AURKA Human

What is AURKA and what are its primary functions in human cells?

AURKA (also known as Aurora-2/BTAK/STK15) is a serine/threonine kinase that regulates multiple cell cycle events occurring from late S-phase through the M phase. Its primary functions include centrosome maturation, mitotic entry, centrosome separation, bipolar spindle assembly, chromosome alignment, cytokinesis, and mitotic exit .

AURKA activity and protein levels both increase from late G2 through the M phase, with peak activity in pro-metaphase . Beyond its well-established mitotic roles, more recent research has identified additional non-mitotic functions during interphase, including roles in neurite elongation and ciliary resorption .

How is AURKA activated and regulated during the cell cycle?

AURKA regulation follows a complex pattern of activation and inactivation through multiple mechanisms:

• Phosphorylation activation: AURKA is activated through phosphorylation of Threonine 288 (T288) on its activation loop .

• Co-factor dependent activation: AURKA binds to and phosphorylates the LIM domain-containing Ajuba protein during G2 phase, resulting in autophosphorylation of Aurora-A in its activating loop .

• TPX2-mediated protection: The microtubule-associated protein TPX2, when released from importins by GTPase Ran, binds to AURKA and protects it from inactivation by protein phosphatase 1 (PP1) .

• Inactivation mechanism: AURKA is inactivated through dephosphorylation of T288 by protein phosphatase 1 (PP1) or protein phosphatase 2A (PP2A) .

The mode of binding between TPX2 and AURKA induces conformational changes that resemble the intramolecular binding and activation of cAMP-dependent kinase .

How does AURKA's structure relate to its functional properties?

AURKA's structure consists of distinct domains that contribute to its function and regulation:

• N-terminal intrinsically disordered region (IDR): Contains the A-box degron motif that is critical for FZR1-dependent mitotic degradation .

• Kinase domain: Contains the activation loop with the critical T288 phosphorylation site required for kinase activity .

• C-terminal domain: Previously thought to contain a D-box degron, but recent research indicates it instead mediates essential structural features of the protein rather than acting as a true degron .

The full-length AURKA's degradation depends on both an intact N-terminal A-box region and proper conformational parameters provided by the C-terminal domain .

What are the molecular mechanisms governing AURKA degradation?

Recent research has revised our understanding of AURKA degradation mechanisms:

• APC/C^FZR1-mediated degradation: AURKA destruction is critically dependent on the anaphase-promoting complex (APC/C^FZR1) during mitotic exit and G1 phase .

• A-box degron function: Contrary to earlier models, the N-terminal intrinsically disordered region containing the A-box is sufficient to confer FZR1-dependent mitotic degradation, while the C-terminal domain does not function as a canonical D-box degron .

• QRVL motif: Both computational prediction and cellular assays suggest that the QRVL short linear interacting motif (SLiM) within the A-box functions as a phospho-regulated D-box .

• Conformational requirements: Degradation of full-length AURKA depends on an intact C-terminal domain because it provides critical conformational parameters that permit both activity and mitotic degradation .

This updated model explains why earlier studies identified both N- and C-terminal regions as important for degradation, even though only the N-terminal region contains a true degron motif.

How do cofactors influence AURKA activity and localization?

AURKA's activity is highly dependent on protein-protein interactions that affect its localization, activation state, and substrate selection:

• TPX2 activation mechanism: TPX2 binding has no effect on the turnover number of AURKA and does not change its reaction mechanism, but rather protects it from dephosphorylation by PP1 . This binding triggers conformational changes in AURKA.

• Targeting to spindle poles: The interaction with TPX2 targets AURKA to spindle microtubules at the poles, ensuring proper spatial regulation .

• Synergistic activation: In vivo, AURKA activation synergistically depends on (auto)phosphorylation within its activation segment (on T288) and TPX2 binding, potentially in combination with microtubule binding .

• Substrate specificity: Different cofactors may alter AURKA's substrate specificity, allowing it to function in multiple cellular contexts and pathways.

Understanding these cofactor relationships is critical for developing more specific inhibitors that could target particular AURKA functions while sparing others.

What is the relationship between AURKA and tumor suppressor pathways?

AURKA interacts with tumor suppressor pathways through multiple mechanisms:

• p53 regulation: AURKA interferes with p53 suppressor function through at least three mechanisms:

  • Direct phosphorylation of p53 at Ser315, facilitating MDM-2 mediated degradation

  • Phosphorylation of p53 at Ser215, inactivating its transcriptional activity

  • Regulation of p53 through the AKT/MDM-2 axis in gastric cancer cells

• TAp73 suppression: AURKA overexpression suppresses TAp73 in p53-deficient cancer cells. TAp73 has significant homology with p53 and plays an essential role in apoptosis induced by cytotoxic agents .

• Pro-survival pathway activation: AURKA up-regulates AKT phosphorylation at Ser473, promoting cell survival and preventing apoptosis .

• GSK-3β and β-catenin regulation: AURKA regulates GSK-3β and β-catenin in gastric cancer cells, potentially affecting Wnt signaling .

These interactions create a network where AURKA overexpression can simultaneously suppress multiple tumor suppressor functions while activating pro-survival pathways.

How does AURKA contribute to tumorigenesis and cancer progression?

AURKA's role in cancer development involves multiple mechanisms:

• Oncogenic transformation: AURKA activation can transform rodent fibroblast cells, establishing it as a bona fide oncogene .

• Genomic instability: AURKA overexpression leads to formation of multipolar mitotic spindles, inducing genome instability .

• Aneuploidy induction: A correlation exists between AURKA overexpression and aneuploidy in gastric cancer; clinical samples with AURKA amplification and overexpression showed aneuploidy and poor prognosis .

• Centrosome abnormalities: AURKA plays an important role in centrosome maturation, and centrosomal anomalies arise at early stages of tumor formation and expand with tumor progression .

• Cell survival promotion: Through regulation of AKT, AURKA supports cancer cell survival and prevents apoptosis .

• Checkpoint override: AURKA has been reported to override the spindle checkpoint activated by paclitaxel (Taxol) and nocodazole, contributing to drug resistance .

What is the prevalence of AURKA alterations across different cancer types?

AURKA gene amplification and/or overexpression patterns vary across cancer types:

AURKA overexpression has been reported to be significantly associated with higher grade tumors and poor prognosis across multiple cancer types .

What methodologies are most effective for analyzing AURKA expression and activity in tumor samples?

When analyzing AURKA in tumor contexts, researchers should consider multiple complementary approaches:

• Gene amplification analysis: FISH (Fluorescence In Situ Hybridization) to detect AURKA gene copy number changes at chromosome 20q13.2 .

• Expression analysis:

  • RT-qPCR for mRNA expression levels

  • Immunohistochemistry (IHC) for protein expression and localization in tissue samples

  • Western blotting for total protein levels in cell lysates

• Activity assessment:

  • Phospho-specific antibodies to detect activated AURKA (pT288)

  • Kinase activity assays using immunoprecipitated AURKA

  • Evaluation of phosphorylation status of downstream targets

• Functional analysis:

  • Centrosome counting to assess centrosome amplification

  • Spindle morphology analysis

  • Chromosome segregation error assessment

  • Aneuploidy measurement through karyotyping or flow cytometry

Correlating these measurements with clinical outcomes and tumor characteristics provides the most comprehensive picture of AURKA's role in a specific cancer context.

What are the current best practices for studying AURKA kinase activity in vitro and in vivo?

Optimal approaches for studying AURKA kinase activity include:

In vitro methods:

• Recombinant protein production: Expression and purification of wild-type and mutant AURKA proteins in bacterial or insect cell systems.

• Kinase assays: Using substrate peptides containing the consensus motif (R/K)X(S/T)(I/L/V) and measuring phosphorylation through:

  • Radioactive ATP incorporation

  • Phospho-specific antibody detection

  • Mass spectrometry analysis

• Co-factor effects: Testing activity with and without TPX2 or other known binding partners to assess co-factor dependence.

In vivo methods:

• FRET-based biosensors: For real-time monitoring of AURKA activity in living cells.

• Phospho-specific antibodies: Detecting T288 phosphorylation or substrate phosphorylation by immunofluorescence or western blotting.

• Genetic manipulation:

  • Conditional knockout models

  • Expression of kinase-dead mutants

  • RNAi-mediated knockdown

  • CRISPR/Cas9 genome editing

• Drug inhibition studies: Using specific AURKA inhibitors with appropriate controls to distinguish from effects on related kinases.

These approaches should be combined for comprehensive characterization of AURKA activity in different biological contexts.

How can researchers effectively distinguish between AURKA and AURKB functions in experimental settings?

Distinguishing between AURKA and AURKB functions requires careful experimental design:

• Subcellular localization analysis:

  • AURKA primarily localizes to centrosomes and spindle poles

  • AURKB is a chromosomal passenger protein with dynamic localization

• Specific inhibitors:

  • MLN8054 and MLN8237 show higher specificity for AURKA

  • ZM-447439 shows greater specificity for AURKB

  • Titration studies with concentration ranges that selectively inhibit one kinase

• Genetic approaches:

  • Selective knockdown with validated siRNAs targeting unique regions

  • Rescue experiments with kinase-specific mutants resistant to siRNA

  • CRISPR/Cas9 genome editing with careful validation

• Substrate analysis:

  • Focus on known specific substrates (e.g., TACC3 for AURKA)

  • Evaluate localization-dependent phosphorylation events

• Timing analysis:

  • Examine effects during specific cell cycle phases when one kinase is predominantly active

Researchers should always use multiple approaches and appropriate controls to confirm kinase-specific effects.

What are the key considerations when designing experiments to study AURKA degradation mechanisms?

When investigating AURKA degradation, researchers should consider:

• Cell cycle synchronization:

  • Methods to precisely arrest cells at specific phases

  • Time-course analysis following release from synchronization

• Protein stability measurements:

  • Cycloheximide chase assays with western blot analysis

  • Pulse-chase experiments with metabolic labeling

  • Live-cell imaging with fluorescently tagged AURKA

• Degron analysis:

  • Mutation of key residues in the A-box region (especially the QRVL motif)

  • Chimeric proteins with degron transfers to stable proteins

  • Structure-function analysis of the C-terminal domain

• APC/C^FZR1 manipulation:

  • FZR1 knockdown or knockout approaches

  • APC/C inhibitor studies (e.g., proTAME)

  • Co-immunoprecipitation to assess physical interaction

• Phosphorylation state:

  • Analysis of phospho-regulation of the A-box

  • Phosphomimetic and phospho-resistant mutations

  • Kinase and phosphatase inhibitor treatments

The study should account for the recent finding that the N-terminal IDR containing the A-box is sufficient for FZR1-dependent mitotic degradation, while the C-terminal domain provides essential structural features rather than acting as a canonical degron .

What are the current AURKA-specific inhibitors available for research, and how do they differ?

Several AURKA inhibitors have been developed with varying specificity profiles:

When selecting an inhibitor for research purposes, consider:

• The desired specificity for AURKA vs. AURKB

• Off-target effects on other kinases

• Compatibility with your experimental system

• Dose-dependent effects that may change selectivity

• Availability and cost for research applications

How can researchers address the challenges of AURKA inhibitor resistance in experimental models?

To investigate and overcome AURKA inhibitor resistance:

• Resistance mechanism characterization:

  • Genomic analysis for mutations in the AURKA ATP-binding pocket

  • Phosphoproteomics to identify compensatory signaling pathways

  • Expression analysis of AURKA binding partners that may affect inhibitor efficacy

• Combination strategies:

  • Target parallel pathways that may compensate for AURKA inhibition

  • Combine with cell cycle checkpoint inhibitors

  • Pair with inhibitors of AURKA-interacting proteins

• Alternative inhibition approaches:

  • Protein-protein interaction disruptors targeting AURKA-TPX2 binding

  • Degradation-inducing chimeric molecules (PROTACs) targeting AURKA

  • Allosteric inhibitors targeting sites outside the ATP-binding pocket

• Genetic verification:

  • CRISPR/Cas9 validation of AURKA dependency in resistant models

  • Overexpression of wild-type vs. mutant AURKA to confirm mechanism

Researchers should design resistance studies with clinically relevant concentrations and exposure times to maximize translational relevance.

What methodologies provide the most robust assessment of AURKA inhibitor efficacy in preclinical models?

A comprehensive evaluation of AURKA inhibitor efficacy should include:

• Biochemical confirmation:

  • In vitro kinase assays with purified AURKA protein

  • Cellular target engagement assays (e.g., cellular thermal shift assay)

  • Phosphorylation status of direct AURKA substrates

• Phenotypic validation:

  • Mitotic index measurements

  • Spindle pole defect quantification

  • Mitotic delay assessment by time-lapse microscopy

  • Cell cycle profile analysis by flow cytometry

• Efficacy in cellular models:

  • Proliferation/viability assays in cancer cell line panels

  • Colony formation assays for long-term effects

  • 3D spheroid growth inhibition

  • Apoptosis measurements

• In vivo evaluation:

  • Pharmacokinetic and pharmacodynamic relationship

  • Tumor growth inhibition in xenograft models

  • Patient-derived xenograft responses

  • Biomarker modulation correlating with efficacy

Attention should be paid to dose scheduling, potential off-target effects, and biomarkers of response that could translate to clinical applications.

What are the most promising emerging areas in AURKA research beyond mitotic functions?

Emerging non-mitotic functions of AURKA represent exciting research frontiers:

• Neurite elongation and neural development:

  • AURKA's role in microtubule dynamics during neurite outgrowth

  • Potential implications for neurodevelopmental disorders

  • Therapeutic relevance for neurodegenerative conditions

• Ciliary resorption and ciliopathies:

  • Mechanisms of AURKA activation in ciliary disassembly

  • Connection to ciliopathies and related disorders

  • Drug targeting potential for ciliary dysfunction diseases

• Metabolic regulation:

  • AURKA's potential roles in controlling cellular metabolism

  • Links to metabolic reprogramming in cancer

  • Relationship with nutrient sensing pathways

• DNA damage response:

  • Emerging functions in DNA repair mechanisms

  • Synthetic lethal interactions with DNA damage response pathways

  • Implications for combination therapies with DNA-damaging agents

• Immune regulation:

  • Potential roles in immune cell function

  • Implications for immuno-oncology approaches

  • Cross-talk with inflammatory signaling pathways

Investigating these non-canonical functions may reveal new therapeutic approaches and explain clinical observations not accounted for by mitotic-only models .

How might advanced structural biology techniques enhance our understanding of AURKA regulation?

Advanced structural approaches offer significant potential to resolve remaining questions about AURKA:

• Cryo-electron microscopy:

  • Structure determination of full-length AURKA including the IDR regions

  • Visualization of AURKA in complex with mitotic regulators

  • Structural basis for multiple active conformations

• Hydrogen-deuterium exchange mass spectrometry:

  • Mapping conformational changes upon activation

  • Identification of allosteric regulatory sites

  • Characterization of protein-protein interaction interfaces

• Single-molecule FRET:

  • Real-time analysis of AURKA conformational dynamics

  • Effects of phosphorylation on structural transitions

  • Influence of binding partners on conformational ensemble

• Molecular dynamics simulations:

  • Prediction of conformational changes upon binding

  • Analysis of intrinsically disordered region dynamics

  • Virtual screening for novel binding sites

• AlphaFold and related AI approaches:

  • Prediction of full-length AURKA structure including disordered regions

  • Modeling of protein-protein complexes

  • Structure-based drug design for novel inhibitors

These approaches could reveal how AURKA's multiple active conformations relate to its diverse functions and guide more specific therapeutic targeting strategies .

What are the challenges and opportunities in developing biomarkers to predict AURKA inhibitor response in patients?

The development of predictive biomarkers for AURKA inhibitor response faces several challenges and opportunities:

Challenges:

• Functional redundancy: Aurora kinases have overlapping functions that may compensate for targeted inhibition.

• Context-dependent roles: AURKA's functions vary across tissue types and genetic backgrounds.

• Multiple conformations: Different active conformations of AURKA may respond differently to inhibitors.

• Technical limitations: Measuring kinase activity directly in patient samples is challenging.

Opportunities:

• Genetic biomarkers:

  • AURKA amplification status as a stratification factor

  • Co-occurring genetic alterations that sensitize to AURKA inhibition

  • Synthetic lethal genetic interactions

• Protein biomarkers:

  • AURKA expression levels by IHC

  • Expression of critical cofactors like TPX2

  • Phosphorylation status of downstream targets

• Functional assays:

  • Ex vivo drug sensitivity testing of patient-derived cells

  • Organoid-based response prediction

  • Phosphoproteomics signatures of AURKA pathway activation

• Imaging biomarkers:

  • Centrosome abnormalities quantification

  • Mitotic index in pre- and post-treatment biopsies

  • PET imaging with selective AURKA inhibitor radiotracers

Integrating multiple biomarker approaches into early-phase clinical trials would accelerate clinical development and patient selection strategies.