Phospho-PDCD4 (Ser67) Antibody
Description
Introduction to PDCD4 and Phosphorylation at Ser67
PDCD4 is a nuclear-cytoplasmic shuttling protein that inhibits tumorigenesis by suppressing AP-1-dependent transcription, translation initiation, and neoplastic transformation . Phosphorylation at Ser67 by kinases like Akt/PKB modulates PDCD4’s stability and function:
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Biological Role:
Key Features of Phospho-PDCD4 (Ser67) Antibody
The antibody is a rabbit polyclonal reagent designed for research applications. Key characteristics include:
Regulatory Mechanisms
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Akt-Mediated Phosphorylation:
Akt phosphorylates PDCD4 at Ser67 and Ser457 in a PI3K-dependent manner, as shown in HEK293 cells . This dual phosphorylation reduces PDCD4’s ability to suppress AP-1-responsive promoters . -
Functional Consequences:
Technical Validation
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Western Blot Data:
The antibody detects phosphorylation of PDCD4 at Ser67 in HEK293 and melanoma cells treated with Akt activators, with signal loss observed in S67A mutants . -
Subcellular Localization:
IF studies using this antibody show nuclear translocation of phosphorylated PDCD4 in response to Akt activation .
Applications in Cancer Research
Product Specs
Target Background
- Research has demonstrated that elevated levels of miR-96 in glioblastoma multiforme (GBM) cells confer resistance to radiation therapy by targeting PDCD4. This finding suggests that PDCD4 could be a potential therapeutic target for treating GBM. PMID: 30066909
- A recent study revealed a dynamic regulatory relationship between PDCD4 and factors crucial for epithelial-mesenchymal transition (EMT). This study established a comprehensive, functional role for PDCD4 in laryngeal carcinoma, suggesting its potential modulation by the STAT3-miR-21 pathway. PMID: 29510060
- Studies indicate that the localization of Pdcd4 to the cytoplasm might be responsible for suppressing target mRNA translation and inducing apoptosis. PMID: 29442268
- PDCD4 and PTEN were identified as functional targets of miR-21. PMID: 30074182
- Multiple studies provide compelling evidence that PDCD4, a novel tumor suppressor gene, is often downregulated or absent in colorectal cancer (CRC), contributing to the suppression of CRC deterioration. [review] PMID: 30243936
- In highly malignant glioma, the expression levels of PDCD4 mRNA and PDCD5 mRNA were significantly reduced compared to low-grade glioma and control groups. These findings suggest that PDCD4 mRNA and PDCD5 mRNA expressions hold promise as targets for glioma diagnosis and treatment. PMID: 29921407
- miR-21 may play a critical role in promoting salivary adenoid cystic carcinoma progression by downregulating PDCD4 and PTEN, while simultaneously upregulating Bcl-2. PMID: 29328455
- Compared to normal cervical tissues, the expression of PDCD4 is decreased in cervical cancer tissues, while miR-150 levels are increased. PMID: 29091902
- Research indicates that lncRNA-XIST, acting as a miRNA sponge, prevents miR-21-5p from suppressing the expression of PDCD4, contributing to the progression of osteosarcoma (OS). This finding suggests that the XIST/miR-21-5p/PDCD4 axis could serve as a potential biomarker or therapeutic target for OS. PMID: 29048648
- miR206 was found to promote the onset of SANFH by inducing apoptosis and suppressing osteoblast proliferation, a process dependent on the inhibition of PDCD4. PMID: 29115490
- Studies have identified PDCD4 as a target gene of miR-93, which directly targets its 3'-UTR and downregulates its expression. Re-expression of PDCD4 was observed to attenuate hepatocellular carcinoma (HCC) cell invasion and migration induced by miR-93. Conversely, knockdown of PDCD4 promoted HCC cell migration and invasion through the EMT pathway. PMID: 28748353
- Reduced expression of PDCD4 was observed in decidual and chorionic tissues, as well as peripheral blood mononuclear cells from patients experiencing missed abortions. PMID: 29017439
- miR503 was found to promote tumor growth and invasion by directly targeting PDCD4. PMID: 28849168
- A novel mechanism of Pdcd4 action as a translation inhibitor and tumor suppressor has been proposed. PMID: 28853972
- Collectively, research highlights a crucial role for miR-23a/b as oncomiRs in gastric cancer through the inhibition of PDCD4 translation. These findings may provide valuable insights into the molecular mechanisms underlying gastric carcinogenesis and pave the way for new treatment strategies for gastric cancer. PMID: 28981115
- lncRNA CASC9 acts as an oncogene by negatively regulating PDCD4 expression. This regulation is achieved by recruiting EZH2, a histone methyltransferase, which alters the H3K27me3 level. This study underscores the significance of lncRNA CASC9 as a potential biomarker for esophageal squamous cell carcinoma (ESCC) diagnosis and prognosis. PMID: 28854977
- Exosomes derived from cisplatin-resistant oral squamous cell carcinoma (OSCC) cells were found to transfer miR-21 to OSCC parental cells, inducing cisplatin resistance by targeting PTEN and PDCD4. PMID: 28910982
- These results emphasize the significant role of the ROS-STAT3-miR-21-PDCD4 signaling axis in arsenic-induced carcinogenesis. PMID: 27876813
- PDCD4 is expressed in the cytoplasm of glandular epithelium of control endometrium and undergoes changes during the menstrual cycle. Compared to the proliferative phase of normal endometrium, PDCD4 expression is downregulated in the proliferative phase of both eutopic and ectopic endometrium. Furthermore, PDCD4 expression lacks cyclic variation in eutopic endometrium of adenomyosis patients, indicating progesterone resistance. PMID: 27765271
- Research confirmed that PDCD4 is downregulated in non-small cell lung cancer (NSCLC). PDCD4 is a functional target for miR-155 at both the transcriptional and post-transcriptional levels. PMID: 28842954
- Studies have shown that miR-208a-3p suppresses apoptosis in gastric cancer cells by targeting PDCD4. PMID: 27634902
- PDCD4 plays a role in negatively controlling the conversion of stromal fibroblasts into cancer-associated fibroblasts. PMID: 27542230
- Research has identified PDCD4 as a novel RSK substrate. Studies demonstrate that RSK-mediated phosphorylation of PDCD4 at S76 promotes PDCD4 degradation. PMID: 27028868
- Research evaluated the relative expression levels of miR-196a2 and three of its selected apoptosis-related targets; ANXA1, DFFA, and PDCD4, in a sample of gastrointestinal cancer patients. PMID: 29091952
- In colorectal cancer tissues, the Sin1 protein, but not its mRNA, was significantly upregulated, while Pdcd4 protein was downregulated. This suggests that the loss of Pdcd4 might correlate with Sin1 protein levels, but not mRNA levels, in colorectal cancer. PMID: 28692058
- miRNA-96 is significantly overexpressed in glioma tissues. Moreover, miRNA-96 plays a critical role in apoptosis by inhibiting the expression of PDCD4 in glioma. PMID: 26846266
- Supporting the clinical relevance of these findings, an inverse correlation was found between ErbB-2/Stat3 nuclear co-expression and PDCD4 expression in ErbB-2-positive primary invasive breast cancer. PMID: 26212010
- This study highlights an oncomiR role for miR-181b in regulating PDCD4 in colorectal cancer, suggesting that miR-181b could be a novel molecular therapeutic target for treating colorectal cancer. PMID: 27647131
- Elevated PDCD4 expression contributes to polycystic ovary syndrome by affecting obesity, insulin resistance, lipid metabolism disorders, and granulosa cell apoptosis. PMID: 26868993
- Research revealed that microRNA 200a inhibits erythroid differentiation by targeting PDCD4 and THRB. PMID: 27734462
- The expression of miR-21 and PDCD4 at the mRNA level was evaluated using quantitative real-time polymerase chain reaction, while the protein level of PDCD4 was determined by Western blotting. Locked nucleic acid-anti-miR-21 transfection was found to be associated with a significant reduction in metastatic properties, as assessed by the in ovo model. PMID: 28347230
- Data indicate that programmed cell death 4 (PDCD4) was identified as a target of ubiquitin-specific protease 4 (USP4), which plays a role as a tumor suppressor. PMID: 27430936
- PDCD4 down-regulation is implicated in the progression of several types of solid tumors. PMID: 27852288
- Results show that exosome-shuttling miR-21 represses PDCD4 protein expression by binding to the 3'-UTR in esophageal cancer cells. PMID: 27035745
- miR-21, acting on PDCD4, which interacts with Twist1 and represses the expression of Twist1, contributes to the EMT induced by arsenite in transformed bronchial epithelial cells. PMID: 25445583
- Data show that microRNA miR-93 directly binds to the 3' untranslated regions (3'-UTR) of the programmed cell death 4 (PDCD4) mRNA transcript and inhibits PDCD4 translation in gastric cancer cells. PMID: 27021515
- miR-21 can confer drug resistance to 5-FU in pancreatic cancer cells by regulating the expression of tumor suppressor genes. The target genes of miR-21, PTEN, and PDCD4, can rescue 5-FU sensitivity and restore phenotypic characteristics disrupted by miR-21. PMID: 26864640
- In HeLa cells, phosphorylation of HuR by ERK8 prevents its binding to PDCD4 mRNA, allowing miR-21-mediated degradation of PDCD4. PMID: 26595526
- miR-183 might function as an oncogene by regulating gastric cancer cell proliferation, apoptosis, and metastasis. The oncogenic effect of miR-183 may relate to its direct targeting of PDCD4. PMID: 26961483
- PDCD4 inhibits cell growth through the PI3K/Akt signaling pathway in non-small cell lung cancer. PMID: 26802652
- This study describes the regulation of PDCD4 specifically in tonsil squamous cell carcinoma by miR-499 and miR-21, documenting the loss of PDCD4 in oropharyngeal squamous cell carcinoma. PMID: 26867589
- Unusually, HuR was also found to bind to miR-21 directly, preventing its interaction with the PDCD4 3'-UTR, thereby preventing the translation repression of PDCD4. PMID: 26189797
- miR-21 plays a role in upregulating PTEN, RECK, and PDCD4 in glioma. PMID: 26284486
- PDGF-BB stimulates cell proliferation through microRNA-21-mediated PDCD4 downregulation, leading to the development of thyroid adenoma. PMID: 26943153
- Results indicate that PDCD4 could be a novel candidate tumor suppressor gene in hepatocellular carcinoma and that promoter hypermethylation is an important mechanism for its downregulation and a good predictor of survival. PMID: 26871813
- These findings suggest that miR-21 and PDCD4 could be potential biomarkers for malignant melanoma and might provide future treatment targets. PMID: 26150475
- Research proposes that SRSF3 could act as a coordinator of PDCD4 protein expression through two mechanisms on two alternatively spliced mRNA isoforms. PMID: 26773498
- Low PDCD4 expression increases osteosarcoma cells' resistance to apoptosis. PMID: 26276504
- These findings support the feasibility of future diagnostic and gene therapy efforts for prostate cancer based on IL-6, miR-21, and PDCD4. PMID: 26252635
- RT-qPCR and Western blotting showed that miR-183 negatively regulated PDCD4 protein expression but had no impact on the mRNA expression of PDCD4. PMID: 26063221
Q&A
What is PDCD4 and what role does phosphorylation at Ser67 play in its function?
PDCD4 (Programmed Cell Death 4) is a tumor suppressor protein that inhibits translation initiation and cap-dependent translation. It functions by:
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Hindering interactions between eIF4A1 and eIF4G
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Inhibiting the helicase activity of eIF4A
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Modulating JUN kinase activation
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Down-regulating MAP4K1 expression to inhibit invasion-driving events
Phosphorylation at Ser67 is a critical regulatory mechanism that affects PDCD4's function and localization. When phosphorylated at Ser67 by Akt kinase, PDCD4 undergoes significant changes:
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Association with 14-3-3 proteins is promoted
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Subsequent degradation via the ubiquitin-proteasome pathway is triggered
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Its ability to interfere with AP-1-responsive promoter transactivation by c-Jun is significantly decreased
This phosphorylation represents a key switch mechanism that controls PDCD4's tumor suppressor activity, with dysregulation of this process implicated in various cancers.
Sample preparation significantly impacts phospho-specific antibody performance. Follow these methodological guidelines:
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Cell Lysis Optimization:
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Use phosphatase inhibitor cocktails during lysis to preserve phosphorylation
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Include protease inhibitors to prevent degradation
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Perform lysis under cold conditions (4°C) to minimize enzymatic activity
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Sample Stimulation Protocols:
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Controls and Validation:
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Include phosphatase-treated samples as negative controls
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Use competitive blocking with phosphopeptides to confirm specificity
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Compare results with total PDCD4 antibodies to normalize phosphorylation levels
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For Western blots specifically, loading 30 μg of protein per lane has been validated for detecting phospho-PDCD4 , though optimization may be required for your specific cell type.
How can researchers resolve contradictory findings regarding kinase-specific phosphorylation of PDCD4?
The literature contains competing models for PDCD4 regulation. To resolve these contradictions:
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Kinase-Specific Phosphorylation Analysis:
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Cell Type Considerations:
In one study, inhibition of mTORC1 or the proteasome prevented refeeding-associated decreases in PDCD4 abundance, while Ser67 phosphorylation appeared insensitive to nutrient status , suggesting complex regulatory mechanisms that vary by context.
PDCD4 phosphorylation status correlates with cancer development and progression:
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Cancer-Specific Phosphorylation Patterns:
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Mechanistic Impact:
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Phosphorylation-induced degradation reduces PDCD4's tumor suppressor activity
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Nuclear localization following phosphorylation alters transcriptional regulation
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Reduced interaction with eIF4A may enhance translation of oncogenic mRNAs
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Therapeutic Implications:
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Inhibiting kinases targeting PDCD4 may restore its tumor suppressor function
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Monitoring phospho-PDCD4 levels may serve as a biomarker for pathway activation
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Developing strategies to stabilize PDCD4 represents a potential therapeutic approach
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Research indicates that oncogenes within the Ras/MAPK pathway may regulate PDCD4 localization in melanoma cells , suggesting a direct mechanism linking oncogenic signaling to PDCD4 inactivation through phosphorylation.
What advanced validation strategies should be employed for phospho-specific PDCD4 antibodies?
For rigorous phospho-antibody validation:
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Specificity Controls:
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Lambda phosphatase treatment of samples should eliminate signal
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Competing peptide assays with phosphorylated vs. non-phosphorylated peptides
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Use of PDCD4 knockout models followed by reconstitution with WT or S67A mutants
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Cross-reactivity testing with other phosphorylated proteins
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Signal Validation:
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Confirm antibody specificity using physiological stimuli known to induce phosphorylation
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Employ multiple antibodies from different sources targeting the same phospho-site
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Use mass spectrometry to confirm phosphorylation status
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Technical Validation Matrix:
| Validation Method | Purpose | Expected Outcome |
|---|---|---|
| Peptide competition | Specificity | Signal reduction with phospho-peptide only |
| Phosphatase treatment | Phospho-specificity | Complete signal loss |
| S67A mutant expression | Site specificity | No detection of mutant protein |
| Signal induction | Physiological relevance | Increased signal with Akt activation |
| Kinase inhibition | Pathway specificity | Reduced signal with appropriate inhibitors |
Some commercial antibodies undergo rigorous validation, including chromatography purification using epitope-specific phosphopeptides and removal of non-phospho-specific antibodies , ensuring high specificity for the phosphorylated state.
How can quantitative phosphoproteomics be integrated with antibody-based detection of PDCD4 phosphorylation?
Integrating phosphoproteomics with antibody-based approaches provides comprehensive insights:
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Complementary Methodologies:
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Use phosphoproteomics to discover novel PDCD4 phosphorylation sites
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Apply antibody-based techniques for targeted validation and functional studies
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Employ SILAC or TMT labeling for quantitative comparison across conditions
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Research Examples:
This integrated approach allows researchers to place PDCD4 phosphorylation within broader signaling networks and contextualize site-specific modifications with global phosphorylation dynamics.
What are the optimal experimental designs for studying dynamic regulation of PDCD4 phosphorylation?
To capture the dynamic regulation of PDCD4 phosphorylation:
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Temporal Analysis:
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Perform time-course experiments after stimulation:
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Short intervals (0-60 min) for immediate phosphorylation events
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Extended periods (1-24 h) for degradation and protein abundance effects
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Use synchronized cell populations to control for cell cycle effects
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Stimulus-Response Relationships:
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Titrate stimulus concentrations to establish dose-response curves
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Compare different stimuli (growth factors, serum, PMA) for pathway specificity
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Include both physiological and pharmacological stimuli
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Multi-site Phosphorylation Analysis:
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Monitor both Ser67 and Ser457 phosphorylation simultaneously
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Create phosphorylation state-specific mutants (S67A, S457A, double mutants)
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Assess functional consequences of site-specific phosphorylation
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Experimental Controls Table:
