LL-K9-3

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.

Amino Acid Sequence

3D Structure (/structures/AF-P10275-F1-model_v6.cif)

Function

Steroid hormone receptors are ligand-activated transcription factors that regulate eukaryotic gene expression and affect cellular proliferation and differentiation in target tissues. Transcription factor activity is modulated by bound coactivator and corepressor proteins like ZBTB7A that recruits NCOR1 and NCOR2 to the androgen response elements/ARE on target genes, negatively regulating androgen receptor signaling and androgen-induced cell proliferation. Transcription activation is also down-regulated by NR0B2. Activated, but not phosphorylated, by HIPK3 and ZIPK/DAPK3.

Amino Acid Sequence

3D Structure (/structures/AF-P01106-F1-model_v6.cif)

Function

Transcription factor that binds DNA in a non-specific manner, yet also specifically recognizes the core sequence 5'-CAC[GA]TG-3'. Activates the transcription of growth-related genes. Binds to the VEGFA promoter, promoting VEGFA production and subsequent sprouting angiogenesis. Regulator of somatic reprogramming, controls self-renewal of embryonic stem cells (By similarity). Functions with TAF6L to activate target gene expression through RNA polymerase II pause release (By similarity). Positively regulates transcription of HNRNPA1, HNRNPA2 and PTBP1 which in turn regulate splicing of pyruvate kinase PKM by binding repressively to sequences flanking PKM exon 9, inhibiting exon 9 inclusion and resulting in exon 10 inclusion and production of the PKM M2 isoform.

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

Previous studies suggest that chaperones and the proteasome are engaged in the HyT mediated degradation process. To investigate whether the ubiquitin-proteasome system is involved in CDK9-cyclin T1 degradation induced by LL-K9-3, immunoprecipitation was conducted with HEK293T cells transfected with plasmids expressing Flag-CDK9, HA-cyclin T1, as well as Myc-ubiquitin. After anti-Flag and anti-HA immunoprecipitation, increased K48-linkage polyubiquitinated CDK9 and cyclin T1 were observed following treatment of LL-K9-3(Figure 4A). Similarly, after purification with anti-Myc beads, immunoblot analysis suggested that the ubiquitination modifications on CDK9 and cyclin T1 were considerably increased after LL-K9-3 treatment (Figure 4B). In addition, the proteasome inhibitor MG132 abrogated the degradation of CDK9-cyclin T1 induced by LL-K9-3(Figure 4C, D). Taken together,LL-K9-3 induced polyubiquitination of CDK9-cyclin T1 and triggered proteasome-mediated degradation

Description

To determine the efficacy of LL-K9-3 on CDK9-cyclin T1 degradation, immunoblot analysis was performed using 22RV1 cells after treating with LL-K9-3 at various concentrations. LL-K9-3 induced dose-dependent degradation of CDK9 and cyclin T1 with comparable efficiency, with DC50of 0.662 and 0.589μM for CDK9 and cyclin T1.

Description

To determine the efficacy of LL-K9-3 on CDK9-cyclin T1 degradation, immunoblot analysis was performed using 22RV1 cells after treating with LL-K9-3 at various concentrations. LL-K9-3 induced dose-dependent degradation of CDK9 and cyclin T1 with comparable efficiency, with DC50of 0.662 and 0.589μM for CDK9 and cyclin T1

Description

Selective CDK9 inhibitors have been reported to repress cell proliferation and tumor growth in CRPC. SNS032, LL-K9-3 and 43 The anti-proliferative effects of Thal-SNS032 5-day cell viability assay, both were then compared. In a LL-K9-3 and Thal-SNS032 showed more potent inhibition of proliferation than and Thal-SNS032 was slightly more potent than (Figure 7A). (A) Cell viability of 22RV1 treated with SNS032, LL-K9-3, and Thal-SNS032 for 5 days. IC50 values are calculated from fitted cel viability curves (n = 3, mean±SD).