Phospho-YAP1 (S127) Recombinant Monoclonal Antibody
Description
Mechanistic Role of YAP1 S127 Phosphorylation
Phosphorylation at S127 by LATS1/2 kinases inactivates YAP1 by promoting its cytoplasmic retention. This prevents its interaction with TEAD transcription factors, thereby suppressing oncogenic gene expression (e.g., CTGF, CYR61) . The antibody serves as a critical tool to monitor this regulatory mechanism.
Key Pathway Interactions
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Hippo Signaling: MST1/2 → LATS1/2 → YAP1 (S127 phosphorylation → cytoplasmic retention) .
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Cancer Implications: Loss of S127 phosphorylation correlates with YAP1 nuclear translocation and tumorigenesis .
Western Blot Validation
Observed vs. Predicted MW:
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Predicted: 54 kDa (YAP1 isoform)
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Observed: 75 kDa (post-translational modifications, dimerization, or cross-reactivity) .
Immunohistochemistry (IHC)
Cholangiocarcinoma (CCA) Models
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S127 Phosphorylation: Reduced in CCA cells, enabling nuclear YAP1 accumulation and oncogene expression (CTGF, MCL-1) .
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SFK Inhibition: Dasatinib (SRC/YES/FYN/LCK/LYN inhibitor) reduces YAP1 Y357 phosphorylation (tyrosine site) but does not affect S127 phosphorylation, indicating independent regulatory pathways .
Key Experiment:
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S127A-YAP1 Mutant: Resistant to LATS1/2 phosphorylation; retains nuclear localization unless treated with SFK inhibitors (e.g., dasatinib), confirming SFK-dependent nuclear retention .
Technical Considerations
Product Specs
The synthesized DNA sequence corresponding to the pS127-YAP1 monoclonal antibody was cloned into the plasmid and then transfected into the cell line for expression. The product was purified through the affinity-chromatography method and obtained the specifically phosphorylated YAP1 recombinant monoclonal antibody. This anti-S127-YAP1 recombinant antibody is a rabbit IgG and has been tested in scientific applications, including ELISA, WB, and IHC. It specifically reacts with human YAP1 phosphorylated at Ser 127 residue.
The transcriptional coactivator YAP1 is involved in cell proliferation, cell-cell interactions, organ size, and tumorigenesis. YAP1's nuclear activity is dependent on post-transcriptional changes and nuclear translocation. Androgen reduces Androgen attenuates the inactivating phospho–Ser-127 modification of YAP1 and promotes YAP1 nuclear abundance and activity. The traditional Hippo kinase cascade is known to phosphorylate YAP1 at Ser127, which is required for its cytoplasmic localization and inactivation.
Target Background
YAP1 is a transcriptional regulator that can act both as a coactivator and a corepressor. It is a critical downstream regulatory target in the Hippo signaling pathway, which plays a pivotal role in organ size control and tumor suppression by restricting proliferation and promoting apoptosis. The core of this pathway is composed of a kinase cascade wherein STK3/MST2 and STK4/MST1, in complex with its regulatory protein SAV1, phosphorylates and activates LATS1/2 in complex with its regulatory protein MOB1, which in turn phosphorylates and inactivates YAP1 oncoprotein and WWTR1/TAZ.
YAP1 plays a key role in tissue tension and 3D tissue shape by regulating cortical actomyosin network formation. It acts via ARHGAP18, a Rho GTPase activating protein that suppresses F-actin polymerization. YAP1 also plays a key role in controlling cell proliferation in response to cell contact. Phosphorylation of YAP1 by LATS1/2 inhibits its translocation into the nucleus to regulate cellular genes important for cell proliferation, cell death, and cell migration.
The presence of TEAD transcription factors are required for YAP1 to stimulate gene expression, cell growth, anchorage-independent growth, and epithelial mesenchymal transition (EMT) induction. YAP1 suppresses ciliogenesis via acting as a transcriptional corepressor of the TEAD4 target genes AURKA and PLK1. In conjunction with WWTR1, YAP1 is involved in the regulation of TGFB1-dependent SMAD2 and SMAD3 nuclear accumulation. Additionally, YAP1 activates the C-terminal fragment (CTF) of ERBB4 (isoform 3).
- Studies indicate that the transcriptional co-activators YAP and TAZ recently emerged as key mediators of the biological effects observed in response to extracellular matrix (ECM) elasticity and cell shape. PMID: 22895435
- Loss of p53 or LKB1 relieves DVL-linked reciprocal inhibition between the Wnt and nuclear YAP activity. PMID: 29895829
- LncRNA B4GALT1-AS1 promotes OS cells stemness and migration via recruiting HuR to enhance YAP activity. PMID: 30182452
- The miR-590-5p/YAP axis may be an important novel mechanism in the pathogenesis of CD and colorectal cancer. PMID: 29912317
- The observed decrease in total YAP levels in endothelial cells exposed to pulsatile flow is due to degradation via a proteasome-independent mechanism. PMID: 29758328
- Disruption of TAZ/YAP activity alleviates tumor burden in Lats1/2-deficient mice and inhibits human malignant peripheral nerve sheath tumors cell proliferation. PMID: 29438698
- Molecular mechanisms of YAP protein in the lung physiological conditions and lung diseases.[review] PMID: 30385178
- Report that YAP is subject to non-proteolytic, K63-linked polyubiquitination by the SCF(SKP2) E3 ligase complex (SKP2), which is reversed by the deubiquitinase OTUD1. The non-proteolytic ubiquitination of YAP enhances its interaction with its nuclear binding partner TEAD, thereby inducing YAP's nuclear localization, transcriptional activity, and growth-promoting function. PMID: 29891922
- Dual governing of YAP and COX-2 may lead to the discovery of promising therapeutic strategies for HCC patients. PMID: 29505957
- YAP messenger RNA (mRNA) and protein expression levels were less in preeclamptic placentas. Yes-associated protein enhanced cell invasion, reduced the cellular apoptotic response, and had no effect on proliferation. PMID: 29303055
- Study indicated that lncRNAATB functions as a ceRNA to promote MM proliferation and invasion by enhancing Yes associated protein 1 expression by competitively sponging microRNA miR5905p. PMID: 29956757
- O-GlcNAcylation of YAP was required for high-glucose-induced liver tumorigenesis. PMID: 28474680
- These results unveil a novel mechanism of YAP activation in cancer and open the possibility to target GR to prevent cancer stem cells self-renewal and chemoresistance. PMID: 28102225
- High YAP1 expression is associated with the pathogenesis of gastric cancer. PMID: 30066917
- YAP1 as a fluid mechanosensor that functions to regulate genes that promote metastasis. PMID: 28098159
- These observations revealed the importance of YAP in promoting TKI-resistance and combined YAP inhibition can be a potential therapy delaying the occurrence of TKI-resistance in lung adenocarcinoma. PMID: 29321482
- High Yap expression is associated with resistance to EGFR inhibitors in colorectal cancer. PMID: 30106444
- High YAP1 expression is associated with malignant melanoma. PMID: 30106445
- Data (including data from studies in knockout mice) suggest that KIBRA plays an important role in regulating HPO activity, YAP signaling, and actin cytoskeletal dynamics in podocytes; expression of KIBRA and YAP plus phosphorylation of YAP are up-regulated in glomeruli of patients with focal segmental glomerulosclerosis. (KIBRA = kidney/brain protein-KIBRA; HPO = hepatopoietin protein; YAP = Yes associated protein-1) PMID: 28982981
- YAP/TAZ mechanotransduction integrates with cell-cell communication pathways for fine-grained orchestration of stem cell decisions. PMID: 28513598
- YAP1 regulates the SOX2 expression in urothelial carcinoma of the bladder.COX2 and YAP1 signaling pathways are connected with each other to induce SOX2 expression, cancer stem cell enrichment, and acquired resistance to chemotherapy in urothelial carcinoma of the bladder. PMID: 29180467
- Our study showed for the first time that MLK7-AS1 interacted with miR-375 to promote proliferation, metastasis, and EMT process in ovarian cancer cells through upregulating YAP1. PMID: 30249278
- Loss of DLG5 expression promoted breast cancer progression by inactivating the Hippo signaling pathway and increasing nuclear YAP. PMID: 28169360
- YAP enhances gastric cancer cell proliferation. PMID: 30021363
- miR-205 targets YAP1 and inhibits proliferation and invasion of thyroid cancer cells. PMID: 29845281
- FAK controls the nuclear translocation and activation of YAP in response to mechanical activation and submit that the YAP-dependent process of durotaxis requires a cell with an asymmetric distribution of active and inactive FAK molecules. PMID: 29070586
- The tumor promoting role of YAP is involved in SHP2 which functions as a tumor promoter in vitro but as a tumor suppressor in vivo. PMID: 29699904
- The combined treatment significantly sensitized the A549/DDP cells to DDPinduced growth inhibition by reducing YAP promoter activity. PMID: 29901163
- These results reveal a novel positive feedback loop involving CD44S and YAP1, in which CD44S functions as both an upstream regulator and a downstream effector of YAP1 in hepatocellular carcinoma. PMID: 29649630
- Hypoxic stress in the hepatocellular carcinoma (HCC)cells promoted YAP binding to HIF-1a in the nucleus and sustained HIF-1a protein stability to bind to PKM2 gene and directly activates PKM2 transcription to accelerate glycolysis. PMID: 30180863
- PTEN lipid phosphatase inactivation abolished the MOB1-LATS1/2 interaction, decreased YAP phosphorylation and finally promoted YAP nuclear translocation, which enhanced the synergistic effect of YAP-TEAD, thus inducing cell proliferation and migration. PMID: 30134988
- Up-regulation of COPB2 inhibited cell apoptosis and promoted cell growth and tumorigenesis through up-regulating YAP1 expression in lung adenocarcinoma. PMID: 29674272
- In the present review, we focus on the functions of YAP/TAZ in cancer, discuss their potential as new therapeutic targets for tumor treatment, and provide valuable suggestions for further study in this field. PMID: 29749524
- HuR acts as a tumor promoter by enhancing YAP expression in osteosarcoma cells. PMID: 29597092
- These results indicate a negative link between miR-622 and YAP1 and further confirm that YAP1 is a direct target of miR-622, suggesting that miR-622 could be a new important therapeutic strategy for gliomas treatment. PMID: 28796324
- YAP1 and LATS1 can be considered as new prognostic factors in clear cell renal cell carcinoma. PMID: 29850494
- Yap1 expression in aggressive thyroid cancer. PMID: 28120182
- These results suggested that silibinin induced glioblastoma cell apoptosis concomitant with autophagy which might be due to simultaneous inhibition of mTOR and YAP and silibinin induced autophagy exerted a protective role against cell apoptosis in both A172 and SR cells. PMID: 29780826
- These observations suggest that Zyxin promotes colon cancer tumorigenesis in a mitotic-phosphorylation-dependent manner and through CDK8-mediated YAP activation. PMID: 29967145
- We discover that Verteporfin (VP) inhibits YAP-induced bladder cancer cell growth and invasion via repressing the target genes' expression of the Hippo signaling pathway. PMID: 29725256
- Myeloid Zinc Finger 1 and GA Binding Protein Co-Operate with Sox2 in Regulating the Expression of Yes-Associated Protein 1 in Cancer Cells. PMID: 28905448
- LncBRM and YAP1 signaling may serve as biomarkers for diagnosis and potential drug targets for hepatocellular carcinoma. PMID: 27905400
- LncARSR interacts with Yes-associated protein (YAP) to block its phosphorylation by LATS1, facilitating YAP nuclear translocation. PMID: 27886176
- YAP contributes to glioma cell migration and invasion by regulating N-cadherin and Twist, as well as cytoskeletal reorganization. PMID: 29306996
- LIFR attenuates tumor metastasis by suppressing YAP expression, suggesting that LIFR may serve as a potential target for clear cell renal cell carcinoma treatment. PMID: 29902078
- Serine/threonine-protein kinase proteins, also known as P21-activated kinase (PAK), and the mechanosensitive factor, Yes-associated protein 1 (YAP-1) are core mediators of pro-fibrotic integrin beta-1 signaling. PMID: 27535340
- The gene transcription and protein expression of YAP may be involved in the development of prostate cancer and may be considered a potential target for the treatment of such cancers. PMID: 29286134
- Upregulated miR-200a enhances treatment resistance via antagonizing TP53INP1 and YAP1 in breast cancer. PMID: 29329575
- Bioinformatics analysis and luciferase reporter assays indicated that miR625 targeted the 3'untranslated region of Yesassociated protein 1 (YAP1). PMID: 29257207
- Functional evaluation of a HTM cell monolayer using a permeability assay demonstrated that the inhibition of YAP and TAZ attenuated the DEX-induced impairment of permeability. These findings suggest that YAP and TAZ play pivotal roles in the DEX-induced cytoskeletal changes of HTM cells, and reveal novel potential mechanisms for the development and progression of glaucoma. PMID: 29115373
Q&A
What is the significance of YAP1 phosphorylation at Ser127 in cellular signaling?
YAP1 (Yes-associated protein 1) serves as a critical transcriptional coactivator within the Hippo signaling pathway, which regulates cell proliferation, organ size, and tumorigenesis. Phosphorylation at Ser127 is a pivotal post-translational modification that modulates YAP1 activity by promoting its cytoplasmic retention and inhibiting nuclear translocation. This phosphorylation event is mediated by the Hippo kinase cascade, particularly LATS1/2 kinases . The cytoplasmic sequestration of phosphorylated YAP1 prevents its interaction with TEAD transcription factors, thereby suppressing its role in gene expression related to cell growth and survival .
Phosphorylation at Ser127 has profound implications for cancer biology, as dysregulation of this modification can lead to aberrant cellular proliferation and metastasis. In addition, androgen signaling has been shown to attenuate Ser127 phosphorylation, facilitating YAP1 nuclear localization and enhancing its transcriptional activity . Understanding this mechanism is crucial for exploring therapeutic interventions targeting YAP1 in cancer treatment.
How can the Phospho-YAP1 (S127) Recombinant Monoclonal Antibody be applied in experimental research?
The Phospho-YAP1 (S127) Recombinant Monoclonal Antibody is a versatile tool validated for use in multiple experimental applications, including Western blotting (WB), immunohistochemistry (IHC), immunofluorescence (IF), and enzyme-linked immunosorbent assays (ELISA) . These methodologies enable researchers to investigate the phosphorylation status of YAP1 under various experimental conditions.
Western Blotting:
Western blot analysis is commonly employed to detect phosphorylated YAP1 in cell lysates. The antibody demonstrates high specificity for Ser127-phosphorylated YAP1 and can differentiate between phosphorylated and non-phosphorylated forms based on molecular weight shifts observed on SDS-PAGE gels . For optimal results, researchers should use recommended dilutions (e.g., 1:1000–1:5000) and include appropriate controls such as untreated or kinase-inhibited samples .
Immunohistochemistry:
In IHC applications, this antibody can be used to visualize the spatial distribution of phosphorylated YAP1 within tissue sections. It is particularly useful for examining the localization of YAP1 in cancerous tissues versus normal tissues . Recommended dilutions range from 1:50 to 1:200, depending on tissue type and fixation method .
Immunofluorescence:
IF enables the visualization of phosphorylated YAP1 at subcellular levels using fluorescence microscopy. This approach is ideal for studying dynamic changes in YAP1 localization during cellular processes such as apoptosis or differentiation . Researchers should titrate antibody concentrations to achieve optimal signal-to-noise ratios.
ELISA:
Quantitative detection of phosphorylated YAP1 can be achieved through ELISA assays using this antibody. This method is particularly useful for high-throughput screening of phosphorylation levels across multiple samples or conditions .
What are the best practices for optimizing antibody performance in experimental setups?
To ensure reliable results when using the Phospho-YAP1 (S127) Recombinant Monoclonal Antibody, researchers should adhere to several best practices:
Sample Preparation:
Proper sample preparation is critical for detecting phosphorylated proteins. For Western blotting, lysates should be prepared using lysis buffers containing phosphatase inhibitors to preserve phosphorylation states . Similarly, tissue fixation methods for IHC must be optimized to prevent loss of antigenicity.
Dilution Optimization:
Antibody dilutions should be optimized based on application type and sample characteristics. For example, WB typically requires higher dilutions (e.g., 1:1000–1:5000), while IHC may require lower dilutions (e.g., 1:50–1:200) .
Validation Controls:
Include positive controls such as cells treated with kinase activators or inhibitors to validate antibody specificity. Negative controls using non-phosphorylated YAP1 or unrelated phosphoproteins can help confirm specificity .
Blocking Conditions:
Blocking buffers containing nonfat dry milk or bovine serum albumin are recommended to minimize nonspecific binding during WB or IHC procedures .
How does phosphorylation at Ser127 affect YAP1's interaction with other signaling molecules?
Phosphorylation at Ser127 alters YAP1's ability to interact with TEAD transcription factors and other signaling molecules within the Hippo pathway. This modification facilitates binding to 14-3-3 proteins, which sequester YAP1 in the cytoplasm . Additionally, phosphorylated YAP1 undergoes ubiquitination via β-transducin repeat-containing E3 ubiquitin ligase complexes, leading to its proteasomal degradation .
These molecular interactions underscore the importance of Ser127 phosphorylation as a regulatory checkpoint within cellular signaling networks. Dysregulation of these processes has been implicated in cancer progression and resistance to therapy.
What experimental models are suitable for studying Phospho-YAP1 (S127)?
Several experimental models have been validated for studying Phospho-YAP1 (S127), including:
Cell Lines:
Human cancer cell lines such as HeLa cells are widely used for investigating YAP1 phosphorylation dynamics under controlled conditions . NIH/3T3 fibroblasts treated with serum starvation or kinase inhibitors also serve as robust models for studying Hippo pathway activation .
Animal Models:
Mouse models genetically engineered to express mutant forms of YAP1 provide valuable insights into the physiological roles of Ser127 phosphorylation in vivo .
Tissue Samples:
Analyzing tissue samples from patients with cancer can reveal correlations between Ser127 phosphorylation levels and disease progression .
How can data contradictions regarding Phospho-YAP1 (S127) be resolved?
Data contradictions often arise due to variability in experimental conditions or differences in antibody specificity. To address these issues:
Standardization:
Standardize experimental protocols across laboratories, including antibody dilutions, incubation times, and detection methods .
Cross-validation:
Use multiple antibodies targeting different epitopes of phosphorylated YAP1 to confirm findings .
Replication:
Replicate experiments using independent samples or alternative detection methods such as mass spectrometry-based phosphoproteomics .
By implementing these strategies, researchers can enhance reproducibility and resolve discrepancies.
