Donovan M, Romano P, Tibbetts M, Hammond C I
Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459-0175.
Yeast. 1994 Jan;10(1):113-24. doi: 10.1002/yea.320100111.
We have isolated two yeast genes, KIN1 and KIN2, by their homology to the protein kinase family of viral oncogenes. Previous studies have identified the yeast KIN1 gene product (pp145KIN1) as a 145 kilodalton (kDa) phosphoprotein with serine/threonine-specific protein kinase activity. To identify and biochemically characterize the KIN2 gene product, antibodies were raised against a bacterial beta-galactosidase/KIN2 fusion polypeptide. In vivo, the KIN2 gene product is a 145 kDa phosphoprotein, pp145KIN2. In immune complexes, pp145KIN2 demonstrates serine/threonine protein kinase activity, transferring phosphate from [gamma-32P]ATP to either itself or the exogenously added substrates alpha-casein, acid-denatured enolase, or phosvitin. In vitro, kinase activity is dependent on either Mn2+ or Mg2+ ions. Both enzymes, pp145KIN1 and pp145KIN2, prefer ATP over GTP as their phosphoryl donor. Since a new class of yeast protein kinases has been identified which are serine/tyrosine-specific, we analysed a wide range of substrates to see if any could be phosphorylated by pp145KIN1 or pp145KIN2 on tyrosine residues. Both enzymes phosphorylate alpha-casein, acid-denatured enolase, and phosvitin on serine and threonine residues. Neither enzyme could phosphorylate tyrosine residues even though good substrates for tyrosine-specific kinases such as enolase, angiotensin II, and the synthetic polymer GLU80TYR20 were used. The biochemical analysis of KIN2 kinase activity shows remarkable similarity to that of its most closely related yeast kinase, KIN1. It remains to be seen if these two yeast protein kinases share any functional relationships or substrates in vivo.
我们通过与病毒癌基因的蛋白激酶家族的同源性，分离出了两个酵母基因KIN1和KIN2。先前的研究已将酵母KIN1基因产物（pp145KIN1）鉴定为一种具有丝氨酸/苏氨酸特异性蛋白激酶活性的145千道尔顿（kDa）磷蛋白。为了鉴定KIN2基因产物并对其进行生化特性分析，我们制备了针对细菌β-半乳糖苷酶/KIN2融合多肽的抗体。在体内，KIN2基因产物是一种145 kDa的磷蛋白，即pp145KIN2。在免疫复合物中，pp145KIN2表现出丝氨酸/苏氨酸蛋白激酶活性，可将[γ-32P]ATP中的磷酸基团转移至自身或外源添加的底物α-酪蛋白、酸变性烯醇化酶或卵黄高磷蛋白上。在体外，激酶活性依赖于Mn2+或Mg2+离子。pp145KIN1和pp145KIN2这两种酶都更倾向于以ATP而非GTP作为磷酰基供体。由于已鉴定出一类新的酵母蛋白激酶，它们具有丝氨酸/酪氨酸特异性，因此我们分析了多种底物，以确定是否有任何底物可被pp145KIN1或pp145KIN2在酪氨酸残基上磷酸化。这两种酶都能在丝氨酸和苏氨酸残基上磷酸化α-酪蛋白、酸变性烯醇化酶和卵黄高磷蛋白。即使使用了酪氨酸特异性激酶的良好底物，如烯醇化酶、血管紧张素II和合成聚合物GLU80TYR20，这两种酶也都不能磷酸化酪氨酸残基。KIN2激酶活性的生化分析显示，它与其关系最密切的酵母激酶KIN1具有显著的相似性。这两种酵母蛋白激酶在体内是否存在任何功能关系或共享底物，还有待观察。
