AURKB Human

What is the primary function of AURKB in human cells?

AURKB functions as the catalytic component of the Chromosomal Passenger Complex (CPC), which orchestrates key mitotic events. Methodologically, researchers studying AURKB function should employ:

• Time-lapse microscopy with fluorescently-tagged AURKB to track dynamic localization (chromosomes in prophase → centromeres in metaphase → central spindle in anaphase → midbody during cytokinesis)

• Phospho-specific antibodies against validated substrates (particularly Histone H3 at Ser10)

• Selective inhibitors (such as AZD1152/Barasertib) with appropriate controls to dissect AURKB-specific functions from other Aurora kinases

• Co-immunoprecipitation studies to analyze interactions with other CPC components (INCENP, Survivin, Borealin)

Cell-type specific expression patterns should be considered when studying AURKB function, as its expression correlates with proliferative capacity, showing highest levels in rapidly dividing cells and tissues.

How are AURKB+ cells detected and quantified in human tissue samples?

Robust detection of AURKB requires complementary approaches:

• Immunostaining: Co-stain with cell-type specific markers (as seen with AURKB+/cTnT+ cardiomyocytes in cardiac research)

• Flow cytometry: For quantitative assessment across large cell populations

• Western blotting: Use validated antibodies with appropriate positive controls (mitotic cell extracts)

• mRNA detection: RT-qPCR with intron-spanning primers or RNA-seq analysis

For accurate quantification of AURKB+ cells, researchers should:

• Include cell cycle markers to differentiate proliferating cells

• Perform statistical analysis across multiple tissue sections

• Use automated imaging software to reduce observer bias

• Validate findings with multiple detection methods

In cardiac research, increased percentages of AURKB+ cardiomyocytes were detected in specific experimental conditions, such as Mettl3-deficient neonatal mice, indicating enhanced proliferative capacity .

What are the key substrates of AURKB in human cells?

AURKB phosphorylates numerous substrates critical for mitotic progression. Methodologically, researchers should:

• Validation approaches:

  • In vitro kinase assays with purified components

  • Phospho-specific antibodies to monitor modification status

  • Site-directed mutagenesis (phospho-mimetic/dead mutations)

  • Functional assays to assess phenotypic consequences

• Key validated substrates include:

  • Histone H3 (Ser10, Ser28): Controls chromosome condensation

  • CENP-A (Ser7): Regulates kinetochore assembly

  • MCAK: Controls microtubule depolymerase activity

  • MgcRacGAP: Regulates cytokinesis completion

  • Vimentin: Coordinates intermediate filament dynamics during division

For comprehensive substrate identification, researchers should combine candidate approaches with unbiased phosphoproteomic screens comparing wild-type versus AURKB-inhibited conditions.

How does AURKB expression change during the cell cycle?

AURKB expression and activity are tightly regulated throughout the cell cycle. To properly study this:

• Synchronization techniques: Use double thymidine block, nocodazole arrest, or mitotic shake-off to obtain cell populations at specific cycle phases

• Live-cell imaging: Employ fluorescent reporters to track AURKB expression/localization in real-time

• Western blotting: Analyze protein levels across synchronized populations

• Flow cytometry: Combine with DNA content analysis to correlate expression with cell cycle phase

Key findings include:

• Low expression in G1 phase

• Increasing levels through S phase

• Peak expression during G2/M transition

• Protein degradation after mitotic exit via APC/C-mediated ubiquitination

Researchers should distinguish between mRNA expression, protein abundance, and kinase activity, as these parameters may not directly correlate throughout the cell cycle.

What experimental tools are available for manipulating AURKB in human cells?

Researchers have multiple options for modulating AURKB function:

• Chemical tools:

  • Small molecule inhibitors: AZD1152/Barasertib (AURKB-selective), VX-680/MK-0457 (pan-Aurora)

  • Degraders: PROTACs targeting AURKB for proteasomal degradation

  • Activity-based probes: For monitoring AURKB activity in situ

• Genetic approaches:

  • siRNA/shRNA: For transient or stable knockdown

  • CRISPR-Cas9: For knockout or endogenous tagging

  • Overexpression systems: Wild-type or mutant variants

• Advanced technologies:

  • Analog-sensitive alleles: For selective inhibition of engineered AURKB

  • Optogenetic tools: Light-inducible activation/inhibition

  • Degron systems: For rapid protein depletion

When selecting tools, researchers should consider temporal dynamics, as complete AURKB inhibition may prevent mitosis, complicating interpretation of results.

How can researchers distinguish between AURKB activity and other Aurora kinases?

Distinguishing AURKB from AURKA and AURKC remains challenging due to sequence similarity. Methodological approaches include:

• Pharmacological strategies:

  • Careful titration of inhibitor concentrations

  • Use of selective inhibitors with verification of target engagement

  • Correlation with substrate phosphorylation patterns

• Genetic approaches:

  • Selective knockdown with validation of specificity

  • Rescue experiments with inhibitor-resistant mutants

  • CRISPR knockout with complementation studies

• Localization-based discrimination:

  • AURKB: Chromosomes → centromeres → midbody

  • AURKA: Centrosomes → spindle poles

  • AURKC: Primarily in meiotic cells

• Substrate specificity:

  • AURKB preferentially phosphorylates H3S10, CENP-A

  • AURKA primarily targets TPX2, TACC3

Researchers should employ multiple complementary approaches and include appropriate controls to confidently attribute observed effects to specific Aurora kinases.

What roles does AURKB play in heart regeneration?

The search results indicate AURKB serves as a marker for proliferating cardiomyocytes during heart regeneration . To investigate this:

• Detection strategies:

  • Co-immunostaining for AURKB and cardiac markers (cTnT)

  • Quantification of AURKB+ cardiomyocytes in different experimental conditions

  • Correlation with other proliferation markers (pH3, Ki67)

• Functional approaches:

  • Cardiac-specific AURKB manipulation (knockout/overexpression)

  • Administration of selective inhibitors in regeneration models

  • Lineage tracing of AURKB+ cardiomyocytes during regeneration

• Molecular mechanism investigation:

  • Analysis of AURKB-mediated phosphorylation events in cardiomyocytes

  • Integration with regulatory pathways (such as Mettl3-mediated m6A modification)

  • Identification of cardiomyocyte-specific AURKB substrates

Research has shown increased percentages of AURKB+ cardiomyocytes in Mettl3-deficient neonatal mice and in models with mutated m6A consensus sequences in Fgf16, corresponding with enhanced regenerative capacity .

How does AURKB dysregulation contribute to chromosomal instability in cancer?

AURKB aberrations promote chromosomal instability through multiple mechanisms. Methodological approaches include:

• Quantifying chromosomal instability parameters:

  • Micronuclei formation assays

  • Fluorescence in situ hybridization for aneuploidy detection

  • Live-cell imaging of chromosome segregation errors

  • Single-cell sequencing for copy number variation analysis

• Mechanistic investigations:

  • Assessment of kinetochore-microtubule attachment stability

  • Measurement of spindle assembly checkpoint function

  • Analysis of sister chromatid cohesion timing

  • Quantification of cytokinesis failure rates

• Expression modulation studies:

  • Titrated overexpression to determine threshold effects

  • Dominant-negative approaches to disrupt function

  • Correlation of expression levels with CIN markers in patient samples

AURKB overexpression can override spindle assembly checkpoint function, while reduced activity impairs error correction mechanisms. Both scenarios promote genomic instability, highlighting the requirement for precise AURKB regulation.

What are the latest methods for mapping AURKB-dependent phosphoproteomes?

State-of-the-art phosphoproteomic approaches include:

• Experimental design considerations:

  • Acute vs. sustained AURKB inhibition comparisons

  • Cell synchronization to capture mitotic phosphorylation events

  • Subcellular fractionation to enrich for relevant compartments

• Advanced techniques:

  • Multiplexed quantitative phosphoproteomics (TMT, iTRAQ)

  • Complementary enrichment strategies (IMAC, TiO2, phospho-antibodies)

  • Data-independent acquisition mass spectrometry

  • Parallel reaction monitoring for targeted analysis

• Bioinformatic analysis:

  • Motif analysis for AURKB consensus sequences (R/K-X-S/T)

  • Integration with protein-protein interaction networks

  • Kinase-substrate enrichment analysis (KSEA)

  • Temporal clustering of phosphorylation events

• Validation approaches:

  • In vitro kinase assays with purified components

  • Phospho-specific antibodies for key substrates

  • Mutational analysis of identified phosphosites

  • Correlation with AURKB localization patterns

Integration of phosphoproteomic data with other omics approaches provides comprehensive insights into AURKB-regulated processes across different cellular contexts.

How can CRISPR-Cas9 be optimized for studying AURKB function?

CRISPR-Cas9 optimization for AURKB studies requires:

• Guide RNA design considerations:

  • Target functional domains (kinase domain, activation loop)

  • Use algorithms that minimize off-target effects

  • Design multiple guides and validate independently

  • Consider exon essentiality and potential for compensatory splicing

• Editing strategies:

  • Complete knockout vs. domain-specific mutations

  • Homology-directed repair for precise modifications

  • Endogenous tagging for visualization/purification

  • Base or prime editing for specific amino acid changes

• Temporal control systems:

  • Inducible Cas9 expression

  • Conditional guide RNA expression

  • Degron-tagging for rapid protein depletion

  • Auxin-inducible degron systems

• Validation requirements:

  • Deep sequencing to confirm edits and assess off-targets

  • Rescue experiments with wild-type or mutant AURKB

  • Phenotypic analysis across multiple independent clones

  • Evaluation of compensatory mechanisms

As AURKB is essential for cell division, researchers should implement conditional approaches rather than constitutive knockout strategies to study dynamic functions.

What are the methodological challenges in developing AURKB-specific inhibitors?

Developing selective AURKB inhibitors presents several challenges:

• Structural similarity obstacles:

  • 70% sequence identity in ATP-binding pockets across Aurora kinases

  • Limited structural differences for selective targeting

  • Shared binding modes with many kinase inhibitors

• Selectivity assessment approaches:

  • Kinome-wide profiling against 300+ kinases

  • Cellular thermal shift assays to confirm target engagement

  • NanoBRET target engagement in live cells

  • Correlation of cellular effects with biochemical potency

• Structure-based design strategies:

  • Fragment-based screening for unique binding modes

  • Targeting allosteric sites rather than ATP-binding pocket

  • Exploiting unique features in activation loop conformations

  • Development of covalent inhibitors for specific cysteines

• Alternative approaches:

  • PROTAC-based degraders with AURKB-selective binding moieties

  • Targeting protein-protein interactions specific to AURKB

  • Substrate-competitive inhibitors

  • Conformation-selective inhibitors

Researchers must balance selectivity with pharmacokinetic properties and cellular penetration to develop effective tools for biological studies and potential therapeutic applications.

How does post-translational modification regulate AURKB activity?

AURKB undergoes multiple post-translational modifications that regulate its function. Methodologically:

• Key modifications to study:

  • Autophosphorylation at T232 (activation loop): Essential for kinase activity

  • Phosphorylation by CDK1 (S331): Primes for activation

  • Ubiquitination: Controls protein stability and degradation

  • SUMOylation: Affects CPC assembly and chromosome targeting

  • Acetylation: Influences chromatin interactions

• Detection strategies:

  • Phospho-specific antibodies for known sites

  • Mass spectrometry for unbiased PTM mapping

  • Western blotting with mobility shift analysis

  • Immunoprecipitation with modification-specific antibodies

• Functional analysis approaches:

  • Site-directed mutagenesis (phospho-mimetic/dead mutations)

  • In vitro reconstitution with modified components

  • Pharmacological inhibition of modifying enzymes

  • Proteomic analysis following perturbation of PTM pathways

• Temporal dynamics assessment:

  • Synchronization methods to capture cell cycle-dependent changes

  • Live-cell imaging with PTM-specific biosensors

  • Rapid isolation techniques to preserve labile modifications

These approaches provide insights into how PTMs collectively regulate AURKB localization, activity, substrate specificity, and protein-protein interactions throughout the cell cycle.