A Kinase-Independent Activity of Cdk9 Modulates Glucocorticoid Receptor-Mediated Gene Induction

Abstract

A gene induction competition assay has recently uncovered new inhibitory activities of two transcriptional cofactors, NELF-A and NELF-B, in glucocorticoid-regulated transactivation. NELF-A and -B are also components of the NELF complex, which participates in RNA polymerase II pausing shortly after the initiation of gene transcription. We therefore asked if cofactors (Cdk9 and ELL) best known to affect paused polymerase could reverse the effects of NELF-A and -B. Unexpectedly, Cdk9 and ELL augmented, rather than prevented, the effects of NELF-A and -B. Furthermore, Cdk9 actions are not blocked either by Ckd9 inhibitors (DRB or flavopiridol) or by two Cdk9 mutants defective in kinase activity. The mode and site of action of NELF-A and -B mutants with an altered NELF domain are similarly affected by wild-type and kinase-dead Cdk9. We conclude that Cdk9 is a new modulator of GR action, that Ckd9 and ELL have novel activities in GR-regulated gene expression, that NELF-A and -B can act separately from the NELF complex, and that Cdk9 possesses activities that are independent of Cdk9 kinase activity. Finally, the competition assay has succeeded in ordering the site of action of several cofactors of GR transactivation. Extension of this methodology should be helpful in determining the site and mode of action of numerous additional cofactors and in reducing unwanted side effects.

Kinetically Defined Mechanisms and Positions of Action of Two New Modulators of Glucocorticoid Receptor-regulated Gene Induction

“…first-order Hill plots) of LUC activity above background versus Dex concentration were constructed to obtain the A max and EC 50 parameters. As reported previously (16), the fits of the data to a first-order Hill plot were very good (R 2 Ն 0.99). In this system, increasing concentrations of BRD4 plasmid generally provoke a decrease in A max and an increase in EC 50 .…”

“…This different approach is made possible by the observation that the dose-response curve for steroid-regulated gene expression is usually non-cooperative with a Hill coefficient of 1, as in a classical Michaelis-Menten curve. For such regulated genes, an experimentally validated chemical kinetic model has been developed that explains ligand regulation of gene induction, gene repression, and the partial agonist activity of antisteroids (13)(14)(15)(16)(17)(18).…”

“…Information about each factor is then obtained from a series of graphs, similar to Lineweaver-Burk graphs in biochemistry, that plot combinations of the A max and EC 50 values from the competition assays. Specifically, the graphs reveal the number of sites at which each factor acts, their kinetically defined mechanism of action, and their relative order of action in the overall sequence leading to changes in gene expression (12)(13)(14)(15)(16)(17)(18)(19)(20)(21). This approach has been extensively validated, and the derived information is novel and not available from other methodologies.…”

“…In addition, several well known transcription factors have been found to modulate GRregulated gene induction, such as components of the transcription negative elongation factor (NELF) complex (20) and CDK9 (16), which is the kinase subunit of P-TEFb. The NELF complex is a negative regulator of RNA polymerase II (Pol II), enforcing a pause in early elongation about 100 bp after transcription initiation.…”

Function of CDK12 in Tumor Initiation and Progression and Its Clinical Consequences

“…Tato CDK, která byla z počátku studována zejm. v souvislosti s regulací replikace viru HIV způso-bujícího onemocnění AIDS [12][13][14], byla později identifi kována jako zcela klíčový transkripční faktor nezbytný pro aktivitu řady dalších transkripčních faktorů a bona fi de onkogenů (NFκB, C-MYC, estrogenový receptor, glukokortikoidový receptor a další), jejichž funkce v procesu kancerogeneze již byla detailně popsána [15][16][17][18]. Způsob, jakým CDK9 pozitivně reguluje podřízené molekuly, spočívá v její vazbě na daný faktor/ onkogen a následné fosforylaci tzv.…”