Characterization of Cdk955 and differential regulation of two Cdk9 isoforms

Positive transcription elongation factor b (P-TEFb) controls the fraction of initiated RNA polymerase II molecules that make full length transcripts. This important factor is a heterodimer of cyclin-dependent kinase 9 (Cdk9) and one of four cyclin partners, cyclin T1, T2a, T2b or K. There are two isoforms of Cdk9 in mammalian cells, Cdk942 and Cdk955. Cdk955 has a 117 residue amino terminal extension not present in Cdk942. An expression vector with a tetracycline-responsive promoter driving FLAG-tagged Cdk955 and a HeLa 37 Tet-Off cell line were constructed. FLAG-tagged Cdk955 was inducibly expressed and was found to be localized to the nucleus by immunofluorescence. Western analysis of murine tissues showed that the relative abundance of the two forms of Cdk9 varied across different tissues with liver having more Cdk955 than Cdk942. During adaptation of primary rat hepatocytes to culture the ratio of the two forms of Cdk9 changed. Initially, Cdk955 was the predominate form, but as the cells began to enter the cell cycle Cdk942 became the major form. During this change, expression of Cdk942 was induced, while Cdk955 remained relatively constant.