Protein-Protein Interactions of Tandem Affinity Purified Protein Kinases from Rice

Eighty-eight rice (Oryza sativa) cDNAs encoding rice leaf expressed protein kinases (PKs) were fused to a Tandem Affinity Purification tag (TAP-tag) and expressed in transgenic rice plants. The TAP-tagged PKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by tandem mass spectrometry. Forty-five TAP-tagged PKs were recovered in this study and thirteen of these were found to interact with other rice proteins with a high probability score. In vivo phosphorylated sites were found for three of the PKs. A comparison of the TAP-tagged data from a combined analysis of 129 TAP-tagged rice protein kinases with a concurrent screen using yeast two hybrid methods identified an evolutionarily new rice protein that interacts with the well conserved cell division cycle 2 (CDC2) protein complex.本研究将88个编码水稻（Oryza sativa）叶片表达型蛋白激酶（PKs）的互补脱氧核糖核酸（cDNA）与串联亲和纯化标签（TAP-tag）融合，并在转基因水稻植株中实现表达。从转基因水稻植株的T1代子代中纯化得到带TAP标签的蛋白激酶及其互作蛋白，并通过串联质谱法完成鉴定。本研究共回收得到45个带TAP标签的蛋白激酶，其中13个可与其他水稻蛋白以高置信度评分发生互作。另有3个蛋白激酶被检测到存在体内磷酸化位点。将129个带TAP标签的水稻蛋白激酶的联合分析数据，与同期采用酵母双杂交技术开展的筛选实验结果进行比对后，本研究鉴定出一种进化上新型的水稻蛋白，该蛋白可与高度保守的细胞分裂周期蛋白2（CDC2）复合物发生互作。