LL-CDK9-12

Amino Acid Sequence

3D Structure (/structures/AF-P50750-F1-model_v6.cif)

Function

Protein kinase involved in the regulation of transcription. Member of the cyclin-dependent kinase pair (CDK9/cyclin-T) complex, also called positive transcription elongation factor b (P-TEFb), which facilitates the transition from abortive to productive elongation by phosphorylating the CTD (C-terminal domain) of the large subunit of RNA polymerase II (RNAP II) POLR2A, SUPT5H and RDBP. This complex is inactive when in the 7SK snRNP complex form. Phosphorylates EP300, MYOD1, RPB1/POLR2A and AR and the negative elongation factors DSIF and NELFE. Regulates cytokine inducible transcription networks by facilitating promoter recognition of target transcription factors. P-TEFb is also involved in cotranscriptional histone modification, mRNA processing and mRNA export. Modulates a complex network of chromatin modifications including histone H2B monoubiquitination (H2Bub1), H3 lysine 4 trimethylation (H3K4me3) and H3K36me3; integrates phosphorylation during transcription with chromatin modifications to control co-transcriptional histone mRNA processing. The CDK9/cyclin-K complex has also a kinase activity towards CTD of RNAP II and can substitute for CDK9/cyclin-T P-TEFb in vitro. Replication stress response protein; the CDK9/cyclin-K complex is required for genome integrity maintenance, by promoting cell cycle recovery from replication arrest and limiting single-stranded DNA amount in response to replication stress, thus reducing the breakdown of stalled replication forks and avoiding DNA damage. In addition, probable function in DNA repair of isoform.

Amino Acid Sequence

3D Structure (/structures/AF-O60563-F1-model_v6.cif)

Function

Regulatory subunit of the cyclin-dependent kinase pair (CDK9/cyclin-T1) complex, also called positive transcription elongation factor B (P-TEFb), which facilitates the transition from abortive to productive elongation by phosphorylating the CTD (C-terminal domain) of the large subunit of RNA polymerase II (RNA Pol II). Required to activate the protein kinase activity of CDK9: acts by mediating formation of liquid-liquid phase separation (LLPS) that enhances binding of P-TEFb to the CTD of RNA Pol II. (Microbial infection) In case of HIV or SIV infections, binds to the transactivation domain of the viral nuclear transcriptional activator, Tat, thereby increasing Tat's affinity for the transactivating response RNA element (TAR RNA). Serves as an essential cofactor for Tat, by promoting RNA Pol II activation, allowing transcription of viral genes. via interaction with KU70/XRCC6. RPB1/POLR2A phosphorylation on 'Ser-2' in CTD activates transcription. AR phosphorylation modulates AR transcription factor promoter selectivity and cell growth. DSIF and NELF phosphorylation promotes transcription by inhibiting their negative effect. The phosphorylation of MYOD1 enhances its transcriptional activity and thus promotes muscle differentiation. Catalyzes phosphorylation of KAT5, promoting KAT5 recruitment to chromatin and histone acetyltransferase activity.

Mechanism

The ubiquitin-proteasome pathway (UPP) constitutes the principal pathway for protein degradation in eukaryotic cells, mediating the breakdown of target proteins through ubiquitin tagging and subsequent proteasomal degradation. In an ATP-dependent manner, the ubiquitin-activating enzyme (E1) activates ubiquitin (Ub) and conjugates it to a ubiquitin-conjugating enzyme (E2). E3 ligase subsequently catalyzes the transfer of Ub to the target protein, leading to the formation of a polyubiquitin chain that completes the ubiquitination mark. The 19S regulatory particle of the 26S proteasome recognizes this ubiquitination signal and, after deubiquitination, mediates the ATP-dependent delivery of the substrate into the 20S proteasome for degradation

Description

To better elucidate the mechanism of the degradation of CDK9 cyclin T1 induced by LL-CDK9-12, we investigated whether the ubiquitin proteasome system was involved. LL-CDK9-12 substantially induced the degradation of CDK9 and cyclin T1 at concentrations of 3 μM and 1 μM, whereas the proteasome inhibitor MG132 reversed the degradation of CDK9 and cyclin T1 (Figure 5A and B). This data suggested that the proteasome was involved in the LL-CDK9-12 induced protein degradation of CDK9-cyclin T1

Description

LL-CDK9-12 was the most effective degrader of the CDK9-cyclin T1 complex and was chosen for further evaluation. LL-CDK9-12 induced efficient and dose-dependent degradation of CDK9 and cyclin T1, with the DC 50 values of 0.362 μM and 0.683 μM, respectively (Figure 4A and B).

Description

LL-CDK9-12 was the most effective degrader of the CDK9-cyclin T1 complex and was chosen for further evaluation. LL-CDK9-12 induced efficient and dose-dependent degradation of CDK9 and cyclin T1, with the DC 50 values of 0.362 μM and 0.683 μM, respectively (Figure 4A and B).

Description

We further analyzed the anti-proliferative effect of LL-CDK9-12 on prostate cancer cells 22RV1. Compared to its parental molecule SNS032, LL-CDK9-12 exhibited a more substantial anti-proliferative effect. In a five-day cell viability assay, LL CDK9-12 and SNS032 inhibited the proliferation of 22RV1 cells, with IC 50 values of 169.47±7.16 nM and 328.07±57.09 nM, respectively (Figure 6A).