Szyszka R, Bou G, Ballesta J P
Centro de Biologia Molecular, UAM, Madrid, Spain.
Biochim Biophys Acta. 1996 Apr 16;1293(2):213-21. doi: 10.1016/0167-4838(95)00246-4.
A new protein kinase, showing a high specificity for the ribosomal acidic P proteins (RAP kinase) has been purified and characterized from Saccharomyces cerevisiae extracts. Purification was carried out by four chromatographic steps, including DEAE-cellulose, phosphocellulose, heparin-Sepharose and P protein-Sepharose. The purified enzyme preparation contains only one polypeptide of around 55 kDa as determined by SDS gel electrophoresis and gradient centrifugation. RAP kinase is different from all previous well-characterized kinases and does not show cross-reaction with antibodies to the 71 kDa 60S ribosomal subunit-specific kinase PK60 previously reported. The enzyme uses ATP as a better phosphate donor and is less sensitive to heparin than casein kinase II but is moderately affected by salt. Among the different substrates tested, ribosomal acidic proteins are preferentially modified by RAP kinase, which phosphorylates only serine residues in the four P proteins as well as the related ribosomal protein P0. Casein is phosphorylated at a much lower level. All the data indicate that RAP kinase might be the enzyme responsible for the phosphorylation of the P proteins, and in this way may also participate in a possible translational regulatory mechanism.
从酿酒酵母提取物中纯化并鉴定出一种对核糖体酸性P蛋白具有高度特异性的新型蛋白激酶（RAP激酶）。纯化过程通过四个色谱步骤进行，包括DEAE-纤维素、磷酸纤维素、肝素-琼脂糖和P蛋白-琼脂糖。经SDS凝胶电泳和梯度离心测定，纯化的酶制剂仅含有一种约55 kDa的多肽。RAP激酶与所有先前已充分表征的激酶不同，并且与先前报道的针对71 kDa 60S核糖体亚基特异性激酶PK60的抗体不发生交叉反应。该酶以ATP作为更好的磷酸供体，对肝素的敏感性低于酪蛋白激酶II，但受盐的影响适中。在测试的不同底物中，核糖体酸性蛋白优先被RAP激酶修饰，RAP激酶仅使四种P蛋白以及相关核糖体蛋白P0中的丝氨酸残基磷酸化。酪蛋白的磷酸化水平要低得多。所有数据表明，RAP激酶可能是负责P蛋白磷酸化的酶，并且可能以此方式参与一种可能的翻译调控机制。
