53R and SNF1-delta N protein showed different elution profiles, consistent with differential formation of high molecular weight complexes. Taken together, the results suggest that the N terminus positively affects the function of the SNF1 kinase and may be involved in interaction with a positive effector other than SNF4. We also showed that the conserved threonine residue 210 in subdomain VIII, which is a phosphorylation site in other kinases, is essential for SNF1 activity. Finally, we present evidence that when the C terminus is deleted, overexpression of the SNF1 kinase domain is deleterious to the cell.