Dailey D, Schieven G L, Lim M Y, Marquardt H, Gilmore T, Thorner J, Martin G S
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Mol Cell Biol. 1990 Dec;10(12):6244-56. doi: 10.1128/mcb.10.12.6244-6256.1990.
Extracts of bakers' yeast (Saccharomyces cerevisiae) contain protein-tyrosine kinase activity that can be detected with a synthetic Glu-Tyr copolymer as substrate (G. Schieven, J. Thorner, and G.S. Martin, Science 231:390-393, 1986). By using this assay in conjunction with ion-exchange and affinity chromatography, a soluble tyrosine kinase activity was purified over 8,000-fold from yeast extracts. The purified activity did not utilize typical substrates for mammalian protein-tyrosine kinases (enolase, casein, and histones). The level of tyrosine kinase activity at all steps of each preparation correlated with the content of a 40-kDa protein (p40). Upon incubation of the most highly purified fractions with Mn-ATP or Mg-ATP, p40 was the only protein phosphorylated on tyrosine. Immunoblotting of purified p40 or total yeast extracts with antiphosphotyrosine antibodies and phosphoamino acid analysis of 32P-labeled yeast proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 40-kDa protein is normally phosphorylated at tyrosine in vivo. 32P-labeled p40 immunoprecipitated from extracts of metabolically labeled cells by affinity-purified anti-p40 antibodies contained both phosphoserine and phosphotyrosine. The gene encoding p40 (YPK1) was cloned from a yeast genomic library by using oligonucleotide probes designed on the basis of the sequence of purified peptides. As deduced from the nucleotide sequence of YPK1, p40 is homologous to known protein kinases, with features that resemble known protein-serine kinases more than known protein-tyrosine kinases. Thus, p40 is a protein kinase which is phosphorylated in vivo and in vitro at both tyrosine and serine residues; it may be a novel type of autophosphorylating tyrosine kinase, a bifunctional (serine/tyrosine-specific) protein kinase, or a serine kinase that is a substrate for an associated tyrosine kinase.
面包酵母（酿酒酵母）提取物含有蛋白酪氨酸激酶活性，该活性可用合成的Glu-Tyr共聚物作为底物检测到（G. 希夫恩、J. 索纳和G.S. 马丁，《科学》231:390 - 393，1986年）。通过将该检测方法与离子交换和亲和层析相结合，从酵母提取物中纯化出一种可溶性酪氨酸激酶活性，纯化倍数超过8000倍。纯化后的活性不利用哺乳动物蛋白酪氨酸激酶的典型底物（烯醇化酶、酪蛋白和组蛋白）。每次制备过程中各个步骤的酪氨酸激酶活性水平与一种40 kDa蛋白质（p40）的含量相关。用Mn-ATP或Mg-ATP孵育纯化程度最高的组分时，p40是唯一在酪氨酸上被磷酸化的蛋白质。用抗磷酸酪氨酸抗体对纯化的p40或总酵母提取物进行免疫印迹分析，以及对经十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分离的32P标记酵母蛋白进行磷酸氨基酸分析，结果表明该40 kDa蛋白质在体内正常情况下在酪氨酸处被磷酸化。通过用基于纯化肽序列设计的寡核苷酸探针从酵母基因组文库中克隆出编码p40的基因（YPK1）。根据YPK1的核苷酸序列推断，p40与已知蛋白激酶同源，其特征与已知蛋白丝氨酸激酶的相似性超过已知蛋白酪氨酸激酶。因此，p40是一种在体内和体外酪氨酸和丝氨酸残基处均被磷酸化的蛋白激酶；它可能是一种新型的自磷酸化酪氨酸激酶、双功能（丝氨酸/酪氨酸特异性）蛋白激酶，或者是一种作为相关酪氨酸激酶底物的丝氨酸激酶。
