Physical interaction between CDK9 and B-Myb results in suppression of B-Myb gene autoregulation

Abstract

B-Myb is a transcription factor belonging to the myb family, whose activity has been associated with augmented DNA synthesis and cell cycle progression. We showed recently that B-Myb autoregulates its own expression through promoter transactivation. We report in this study that CDK9, the cyclin T associated kinase, which phosphorylates and activates RNA-Polymerase II, suppresses B-Myb autoregulation through direct interaction with the carboxyl-terminus of the B-Myb protein. Downregulation of the transactivating ability of B-Myb is independent of the kinase activity of CDK9, because a kinase de®cient mutant (dn-CDK9) also represses B-myb gene autoregulation. Overexpression of CDK9 did not result in suppression of p53-dependent transactivation or inhibition of the basal activity of the promoters tested so far, demonstrating that CDK9 is a B-Myb-speci®c repressor. Rather, transfection of the dominant negative dn-CDK9 construct inhibited the basal activity of the reporter genes, con®rming an essential role for CDK9 in gene transcription. In addition, Cyclin T1 restores B-Myb transactivating activity when co-transfected along with CDK9, suggesting that the down-regulatory e ect observed on B-Myb is speci®cally due to CDK9 alone. Thus, our data suggest that CDK9 is involved in the negative regulation of activated transcription mediated by certain transcription factors, such as B-Myb. This may indicate the existence of a feedback loop, mediated by the di erent activities of CDK9, which links basal with activated transcription.

Inhibition of cyclin A/Cdk2 phosphorylation impairs B-Myb transactivation function without affecting interactions with DNA or the CBP coactivator

“…While the generation of transiently active truncated B-Myb species in response to cyclin Amediated phosphorylation would clearly provide a rationale for the presence of an inhibitory C-terminal domain, it also remains possible that B-Myb hyperphosphorylation a ects interactions with as yet unrecognized regulatory proteins. In this regard, several B-Myb-binding proteins have now been identi®ed which inhibit its transactivation function, for example p107, Cdk9 and cyclin D1 (Sala et al, 1996;De Falco et al, 2000;Horstmann et al, 2000), and it will be of further interest to determine whether phosphorylation counteracts these e ects.…”

Cdk9, a member of the cdc2-like family of kinases, binds to gp130, the receptor of the IL-6 family of cytokines

“…The polyclonal anti-HA Tag (Santacruz) was used at a dilution 1 : 3000 in Western blot. Immunoprecipitation and Western blotting conditions have been described (De Falco et al, 2000).…”

“…The antibody (Ab) was used at a dilution of 1 : 1000 in Western blot. Anti-Cdk9 has already been described (De Falco et al, 2000). The antibody was affinity purified and used at a dilution 1 : 300 in Western blot.…”

Physical interaction between pRb and cdk9/cyclinT2 complex

“…These data indicate functional di erences between cycT1 and cycT2, even if cycT2 function remains less understood. The catalytic activity of the cdk9 immunocomplexes results only from cdk9 and it is abolished using a dominant negative form (kinase inactive mutant, cdk9dn) (De Falco et al, 2000), which consists of a point mutation within the ATP-binding domain that changes the Asp to Asn at residue 167.…”