Cyclin-dependent kinase 11^p110 activity in the absence of CK2

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.