RASSF1A Antibody

What is RASSF1A and what makes it structurally unique?

RASSF1A is a tumor suppressor protein encoded by the RASSF1 gene located at chromosome 3p21.3. The protein contains a Ras Association Domain (RA) that enables binding to Ras in a GTP-dependent manner to mediate apoptosis. RASSF1A features an amino-terminal cysteine-rich region similar to the diacylglycerol binding domain (C1 domain) found in protein kinase C family proteins and a carboxy-terminal putative Ras-association (RA) domain. Unlike RASSF1C, which is a smaller isoform, RASSF1A contains the full amino-terminal C1 domain critical for its tumor suppressor function . Seven alternatively spliced transcript variants of the RASSF1 gene have been reported, encoding distinct isoforms . RASSF1A functions as a scaffold protein without enzymatic activity, connecting various signaling pathways including HIPPO and apoptotic regulation.

How does RASSF1A regulate cell cycle progression?

RASSF1A acts as a cell cycle regulator primarily at the G1/S-phase transition. Research demonstrates that RASSF1A inhibits cell cycle progression by preventing the accumulation of cyclin D1 protein, which is essential for cells to pass through the Rb family cell cycle restriction point and enter S phase . This regulation occurs post-transcriptionally, likely at the level of translational control rather than through direct transcriptional repression of the cyclin D1 gene . Experimental evidence shows that ectopic expression of RASSF1A in lung and breast tumor-derived epithelial cells results in growth arrest by significantly reducing BrdU incorporation (only 20±5% of RASSF1A-expressing cells were BrdU-positive compared to 67±8% for RASSF1C-expressing cells) . Importantly, this cell cycle arrest can be relieved by artificially driving expression of cyclin A or expression of the viral Rb family inhibitor E7, confirming the mechanism operates through the cyclin D1/Rb pathway .

What epigenetic mechanisms regulate RASSF1A expression in cancer?

Epigenetic inactivation of RASSF1A through hypermethylation of its CpG-island promoter region is one of the most common events in human cancers . This silencing mechanism has been documented in 80-100% of small cell lung cancer (SCLC) cell lines and tumors, 30-40% of non-small cell lung cancer (NSCLC), 49-62% of breast cancers, 67-70% of primary nasopharyngeal cancers, 91% of primary renal cell carcinomas, and 100% of renal cell carcinoma lines . In stem cells, epigenetic regulation of the Rassf1A promoter maintains stemness by preventing RASSF1A expression, which would otherwise drive differentiation . During normal differentiation, promoter demethylation allows GATA1-mediated RASSF1A expression . These findings provide critical insights for researchers designing experiments to study RASSF1A reactivation or developing therapeutic approaches targeting epigenetic modifications.

What are the validated applications for RASSF1A antibodies in research?

RASSF1A antibodies have been validated for several critical research applications. Specifically, the eB114-10H1 monoclonal antibody has been reported and tested for:

• Western blotting (WB): Detects RASSF1A as a 45 kDa band in HeLa cell extracts at a recommended concentration of 1-10 μg/mL .

• Immunoprecipitation (IP): Successfully pulls down RASSF1A and its interacting partners .

• Protein complex analysis: Can be used to study RASSF1A interactions with proteins like MOAP-1, MST kinases, and components of the HIPPO pathway .

• Phosphorylation studies: Useful for investigating ATM-mediated phosphorylation at Ser131 and other post-translational modifications .

When designing experiments, researchers should note that the eB114 antibody specifically recognizes the RASSF1A isoform through a synthetic peptide corresponding to the splice junction region of RASSF1A, enabling discrimination between RASSF1A and other isoforms like RASSF1C . Proper titration of the antibody is recommended for optimal performance in each specific assay.

How can I optimize Western blotting protocols for RASSF1A detection?

For optimal RASSF1A detection via Western blotting, follow these research-validated approaches:

• Lysate preparation: Use a lysis buffer containing 1% NP-40, 10 mM Tris (pH 7.5), 0.25 mM sodium deoxycholate, 1 mM MgCl₂, 1 mM EGTA, 5 mM β-mercaptoethanol, 10% glycerol, 150 mM NaCl, with phosphatase inhibitors (50 mM sodium fluoride, 1 mM orthovanadate, 80 mM β-glycerophosphate) and protease inhibitor cocktail .

• Antibody concentration: The eB114-10H1 antibody works optimally at 1-10 μg/mL concentration, but should be carefully titrated for your specific cell type or tissue .

• Molecular weight verification: Confirm RASSF1A detection at approximately 45 kDa to ensure specificity .

• Controls: Include both positive controls (HeLa cell extract is recommended) and negative controls (RASSF1A-null cells or RASSF1A knockdown samples via RNAi) .

• Signal enhancement: For low abundance samples, consider immunoprecipitation before Western blotting to concentrate the protein.

By following these methodological guidelines, researchers can achieve consistent and specific detection of RASSF1A in various experimental systems.

What are recommended protocols for studying RASSF1A phosphorylation?

RASSF1A undergoes phosphorylation by ATM kinase at Ser131 upon DNA damage, which is critical for its tumor suppressor function . To study RASSF1A phosphorylation:

• Metabolic labeling: Conduct ³²P labeling by washing cells 48 hours post-transfection and preincubating with phosphate-free MEM for 10 minutes before adding 0.5 mCi of ³²P to each 35-mm culture plate. After 4 hours of incubation, lyse cells and immunoprecipitate RASSF1A variants using anti-Myc or RASSF1A-specific antibodies .

• Phospho-specific antibodies: While not explicitly mentioned in the search results, phospho-specific antibodies against Ser131 would be valuable for detecting ATM-mediated phosphorylation.

• Mutation analysis: Compare wild-type RASSF1A with phosphorylation site mutants (such as S131F or A133S) to understand the functional significance of phosphorylation .

• Kinase inhibitors: Use ATM inhibitors to confirm the kinase responsible for phosphorylation.

• Functional assays: Correlate phosphorylation status with functional outcomes such as cell cycle arrest, p73-dependent apoptosis, or protein-protein interactions .

These methodological approaches allow researchers to comprehensively investigate how phosphorylation affects RASSF1A's tumor suppressor functions and signaling capabilities.

How does RASSF1A connect RAS and HIPPO signaling pathways?

RASSF1A serves as a critical molecular bridge between RAS and HIPPO signaling pathways through several mechanisms:

• RAS binding: RASSF1A contains a RAS Association domain that enables it to form an endogenous complex with activated K-RAS. This interaction is GTP-dependent and positions RASSF1A as a K-RAS-regulated scaffold protein .

• HIPPO pathway activation: Under the influence of K-RAS, RASSF1A binds the MST (HIPPO) kinases, connecting RAS to regulation of the HIPPO pathway. This interaction ultimately regulates the activity of the transcriptional co-activating proteins YAP and TAZ .

• Apoptotic signaling: RASSF1A connects K-RAS to multiple proapoptotic signaling pathways. When bound to K-RAS, RASSF1A binds the protein MOAP-1, which can then bind and activate the proapoptotic executor BAX .

• Stem cell regulation: RASSF1A acts as a functional switch between pluripotency and differentiation by regulating YAP activity. In the absence of RASSF1A (as in stem cells), YAP functions within a quaternary association of YAP-TEAD and β-catenin-TCF3 complexes on the Oct4 distal enhancer, promoting pluripotency. When RASSF1A is expressed during differentiation, it prevents YAP from contributing to this complex and instead promotes a YAP-p73 transcriptional program enabling differentiation .

• Pathway balance: Transgenic mouse models show that RASSF1A deficiency not only reduces HIPPO pathway activity but also causes upregulation of RAS mitogenic signaling pathways in tumors and even in normal tissue without activated RAS .

Understanding these complex interactions provides researchers with multiple experimental approaches to study RASSF1A's role in integrating these critical signaling pathways.

How can I use transgenic models to study RASSF1A function in cancer?

Transgenic models provide powerful systems to investigate RASSF1A's tumor suppressor function:

• K-RAS/RASSF1A compound models: Researchers have demonstrated the utility of combining K-RAS activation (K-RAS12D) with RASSF1A deletion. This experimental approach revealed that RASSF1A deficiency dramatically enhances K-RAS-driven lung tumor development . Specifically, mice with both K-RAS activation and RASSF1A deletion (RAS12D+/RASSF1A-) showed:

  • Reduced HIPPO pathway activity

  • Upregulation of inflammation

  • Enhanced RAS mitogenic signaling in both tumors and normal tissue

• Experimental validation: When designing similar studies, researchers should include appropriate controls:

  • Wild-type (RASSF1A+/+)

  • RASSF1A knockout alone (RASSF1A-/-)

  • K-RAS activation alone (RAS12D+/RASSF1A+)

  • Combined model (RAS12D+/RASSF1A-)

• Molecular analysis techniques: To comprehensively assess pathway alterations, employ:

  • Immunohistochemistry for tissue-specific expression patterns

  • Western blotting for protein expression levels

  • RNA-seq for transcriptional changes

  • Phosphoproteomic analysis for signaling pathway activation

• Functional readouts: Assess tumor burden, proliferation rates, apoptotic indices, and inflammatory markers in these models to understand the functional consequences of RASSF1A loss.

These experimental approaches confirm the importance of RASSF1A inactivation in K-RAS-driven lung tumor development and provide a framework for investigating therapeutic strategies targeting these pathways .

What is the significance of RASSF1A polymorphisms in cancer research?

RASSF1A polymorphisms can significantly impact its tumor suppressor function and provide valuable research tools:

• Key polymorphisms: Several critical RASSF1A variants have been identified:

  • S131F mutation: Found in tumor cell lines, this variant fails to block cyclin D1 accumulation and cell cycle progression. In experimental studies, cells expressing RASSF1A(S131F) showed 65±11% BrdU incorporation compared to just 20±5% for wild-type RASSF1A .

  • A133S mutation: Another variant isolated from tumor cell lines that cannot properly inhibit cell proliferation. Cells expressing this variant showed 55±5% BrdU incorporation .

  • p.Ala133Ser polymorphism: A single-nucleotide polymorphism located near the ATM activation site, converting alanine (G allele) to serine (T allele) at residue 133. Secondary protein structure prediction studies suggest this changes disrupts an alpha helix containing the ATM recognition site .

• Functional implications: These polymorphisms alter a putative mTOR/ATM family kinase substrate site and inhibit phosphorylation of RASSF1A, suggesting that RASSF1A activity is phosphorylation-dependent .

• Experimental applications: These variants serve as valuable negative controls in functional studies and can help elucidate structure-function relationships of RASSF1A.

The table below summarizes how these polymorphisms affect cellular functions in experimental models:

TABLE 1. RASSF1A Variant Effects on Cell Cycle and Cyclin D1 Expression

These data demonstrate how polymorphisms can effectively nullify RASSF1A's ability to inhibit cell cycle progression and cyclin D1 accumulation .

How can I design experiments to study RASSF1A-mediated regulation of cyclin D1?

To investigate RASSF1A's regulation of cyclin D1, researchers should consider these methodological approaches:

• RNAi experiments: Employ RNA interference to downregulate endogenous RASSF1A expression. Previous studies have shown that RNAi-mediated inhibition of RASSF1A results in abnormal accumulation of native cyclin D1 protein without detectable changes in cyclin D1 mRNA levels, suggesting translational regulation .

• Expression studies: Compare the effects of wild-type RASSF1A versus cancer-associated mutants (S131F, A133S) on cyclin D1 levels using both immunoblotting and immunofluorescence techniques. Cancer-associated RASSF1A mutants fail to suppress cyclin D1 accumulation .

• BrdU incorporation assays: Measure cell cycle progression using bromodeoxyuridine (BrdU) incorporation experiments. After transfection with RASSF1A constructs, add 30 μM BrdU to cell cultures for 24 hours, then fix, permeabilize, and treat with 2M HCl before immunostaining with anti-BrdU antibodies and FITC-conjugated secondary antibodies .

• Rescue experiments: Test if ectopic expression of cyclin D1 can overcome RASSF1A-induced cell cycle arrest. This approach helps confirm the causal relationship between RASSF1A's effect on cyclin D1 and cell cycle arrest .

• Translation analysis: Investigate if RASSF1A affects cyclin D1 mRNA translation using polysome profiling or reporter assays with the cyclin D1 5' and 3' UTRs.

These experimental approaches provide comprehensive tools for dissecting the mechanisms by which RASSF1A regulates cyclin D1 and cell cycle progression in normal and cancer cells.

What are the best methods to study RASSF1A's role in stem cell differentiation?

RASSF1A plays a crucial role in stem cell differentiation by regulating the transition from pluripotency to differentiation. Researchers can investigate this function using these methodological approaches:

• Epigenetic analysis: Examine methylation status of the RASSF1A promoter during differentiation using bisulfite sequencing or methylation-specific PCR. Research shows that the RASSF1A promoter is methylated in pluripotent cells but undergoes demethylation during differentiation, allowing GATA1-mediated expression .

• Transcription factor binding studies: Use chromatin immunoprecipitation (ChIP) to analyze the association of:

  • YAP-TEAD and β-catenin-TCF3 complexes on the Oct4 distal enhancer in pluripotent cells

  • GATA1 binding to the RASSF1A promoter during differentiation

  • YAP-p73 complexes forming after RASSF1A expression

• Protein complex analysis: Employ co-immunoprecipitation and proximity ligation assays to investigate how RASSF1A expression affects the quaternary association of YAP-TEAD and β-catenin-TCF3 complexes that maintain pluripotency .

• Functional differentiation assays: Compare differentiation capacity in wild-type versus RASSF1A-depleted stem cells, and test whether ectopic RASSF1A expression accelerates differentiation.

• Transcriptional profiling: Use RNA-seq to identify genes differentially regulated during differentiation in the presence or absence of RASSF1A, with particular focus on YAP-p73 target genes that enable differentiation .

These methodological approaches enable researchers to dissect how RASSF1A acts as a functional "switch" between pluripotency and initiation of differentiation by mediating transcription factor selection of YAP in stem cells.

How can I investigate RASSF1A phosphorylation's impact on p53/p73 activation?

RASSF1A phosphorylation, particularly at Ser131 by ATM kinase, is critical for its ability to activate p73-dependent apoptotic responses. To investigate this relationship:

• Phosphorylation site mutants: Compare wild-type RASSF1A with phosphorylation-deficient mutants (S131F, A133S) in their ability to activate p53/p73-dependent transcription. Research has shown that the polymorphism near the ATM activation site disrupts the alpha helix containing this recognition site, affecting p53/p73 activation .

• DNA damage response: Induce DNA damage using radiation or genotoxic agents to activate ATM-mediated phosphorylation of RASSF1A at Ser131, then measure:

  • RASSF1A phosphorylation status using phospho-specific antibodies or radioactive labeling

  • p73 activation using activity assays or target gene expression

  • Apoptotic responses using TUNEL assays or annexin V staining

• Co-immunoprecipitation studies: Investigate how RASSF1A phosphorylation affects its interaction with:

  • p73 and/or p53

  • MDM2 (which may regulate p53/p73 stability)

  • HIPPO pathway components that might influence p73 activity

• ATM manipulation: Use ATM inhibitors or ATM-deficient cells to confirm the role of this kinase in RASSF1A-mediated p73 activation.

• Transcriptional analysis: Employ reporter assays with p53/p73-responsive promoters to quantify how RASSF1A phosphorylation affects transcriptional activation.

These methodological approaches will provide insights into how RASSF1A phosphorylation connects DNA damage responses to p73-dependent apoptotic outcomes, offering potential therapeutic avenues for cancers with dysregulated RASSF1A function.