Cyclin K Functions as a CDK9 Regulatory Subunit and Participates in RNA Polymerase II Transcription

Abstract

Important progress in the understanding of elongation control by RNA polymerase II (RNAPII) has come from the recent identification of the positive transcription elongation factor b (P-TEFb) and the demonstration that this factor is a protein kinase that phosphorylates the carboxyl-terminal domain (CTD) of the RNAPII largest subunit. The P-TEFb complex isolated from mammalian cells contains a catalytic subunit (CDK9), a cyclin subunit (cyclin T1 or cyclin T2), and additional, yet unidentified, polypeptides of unknown function. To identify additional factors involved in P-TEFb function we performed a yeast two-hybrid screen using CDK9 as bait and found that cyclin K interacts with CDK9 in vivo. Biochemical analyses indicate that cyclin K functions as a regulatory subunit of CDK9. The CDK9-cyclin K complex phosphorylated the CTD of RNAPII and functionally substituted for P-TEFb comprised of CDK9 and cyclin T in in vitro transcription reactions.Accumulating evidence indicates that the expression of many protein coding genes is regulated at the level of transcription elongation. Understanding of elongation control by RNAPII has been hampered by slow progress in the identification of factors involved in transcription elongation. An emerging model is that the interplay of positive and negative elongation factors determines the elongation potential of RNAPII 1 in different promoters (1). Support for this view comes from the recent identification of the negative elongation factors 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB) sensitivityinducing factor, negative elongation factor, and factor 2 and the positive elongation factor P-TEFb. Factor 2 is an ATP-dependent termination factor that releases transcripts associated with stalled RNAPII molecules (2). DSIF is a repressor of elongation that was identified as a factor that renders in vitro transcription reactions sensitive to the drug DRB (3). NELF works in conjunction with DSIF to repress RNA polymerase II elongation (4). P-TEFb is a DRB-sensitive kinase that is believed to stimulate the elongation potential of RNAPII by phosphorylating the CTD of RNAPII molecules that are engaged in early transcription elongation (5, 6). It was recently suggested that P-TEFb-mediated phosphorylation of the CTD prevents the association of DSIF with RNAPII and thereby overcomes DSIFdependent repression (7). Although it has been long accepted that CTD phosphorylation plays a critical role in transcription, it has been difficult to ascertain the mammalian kinases responsible for CTD phosphorylation in vivo. The observation that the ability of several drugs to block CTD phosphorylation in vivo correlates with the ability of these compounds to inhibit P-TEFb in vitro strongly suggests that P-TEFb might indeed function as a CTD kinase in vivo (8). Additional evidence showing that PTEFb kinase functions as a positive elongation factor in vivo comes from studies with the HIV Tat protein that have shown that the catalytic activity of P-TEFb is required for Tat-dependent stimulation of tra...

Regulation of RNA polymerase II activity by CTD phosphorylation and cell cycle control

“…P-TEFb is a positive-acting RNAP II transcription elongation factor, originally characterized in Drosophila embryonic extracts (Marshall et al, 1996;Price, 2000), that counteracts inhibition of RNAP II transcription elongation by two negative-acting factors factors, DSIF and NELF (Wada et al, 1998;Garber and Jones, 1999;Yamaguchi et al, 1999;Zorio and Bentley, 2001). CDK9 can associate with four different cyclin subunits, three different T-type cyclins (cyclin T1, T2a, and T2b) (Peng et al, 1998b) and cyclin K. Interestingly, cyclin K was originally identi®ed in a yeast screen based on its ability to restore cell cycle progression and to rescue the lethality of G1 cyclin gene deletions (Edwards et al, 1998;Fu et al, 1999). Similar to CDK7 and CDK8, CTD kinase activity and protein levels of the CDK9-cyclin T complex appear to not signi®cantly¯uctuate during the cell cycle in most eukaryotic cells (Garriga et al, 1998).…”

Cyclin K Inhibits HIV-1 Gene Expression and Replication by Interfering with Cyclin-dependent Kinase 9 (CDK9)-Cyclin T1 Interaction in Nef-dependent Manner

“…Nef Induces Cyclin K Interaction with CDK9-The CDK9-CycT1 complex (P-TEFb complex) plays a central role in HIV-1 LTR-driven gene expression (46). Both CycK and CycT1 are known to bind CDK9 through their N-terminal cyclin box of which 53 of 80 residues are identical (17,44). Based on this report, we initiated studies to elucidate the mechanism of CycK-mediated reduction in viral gene expression.…”

“…The kinase partner of CycK was identified as CDK9 (17), although a recent report also indicates CycK interaction with CDK12 and -13 (31). The complex between CycK and CDK9 was also shown to function as a CTD kinase in vitro (17). Although Nef was earlier reported as a negative factor for viral replication (32), recent evidence has convincingly demonstrated Nef as an enhancer of viral replication (16,22,(33)(34)(35)(36).…”

“…CycK was first identified as a protein that could restore progression of the cell cycle and was most closely related to human Cyclins C and H (30). The kinase partner of CycK was identified as CDK9 (17), although a recent report also indicates CycK interaction with CDK12 and -13 (31). The complex between CycK and CDK9 was also shown to function as a CTD kinase in vitro (17).…”

“…Several cellular factors have been identified for their roles in this process, including positive transcription elongation factor b (P-TEFb) complex. Different P-TEFb complexes isolated from mammalian cells contain a common catalytic subunit, cyclin-dependent kinase 9 (CDK9), and one of the regulatory cyclins, CycT1, CycT2a, CycT2b, or CycK (17)(18)(19). However, CycT1-containing P-TEFb complex is the key complex responsible for HIV-1 transcription elongation (19).…”