Sachs Nancy A, Vaillancourt Richard R
Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721-0207, USA.
Biochim Biophys Acta. 2003 Dec 5;1624(1-3):98-108. doi: 10.1016/j.bbagen.2003.10.001.
Cyclin-dependent kinase (CDK)11(p110), formerly known as PITSLRE, is a serine/threonine kinase whose catalytic activity has been associated with transcription and RNA processing. To further evaluate the regulation of CDK11(p110) catalytic activity, interacting proteins were identified by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Following the immunoprecipitation of CDK11(p110) from COS-7 cells, the serine/threonine kinase CK2 was identified by LC-MS/MS. These results were extended through the observation that CDK11(p110) serves as a substrate for CK2 and the identification of a phosphorylation site on CDK11(p110) at Ser227 by LC-MS/MS. To obtain CDK11(p110) devoid of CK2, CDK11(p110) was expressed in High Five insect cells and secreted into the media due to the presence of a honeybee melittin signal sequence encoded at the amino-terminus of CDK11(p110). Recombinant CDK11(p110) was purified from the media and phosphorylation of histone H1 subsequently demonstrated. After demonstrating retention of CDK11(p110) kinase activity, it was evaluated for activity on the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II), but only CK2 was found to phosphorylate the CTD.
细胞周期蛋白依赖性激酶（CDK）11（p110），以前称为PITSLRE，是一种丝氨酸/苏氨酸激酶，其催化活性与转录和RNA加工有关。为了进一步评估CDK11（p110）催化活性的调节，通过液相色谱和串联质谱（LC-MS/MS）鉴定相互作用蛋白。从COS-7细胞中免疫沉淀CDK11（p110）后，通过LC-MS/MS鉴定出丝氨酸/苏氨酸激酶CK2。通过观察CDK11（p110）作为CK2的底物以及通过LC-MS/MS鉴定CDK11（p110）上Ser227处的磷酸化位点，扩展了这些结果。为了获得不含CK2的CDK11（p110），CDK11（p110）在High Five昆虫细胞中表达，并由于在CDK11（p110）氨基末端编码的蜜蜂蜂毒肽信号序列的存在而分泌到培养基中。从培养基中纯化重组CDK11（p110），随后证明组蛋白H1的磷酸化。在证明CDK11（p110）激酶活性保留后，评估其对RNA聚合酶II（RNAP II）最大亚基的羧基末端结构域（CTD）的活性，但仅发现CK2使CTD磷酸化。
