Phospho-AURKA (S342) Antibody
Description
Specificity and Validation
The antibody is specifically designed to detect endogenous levels of AURKA protein only when phosphorylated at S342 . Validation studies typically employ phosphopeptide blocking experiments to confirm specificity, where the signal in immunohistochemistry or ELISA assays is blocked by pre-incubation with the phosphorylated peptide but not with the non-phosphorylated version . This demonstrates the antibody's ability to specifically recognize the phosphorylated epitope around S342.
Biological Significance of AURKA S342 Phosphorylation
Understanding the significance of S342 phosphorylation requires contextualizing it within AURKA's broader regulatory mechanisms and cellular functions.
AURKA Structure and Function
Aurora kinase A is a serine/threonine protein kinase of approximately 45.8 kDa that plays critical roles in mitotic progression. It regulates centrosome maturation, mitotic entry, bipolar spindle formation, and cytokinesis . The protein's activity is tightly controlled through multiple phosphorylation events that either activate or inhibit its kinase function.
Inhibitory Role of S342 Phosphorylation
While AURKA is commonly known to be activated by phosphorylation at T288 in its activation loop, research has revealed that phosphorylation at S342 on the αG helix of the C-terminal domain acts as an inhibitory mechanism . Studies in Xenopus models demonstrated that:
-
Auto-phosphorylation of Aurora A on residue S342 limits Aurora A activity
-
An S342D mutation (mimicking constitutive phosphorylation) completely blocks Aurora A activity
-
An S342A mutation renders Aurora A resistant to inhibition following DNA damage
This inhibitory phosphorylation provides a fine-tuning mechanism for regulating active Aurora A during mitosis. Intriguingly, Sarkissian et al. showed that the absence of phosphorylation at S342 predicted the activity of Aurora A better than did phosphorylation at the commonly studied T288 activation site .
Priming for S342 Phosphorylation
The auto-phosphorylation of Aurora A on S342 is induced by glycogen synthase kinase 3 (GSK-3), which places a "priming" phosphorylation on residues S283 and S284 located on the activation loop just prior to T288 . This creates a regulatory cascade where:
-
Serine to alanine substitutions at the S283 and S284 priming sites prevent their phosphorylation, resulting in a constitutively active form of Aurora A dephosphorylated on S342
-
Substitution of serine with aspartic acid at the priming sites mimics their constitutive phosphorylation and results in completely inactive Aurora A auto-phosphorylated on S342
Multiple Regulatory Phosphorylation Sites of AURKA
Aurora A activity is regulated through a complex interplay of multiple phosphorylation sites, with S342 representing just one important regulatory node.
Comparison of AURKA Phosphorylation Sites
The following table summarizes key phosphorylation sites on AURKA and their functions:
| Phosphorylation Site | Effect on AURKA | Function | Kinase Responsible |
|---|---|---|---|
| T288 | Activation | Increases kinase activity | Auto-phosphorylation, TPX2-induced |
| S342 | Inhibition | Decreases kinase activity | Auto-phosphorylation, GSK-3 primed |
| S51 | Protection | Protects from degradation | Ca²⁺/calmodulin-induced |
| S89 | Activation | Required for CDC25B phosphorylation | Nucleophosmin (B23) induced |
| S283/S284 | Priming | Primes for S342 phosphorylation | GSK-3 |
This complex phosphorylation pattern allows for precise spatial and temporal control of AURKA activity during cell cycle progression .
Interplay with Binding Partners
AURKA's activity is further modulated through interactions with various binding partners that can influence its phosphorylation state:
-
TPX2 binding promotes T288 auto-phosphorylation and protects from dephosphorylation
-
Ca²⁺/calmodulin binding induces phosphorylation at S51 and nearby residues
-
PAK kinases can phosphorylate both activating T288 and inhibitory S342 sites
These interactions create a complex regulatory network that fine-tunes AURKA activity in different cellular contexts.
Applications in Research
The Phospho-AURKA (S342) Antibody serves as a valuable tool for investigating the inhibitory regulation of Aurora kinase A in various research contexts.
Experimental Applications
This antibody has been validated for several experimental applications:
-
Immunohistochemistry (IHC): For detecting phosphorylated AURKA in tissue sections, typically at dilutions of 1:100-1:300
-
Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative analysis, typically at dilutions of 1:10000
-
Immunofluorescence (IF): For cellular localization studies, typically at dilutions of 1:50-200
These applications enable researchers to monitor the inhibitory phosphorylation state of AURKA across different experimental conditions and cellular states.
Research Considerations
When using Phospho-AURKA (S342) antibodies, several considerations should be taken into account:
-
Antibody quality variability: As with many phospho-specific antibodies, batch-to-batch variation may occur
-
Cross-reactivity: While commercial antibodies claim specificity, validation in the researcher's specific system is recommended
-
Comparison with total AURKA: Experiments often benefit from parallel detection of total AURKA protein to calculate phosphorylation ratios
-
Treatment conditions: Phosphatase inhibitors should be included in sample preparation to preserve phosphorylation states
Role in Cancer and Disease Studies
Aurora A kinase is frequently dysregulated in cancer, making the study of its inhibitory phosphorylation mechanisms particularly relevant to oncology research.
AURKA in Cancer
AURKA is considered an oncogene, with its chromosomal region often amplified in cancers of the breast, colon, pancreas, ovaries, bladder, liver, and stomach . Overexpression of AURKA is associated with genomic instability, centrosome amplification, and improper chromosome segregation during mitosis, contributing to aneuploidy and cancer progression.
Therapeutic Implications
Understanding the inhibitory role of S342 phosphorylation has important implications for cancer therapeutics:
-
Development of Aurora kinase inhibitors: Several inhibitors have been developed to target AURKA kinase activity, with the most advanced being alisertib
-
Potential for biomarker use: The phosphorylation status of S342 might serve as a biomarker for AURKA activity in tumors, potentially helping predict response to Aurora kinase inhibitors
-
Mechanism-based drug design: Knowledge of inhibitory phosphorylation could inspire new therapeutic approaches that promote S342 phosphorylation rather than directly inhibit kinase activity
Methodological Considerations in AURKA Research
When studying AURKA phosphorylation, researchers must be aware of several technical considerations that affect interpretation of results.
Antibody Quality Issues
Research on Aurora kinase A has been complicated by issues with antibody quality. In particular, phospho-T288 Aurora-A antibodies have shown variable quality and may cross-react with Aurora-B under some conditions . The quality of these antibodies has reportedly deteriorated over time, with multiple cross-reacting bands appearing in Western blots, raising cautions about interpretation of immunohistochemistry assessments .
Alternative Regulatory Mechanisms
Recent studies have identified alternative mechanisms of Aurora-A regulation beyond T288 phosphorylation, including:
-
Allosteric regulation by binding partners
-
Phosphorylation on alternative activating residues (S51, S98)
-
Dephosphorylation on inhibitory sites like S342
-
T288 phosphorylation by alternative kinases such as PAK enzymes
These findings highlight why focusing solely on T288 phosphorylation may provide an incomplete picture of AURKA regulation, making antibodies against other sites, including Phospho-AURKA (S342), important for comprehensive research.
Current Research and Future Directions
Research on AURKA phosphorylation at S342 continues to evolve, with several promising directions for future investigation.
Recent Advances
Recent studies have expanded our understanding of AURKA regulation beyond the canonical T288 phosphorylation site:
-
Recognition that absence of S342 phosphorylation may be a better predictor of AURKA activity than T288 phosphorylation
-
Identification of the priming role of GSK-3 in the phosphorylation cascade leading to S342 phosphorylation
-
Discovery of potential cross-talk between different phosphorylation sites, with some sites being phosphorylated by the same kinases that phosphorylate S342, such as PAK kinases
Future Research Directions
Several important questions remain to be addressed:
-
How is S342 phosphorylation spatially and temporally regulated during normal cell cycle progression?
-
What is the structural basis for the inhibitory effect of S342 phosphorylation?
-
How does S342 phosphorylation status correlate with response to Aurora kinase inhibitors in clinical settings?
-
Can promoting S342 phosphorylation serve as an alternative therapeutic strategy to direct kinase inhibition?
The Phospho-AURKA (S342) Antibody will continue to serve as an essential tool for investigating these and other questions related to the complex regulation of this critical mitotic kinase.
Product Specs
Target Background
- Cells deficient in ARID1A exhibit enhanced AURKA transcription, leading to persistent activation of CDC25C, a key protein for G2/M transition and mitotic entry. PMID: 30097580
- AURKA protein is overexpressed in almost all dermatofibrosarcoma protuberans tissues, and its levels correlate significantly with CD34 protein levels. PMID: 29682829
- Aurora A-dependent phosphorylation of CENP-A at the inner centromere safeguards chromosomes against tension-induced cohesion fatigue until the last kinetochore is attached to spindle microtubules. PMID: 29760389
- Aurora A kinase regulates kinetochore-microtubule dynamics of metaphase chromosomes, and Hec1 S69, a previously uncharacterized phosphorylation target site in the Hec1 tail, is a crucial Aurora A substrate for this regulation. PMID: 29187526
- Upon treatment with phorbol 12-myristate 13-acetate, THP-1 cells differentiate into monocytes by downregulating AURKA, leading to a reduction in H3S10 phosphorylation. The AURKA inhibitor alisertib accelerates the expression of the H3K27 demethylase KDM6B, dissociating AURKA and YY1 from the KDM6B promoter region and inducing differentiation. PMID: 29477140
- The two zinc fingers of BuGZ directly bind to AurA, and BuGZ coacervation appears to promote AurA activation during spindle assembly. PMID: 29074706
- Findings suggest that ATP/GTP binding protein like 2 (AGBL2) plays a critical oncogenic role in the pathogenesis of hepatocellular carcinoma (HCC) through modulation of immunity-related GTPase family, M protein (IRGM)-regulated autophagy and aurora kinase A (Aurora A) activity. PMID: 29126912
- Polymorphisms of the Aurora Kinase a Gene are associated with Breast Cancer Risk. PMID: 28647900
- A study suggests AURKA and TPX2 as potential stratification markers for taxane-based radiochemotherapy. In a lung adenocarcinoma cohort, high expression levels of AURKA and TPX2 were associated with improved overall survival specifically upon taxane-based radiochemotherapy. PMID: 28869599
- These data indicate that Aurora A plays a pivotal role in the regulation of Androgen receptor variant 7 expression and represents a novel therapeutic target in castrate-resistant prostate cancer. PMID: 28205582
- The inverse correlation between the VHL gene expression profile and alisertib sensitivity was further confirmed in human cancer xenograft models. These results collectively suggest that VHL loss could potentially serve as a biomarker for predicting the efficacy of AURKA inhibitors. PMID: 29845253
- LKB1 undergoes AURKA-mediated phosphorylation, which significantly compromises the LKB1/AMPK signaling axis, ultimately leading to increased proliferation, invasion, and migration of non-small cell lung cancer cells. PMID: 28967900
- Epithelial ovarian cancer (EOC) cell apoptosis rate was repressed after treatment with lncRNA TUG1 mimic and promoted after treatment with lncRNA TUG1 inhibitor. AURKA expression, but not CLDN3, SERPINE1, or ETS1 expression, was inversely regulated by lncRNA TUG1 mimic and inhibitor. In conclusion, lncRNA TUG1 promotes cell proliferation and inhibits cell apoptosis by regulating AURKA in EOC cells. PMID: 30200102
- Metformin disrupts the malignant behavior of oral squamous cell carcinoma via a novel signaling pathway involving Late SV40 factor/Aurora-A. Findings indicate that this novel Late SV40 Factor and Aurora-A-signaling inhibition supports the rationale for using metformin as a potential therapeutic agent for oral squamous cell carcinoma. PMID: 28465536
- The present study confirms the importance of pAURKA in the development of gastric adenocarcinoma and reveals a novel functional link between PTEN, AURKA, and pAURKA activation. PMID: 29512701
- The role of four AURKA single nucleotide polymorphisms on hepatocellular carcinoma susceptibility PMID: 29333101
- AURKA overexpression is associated with chronic myeloid leukemia. PMID: 29387948
- The data suggest that AKA is the vertebrate ancestral gene, and that AKB and AKC resulted from gene duplication in placental mammals. PMID: 29283376
- Expression of AURKA and CHEK1 was linked with unfavorable outcome in patients. Our data describe a synthetic lethality interaction between CHEK1 and AURKA inhibitors with potential translational applications in clinical settings. PMID: 28847989
- These findings suggest that Aurora A SNP at codon 57 may predict disease outcome and response to alisertib in patients with solid tumors. PMID: 29122619
- lncRNA TUG1 is associated with advanced disease and worse prognosis in adult AML patients, and it induces AML cell proliferation and represses cell apoptosis by targeting AURKA. PMID: 29654398
- Aurora A can individually shorten cilia when they are growing, but requires interaction with never in mitosis-kinase 2 (Nek2) when cilia are being absorbed. Inhibition of Aurora A increases cilia number. PMID: 29141582
- In patients who received alisertib for advanced or metastatic urothelial carcinoma, longer progression-free survival was observed in carriers of the minor allele A of rs2273535 in AURKA than in patients homozygous for the major allele T. PMID: 28155045
- This combination also reduces the growth of PDAC xenografts in vivo. Mechanistically, inhibiting methyltransferases of the H3K9 pathway in cells arrested in G2-M after targeting AURKA decreases H3K9 methylation at centromeres, induces mitotic aberrations, triggers an aberrant mitotic check point response, and ultimately leads to mitotic catastrophe. PMID: 28442587
- Prostate cancer cells expressing an S273A mutant of CHIP have attenuated AR degradation upon 2-ME treatment compared with cells expressing wild-type CHIP, supporting the idea that CHIP phosphorylation by Aurora A activates its E3 ligase activity for the AR. PMID: 28536143
- Our results indicate that AURKA plays an important role in the activation of EIF4E and cap-dependent translation. Targeting the AURKA-EIF4E-c-MYC axis using alisertib is a novel therapeutic strategy applicable to everolimus-resistant tumors and/or subgroups of cancers that exhibit overexpression of AURKA and activation of EIF4E and c-MYC. PMID: 28073841
- Aurora-A may serve as a predictive biomarker of radiation response and a therapeutic target to reverse radiation therapy resistance. PMID: 28404933
- We also propose a model for the stabilization mechanism in which binding to Aurora-A alters how N-Myc interacts with SCF(FbxW7) to disfavor the generation of Lys48-linked polyubiquitin chains. PMID: 27837025
- Results identified AURKA to be significantly upregulated in the lung squamous cell carcinoma tissues of smoking patients and may play an important role in diagnosis and prognosis. PMID: 28949095
- Authors conclude that AURKA may revive dormant tumor cells via FAK/PI3K/Akt pathway activation, thereby promoting migration and invasion in laryngeal cancer. PMID: 27356739
- Our identification of the novel interaction between Aurora A and H-Ras as a mechanism by which Aurora A can activate Ras-MAPK signaling opens the way for studies into perturbation of the Aurora A/H-Ras interaction and its effect on Ras-MAPK signaling. PMID: 28177880
- MiR-124-3p has a significant impact on the proliferation, migration, and apoptosis of bladder cancer cells by targeting AURKA. PMID: 28269755
- Taken together, our data suggest that Aurora-A plays an important role in the suppression of autophagy by inhibiting the phosphorylation of Akt, which in turn prevents autophagy-induced apoptosis in prostate cancer. PMID: 28269749
- Results show that overexpression of Aurora-A and PTGS2 occurs in colon polyps and has a reverse correlation with miR-137 in both colon polyps and colorectal cancer tissue, suggesting that AURKA and PTGS2 expression is under the regulation of mir-137. PMID: 27764771
- SIX3 is a novel negative transcriptional regulator and acts as a tumor suppressor that directly represses the transcription of AURKA and AURKB in astrocytoma. PMID: 28595628
- This report provides clear evidence that overexpression of the AURKA, SKA3, and DSN1 genes strongly correlates with the progression of colorectal adenomas to colorectal cancer. PMID: 27329586
- Although research biopsies were obtained on only a few patients, they did confirm pharmacodynamic effects of the drug. These effects, however, suggest inhibition of Aurora B rather than Aurora A, which is consistent with pre-clinical data that show dose-dependent effects on both. PMID: 27502708
- Aurora A kinase is hyperphosphorylated in early mitosis under oxidative stress, which may disrupt the function of Aurora A in mitotic spindle formation. PMID: 28017898
- Our findings suggest that AURKA (rs911160) and AURKB (rs2289590) polymorphisms could affect GC risk. Further validation studies in larger and multi-ethnic populations are needed to elucidate their functional impact on the development of GC. PMID: 28843004
- Possible models of regulation of Lck by Aurora-A during T cell activation are described in the review. PMID: 27910998
- Our study demonstrates that KCTD12 binds to CDC25B and activates CDK1 and Aurora A to facilitate the G2/M transition and promote tumorigenesis. Aurora A phosphorylates KCTD12 at serine 243 to trigger a positive feedback loop, thereby potentiating the effects of KCTD12. Therefore, the KCTD12-CDC25B-CDK1-Aurora A axis has important implications for cancer diagnoses and prognoses. PMID: 28869606
- Our findings revealed novel regulatory mechanisms of p53 in regulating Aurora-A gene expression in non-small cell lung carcinoma cells. PMID: 28884479
- HIP2 regulates mitotic spindle alignment. SHIP2 is expressed in G1 phase, whereas Aurora A kinase is enriched in mitosis. SHIP2 binds Aurora A kinase and the scaffolding protein HEF1, promoting their basolateral localization at the expense of their luminal expression connected with cilia resorption. PMID: 27926875
- Aurora kinase inhibitor CCT137690 induces necrosis-like death in pancreatic ductal adenocarcinoma cells via RIPK1, RIPK3, and MLKL signaling. PMID: 28764929
- Our data indicate that hnRNP Q1 is a novel trans-acting factor that binds to Aurora-A mRNA 5'-UTRs and regulates its translation, which increases cell proliferation and contributes to tumorigenesis in colorectal cancer. PMID: 28079881
- A central role of Aurora kinase A (AURKA) in promoting Epithelial-to-mesenchymal transition and cancer stem cell phenotypes via ALDH1A1. PMID: 28193222
- Switching Aurora-A kinase on and off at an allosteric site has been documented. (Review) PMID: 28342286
- This is the first report of F31I and V57I polymorphisms in AURKA gene in breast cancer in Iran. PMID: 28906374
- High Aurora A kinase expression is associated with triple-negative breast cancer. PMID: 27593935
- Results provide evidence that AURKA is a target for the VHL E3 ligase ubiquitination. PMID: 28114281
