Recombinant Bovine Cyclin-dependent kinase 12 (CDK12), partial

What is the primary function of CDK12 and how is it conserved across species?

CDK12 functions primarily as a transcriptional regulator through its ability to phosphorylate RNA polymerase II (RNAPII) on serine 2 of the carboxyl-terminal repeat domain (CTD). This phosphorylation is critical for transcriptional elongation and proper RNA processing. CDK12 plays essential roles in regulating the expression and processing of genes involved in cell cycle progression and DNA damage signaling and repair .

While most studies have focused on human CDK12, the high conservation of CDK protein families across mammals suggests similar core functions in bovine systems. Researchers should note that CDK12 forms an active complex with Cyclin K, which is necessary for its kinase activity .

How does partial recombinant bovine CDK12 differ functionally from the full-length protein?

Partial recombinant bovine CDK12 typically refers to constructs containing the catalytic kinase domain without some regulatory regions. When working with partial constructs:

• The catalytic activity may be preserved, particularly for phosphorylation of RNAPII

• Regulatory interactions may be altered or absent

• Localization signals might be missing, affecting experimental interpretation

For accurate functional studies, researchers should validate which domains are present in their partial construct and compare activity to full-length controls where possible. Phosphorylation assays using RNAPII CTD substrates can assess whether the partial protein retains catalytic function.

What protein targets does CDK12 primarily phosphorylate?

CDK12 primarily phosphorylates:

In research with bovine CDK12, it's important to verify substrate conservation through sequence alignment before designing phosphorylation assays.

What expression systems are most effective for producing functional recombinant bovine CDK12?

Based on experience with human CDK proteins, bacterial expression systems offer advantages of speed and simplicity for producing recombinant CDK12 . The following approaches are recommended:

• E. coli expression with chaperone co-expression: Similar to strategies used for human cyclin proteins, co-expression with molecular chaperones can significantly improve the solubility and proper folding of recombinant bovine CDK12 .

• Auto-induction protocols: These can yield higher protein expression compared to IPTG induction while requiring less hands-on time .

• Baculovirus-insect cell systems: For cases where bacterial expression yields inactive protein, insect cell expression often produces properly folded mammalian kinases with post-translational modifications.

A recommended starting approach is to test expression in E. coli BL21(DE3) cells co-transformed with chaperone-expressing plasmids under auto-induction conditions at reduced temperatures (16-18°C).

What purification strategies maintain the catalytic activity of recombinant bovine CDK12?

For effective purification while preserving activity:

• Affinity chromatography: His-tagged constructs can be purified using immobilized metal affinity chromatography (IMAC) as a first step .

• Buffer optimization: Include the following components:

  • 20-50 mM Tris-HCl or HEPES (pH 7.5-8.0)

  • 150-300 mM NaCl

  • 10% glycerol (stabilizer)

  • 1-5 mM DTT or 0.5-1 mM TCEP (reducing agent)

  • 0.5-1 mM EDTA (to inhibit metalloproteases)

  • Protease inhibitor cocktail

• Activity preservation: Avoid freeze-thaw cycles and maintain protein at 4°C during purification.

• Quality control: Verify kinase activity through in vitro kinase assays using RNA Pol II CTD peptides as substrates.

How can you validate the proper folding and activity of purified recombinant bovine CDK12?

Multiple complementary approaches are recommended:

• In vitro kinase assays: Using ATP, appropriate substrates (RNAPII CTD peptides), and measuring phosphorylation via:

  • Radioactive [γ-32P]ATP incorporation

  • Phospho-specific antibodies

  • Mass spectrometry

• Thermal shift assays: To evaluate protein stability and proper folding.

• Circular dichroism: To assess secondary structure content.

• Size-exclusion chromatography: To confirm monomeric state or complex formation with Cyclin K.

• Inhibitor binding studies: Using known CDK12 inhibitors like THZ531 and measuring binding affinity through thermal shift or activity inhibition .

How can recombinant bovine CDK12 be used to study DNA damage response mechanisms?

CDK12 plays a critical role in the DNA damage response pathway. For research applications:

• Construct preparation:

  • Wild-type bovine CDK12

  • Kinase-dead mutants (typically D877N based on human CDK12 homology)

  • Phospho-mimetic variants

• Experimental approaches:

  • Complementation studies in CDK12-knockout cell lines

  • Pull-down assays to identify interacting partners after DNA damage

  • ChIP-seq to map genome-wide binding sites following damage

  • RNA-seq to profile transcriptional changes upon CDK12 inhibition or depletion

• Key measurements:

  • γH2AX and phospho-KAP1 levels as markers of DNA damage response

  • BrdU incorporation to assess DNA synthesis and cell cycle effects

  • Proximity ligation assays to detect CDK12 recruitment to DNA damage foci

CDK12 inhibition or depletion leads to persistent transcription at damaged genes and exacerbates transcription-replication conflicts, particularly in cells with oncogene activation like MYC .

What mechanisms explain CDK12's role in transcriptional regulation of DNA repair genes?

CDK12 regulates transcription through multiple mechanisms:

• RNAPII phosphorylation: CDK12 phosphorylates Ser2 of the RNAPII CTD, promoting transcriptional elongation .

• Damage-induced transcriptional repression: Upon DNA damage, CDK12:

  • Is recruited to damaged genes via PARP-dependent DDR signaling

  • Interacts with elongation-competent RNAPII

  • Represses transcription at damaged sites to allow proper repair

• Regulation of RNA processing: CDK12 prevents premature transcriptional termination at cryptic poly-A sites and regulates alternative splicing .

When studying bovine CDK12, researchers should design experiments to differentiate between these mechanisms, using specific inhibitors like THZ531 to distinguish kinase-dependent from scaffolding functions.

How does CDK12 deficiency impact cellular metabolism, and what research models best capture these effects?

Recent research has shown that CDK12 deficiency leads to significant metabolic reprogramming in cancer cells:

• Enhanced mitochondrial respiration: CDK12-deficient cells show increased electron transport chain (ETC) activity and ATP synthesis .

• Altered lipid metabolism: CDK12 deficiency downregulates ACSL4 expression, which affects polyunsaturated fatty acid incorporation into cell membranes and modulates ferroptosis susceptibility .

• Research models:

  • CRISPR-Cas9-mediated CDK12 knockout cell lines

  • CDK12 inhibitor (THZ531) treatment

  • Patient-derived xenografts from CDK12-mutated tumors

• Key analytical approaches:

  • Mass spectrometry-based metabolomics

  • Mitochondrial function assays (Seahorse analyzer)

  • Measurements of reactive oxygen species

  • ACSL4 expression and stability analysis

  • Ferroptosis sensitivity testing

For bovine CDK12 research, investigators should validate whether these metabolic effects are conserved across species before developing species-specific models.

What are the most effective strategies for studying CDK12-regulated transcription?

To effectively study CDK12's role in transcription:

• Genome-wide approaches:

  • RNA-seq comparing wild-type and CDK12-depleted/inhibited cells

  • ChIP-seq for phospho-Ser2 RNAPII to identify actively transcribed genes

  • EU incorporation assays to measure global RNA synthesis rates

• Locus-specific approaches:

  • Reporter assays with DNA damage-inducible components

  • Site-specific DNA damage induction (e.g., I-SceI system)

  • RNA FISH to visualize transcription at specific loci

• Validation experiments:

  • qRT-PCR for selected differentially expressed genes

  • Western blotting for protein-level changes

  • mRNA stability assays using actinomycin D treatment

When analyzing results, distinguish between direct transcriptional effects and secondary consequences of CDK12 inhibition.

How can researchers distinguish between CDK12 and CDK13 functions in transcriptional regulation?

CDK12 and CDK13 are paralogs with partially overlapping functions, creating experimental challenges:

• Specific tools for differentiation:

  • Selective inhibitors (validate specificity biochemically)

  • Isoform-specific antibodies (validate with knockout controls)

  • siRNAs with demonstrated specificity

  • CRISPR-Cas9 single and double knockouts

• Experimental design considerations:

  • Compare phenotypes of CDK12, CDK13, and double knockdowns

  • Identify isoform-specific interacting partners through IP-MS

  • Perform rescue experiments with wild-type and kinase-dead mutants

• Data interpretation guidelines:

  • Genes affected by both CDK12 and CDK13 depletion suggest redundant functions

  • Genes uniquely affected by either suggest specific roles

  • Synergistic effects in double knockdowns indicate partial redundancy

What technical challenges arise when expressing recombinant bovine CDK12, and how can they be addressed?

Common challenges with CDK12 expression include:

For bovine CDK12 specifically, sequence-specific optimization of codons for E. coli expression may improve yields.

How do transcription-replication conflicts (TRCs) arise in CDK12-deficient cells, and what methods can detect them?

CDK12 deficiency leads to transcription-replication conflicts through these mechanisms:

• Failure to repress transcription at damaged genes: CDK12 normally represses transcription at damaged loci, but its loss leads to persistent transcription that interferes with DNA replication .

• Methods to detect TRCs:

  • DNA-RNA hybrid (R-loop) immunoprecipitation using S9.6 antibody

  • Nascent strand sequencing (NS-seq) to identify replication fork stalling

  • Proximity ligation assays between replication and transcription machinery

  • γH2AX ChIP-seq to map damage occurring at transcribed regions

  • Double-strand break mapping techniques

• Key findings in CDK12-deficient cells:

  • Double-strand breaks occur preferentially between co-directional early-replicating regions and transcribed genes

  • MYC overexpression exacerbates these conflicts

  • Cell lines with deregulated MYC show enhanced sensitivity to CDK12 inhibition

These approaches can be adapted for studying bovine CDK12 in appropriate cell systems.

What is the relationship between CDK12 function and ferroptosis, and how can this be experimentally investigated?

Recent research has uncovered an unexpected link between CDK12 and ferroptosis sensitivity:

• Mechanistic relationship:

  • CDK12 phosphorylates RNA Pol II to ensure transcription of ACSL4

  • ACSL4 regulates incorporation of polyunsaturated fatty acids into cell membranes

  • CDK12 deficiency downregulates ACSL4, protecting cells from ferroptosis despite increased oxidative stress

• Experimental approaches:

  • mRNA stability assays for ACSL4 using actinomycin D treatment

  • ChIP-qPCR to assess CDK12 binding to the ACSL4 promoter

  • Ferroptosis induction using erastin or RSL3 in CDK12-deficient versus wild-type cells

  • Lipidomic analysis to measure PUFA incorporation into membranes

  • Cellular ROS measurements using specific probes

• Critical controls:

  • ACSL4 rescue experiments in CDK12-deficient cells

  • Ferroptosis inhibitors (ferrostatin-1, liproxstatin-1) to confirm specificity

  • Comparison with other CDK family members to establish specificity

This emerging area offers significant potential for therapeutic exploitation in CDK12-deficient cancers.

How can CDK12 inhibition be exploited therapeutically, and what model systems best represent drug efficacy?

CDK12 inhibition offers several therapeutic opportunities:

• Therapeutic mechanisms:

  • Synthetic lethality in cells with MYC overexpression

  • Enhanced sensitivity to DNA-damaging agents

  • Metabolic vulnerabilities, particularly dependency on the electron transport chain

• Experimental models:

  • Cell lines with defined genetic backgrounds (MYC-overexpressing, BRCA-deficient)

  • Patient-derived xenografts from CDK12-mutated tumors

  • Genetically engineered mouse models with tissue-specific CDK12 knockout

• Combination approaches:

  • CDK12 inhibitors (e.g., THZ531) with DNA-damaging agents

  • CDK12 inhibitors with PARP inhibitors

  • CDK12 inhibitors with ETC inhibitors (e.g., IACS-010759)

• Response biomarkers:

  • Genomic signatures of CDK12 deficiency (tandem duplications)

  • Expression changes in DDR genes

  • Metabolic profiling

  • γH2AX and phospho-KAP1 induction

Bovine models may provide valuable insights for veterinary applications while informing human therapeutic development.

What are the optimal methods for screening CDK12 inhibitors using recombinant bovine CDK12?

For effective inhibitor screening using recombinant bovine CDK12:

• Assay formats:

  • In vitro kinase assays using:

    • Synthetic RNAPII CTD peptides

    • ATP consumption measurements (ADP-Glo)

    • Phospho-specific antibodies

  • Thermal shift assays to measure inhibitor binding

  • Surface plasmon resonance for binding kinetics

• Key controls:

  • Known CDK12 inhibitors (THZ531) as positive controls

  • Kinase-dead CDK12 mutants as negative controls

  • Other CDK family members to assess selectivity

• Screening cascade:

  • Primary biochemical screen → cell-based validation → target engagement → phenotypic profiling

• Phenotypic endpoints:

  • γH2AX and phospho-KAP1 induction

  • Transcriptional effects on DNA repair genes

  • Cell cycle analysis and BrdU incorporation

  • Mitochondrial function assays

These approaches enable comprehensive evaluation of potency, selectivity, and mechanism of action for CDK12 inhibitors.