RPS6KA2 Antibody

What is RPS6KA2 and what are its key biological functions?

RPS6KA2 (Ribosomal Protein S6 Kinase A2) is a member of the RSK (ribosomal S6 kinase) family of serine/threonine kinases. It contains two non-identical kinase catalytic domains and phosphorylates various substrates, including members of the mitogen-activated kinase (MAPK) signaling pathway . Functionally, RPS6KA2 acts downstream of ERK (MAPK1/ERK2 and MAPK3/ERK1) signaling and mediates mitogenic and stress-induced activation of transcription factors, regulates translation, and mediates cellular proliferation, survival, and differentiation . The activity of this protein has been implicated in controlling cell growth and differentiation, and research suggests it may function as a tumor suppressor in epithelial ovarian cancer cells .

RPS6KA2 has been implicated in several pathological conditions. Diseases associated with RPS6KA2 include Coffin-Lowry Syndrome and Rasopathy . The loss of RSK2 activity (a related family member) in humans leads to Coffin-Lowry syndrome, which is characterized by mental retardation and growth deficits . Recent research has also identified RPS6KA2 as a potential tumor suppressor in ovarian cancer, where it shows significantly lower expression in cancerous tissues compared to normal tissues .

Experimental Applications of RPS6KA2 Antibodies

For investigating RPS6KA2's tumor suppressor role in cancer models, researchers should consider:

• Tissue expression profiling: Use immunohistochemistry with validated antibodies to compare RPS6KA2 expression between normal and cancerous tissues. Studies have demonstrated that RPS6KA2 shows significantly lower expression in ovarian cancer tissues compared to normal tissues, with histochemistry scores (H-scores) in normal tissues being 13.5 and 3.8 times higher than in tumor tissues .

• Functional validation: Combine RPS6KA2 antibodies with overexpression or knockdown approaches. For example, studies have shown that overexpression of RPS6KA2 suppresses cell proliferation in ovarian cancer cells, whereas knockdown enhances proliferation and colony formation capabilities .

• Signaling pathway analysis: Use RPS6KA2 antibodies in combination with other antibodies targeting p38/MAPK pathway components to elucidate the molecular mechanisms. Research has demonstrated that RPS6KA2 inhibits proliferation in ovarian cancer via the p38/MAPK signaling pathway .

• In vivo validation: Utilize RPS6KA2 antibodies for analyzing tumor samples from xenograft models. Studies have shown that tumor volume and weight were inhibited with overexpression of RPS6KA2 and promoted with knockdown of RPS6KA2 in mouse models .

What factors should be considered when selecting an RPS6KA2 antibody for specific applications?

When selecting an RPS6KA2 antibody, researchers should consider:

• Antibody type: Both polyclonal and monoclonal antibodies are available. Polyclonal antibodies may offer higher sensitivity but potentially lower specificity compared to monoclonals .

• Target epitope: Different antibodies target different regions of RPS6KA2. For example, some antibodies are generated against C-terminal regions , while others may target other domains. Select antibodies based on your research question and whether specific domains are relevant.

• Species reactivity: Verify cross-reactivity with your experimental model. Available antibodies show reactivity with human, mouse, and rat samples, though sensitivity may vary .

• Validated applications: Ensure the antibody has been validated for your specific application. Some antibodies are validated for multiple applications (WB, IHC, IF), while others may be optimized for specific techniques .

• Isoform recognition: Consider whether the antibody can detect various isoforms or splice variants of RPS6KA2. Research has identified multiple protein isoforms that may appear as duplets or triplets on immunoblots .

How can the specificity of RPS6KA2 antibodies be validated?

To ensure antibody specificity for RPS6KA2:

How do alternative splicing and protein isoforms affect RPS6KA2 antibody detection and biological interpretation?

Research has revealed complexity in RPS6KA2/RSK4 transcript variants and protein isoforms that can significantly impact antibody detection and interpretation:

• Multiple transcription initiation sites: Studies have identified alternative transcription initiation sites and splice variants resulting in mRNA variants with four possible first start codons .

• Alternative splicing events: Several alternative splice sites have been identified, including splicing of the first 15 nucleotides of exon 22 in mouse and alternative splicing of the penultimate exon in both human and mouse . These variations can affect epitope presence or accessibility.

• Protein isoform complexity: In cancer cell lines, the 90-kD wild-type RPS6KA2/RSK4 is often sparse, while several isoforms at or smaller than 72-kD are expressed. Each smaller isoform often appears as a duplet or triplet on immunoblots .

• Context-dependent expression: The levels of different isoforms vary greatly among different cell lines and culture conditions. Factors like cyclin D1 expression, c-Myc levels, and serum conditions can influence expression patterns .

• Functional diversity: Different mRNA variants or protein isoforms may have distinct functional roles. The effects of RPS6KA2/RSK4 on cell growth, death, and chemoresponse depend on which variant or isoform is expressed .

When designing experiments and interpreting results, researchers should consider using antibodies that can detect multiple isoforms or specifically target isoforms of interest, depending on their research questions.

What are the methodological considerations for studying RPS6KA2 phosphorylation status using antibodies?

Studying the phosphorylation status of RPS6KA2 requires careful experimental design:

• Phospho-specific antibodies: Select antibodies that specifically recognize phosphorylated residues of interest. RPS6KA2 contains multiple phosphorylation sites that regulate its activity.

• Sample preparation: Preserve phosphorylation status by using phosphatase inhibitors (e.g., sodium orthovanadate, sodium fluoride, and β-glycerophosphate) during cell/tissue lysis. Process samples quickly and maintain cold temperatures to minimize dephosphorylation.

• Positive controls: Include samples treated with agents known to activate the MAPK pathway, such as phorbol esters or growth factors, to induce RPS6KA2 phosphorylation.

• Detection methods: Consider using Phos-tag™ SDS-PAGE for enhanced separation of phosphorylated from non-phosphorylated forms, followed by immunoblotting with total RPS6KA2 antibodies.

• Functional validation: Combine phosphorylation analysis with kinase activity assays to correlate phosphorylation status with functional outcomes.

• Context awareness: RPS6KA2/RSK4 has been reported to be constitutively activated (fully phosphorylated) even in serum-starved cells in some contexts , suggesting unique regulatory mechanisms compared to other RSK family members.

What are common challenges when using RPS6KA2 antibodies for Western blotting and how can they be addressed?

Researchers may encounter several challenges when using RPS6KA2 antibodies for Western blotting:

• Multiple bands/isoforms: RPS6KA2 can appear as multiple bands due to isoforms, post-translational modifications, or degradation products. Studies have shown that smaller isoforms (~72 kDa) may be more abundant than the full-length 90 kDa protein in some cancer cell lines . Solution: Use positive controls with known expression patterns and consider using loading controls specific to cellular compartments.

• Low signal intensity: The endogenous expression of RPS6KA2 may be low in certain cell types. Solution: Increase protein loading, extend antibody incubation times, use more sensitive detection methods (e.g., enhanced chemiluminescence), or consider immunoprecipitation before Western blotting to concentrate the protein.

• High background: Non-specific binding can obscure specific signals. Solution: Optimize blocking conditions (consider different blocking agents like BSA, milk, or commercial blockers), increase washing duration/frequency, and test different antibody dilutions (typically 1:500-1:1000) .

• Variability between cell lines: The expression and isoform profile of RPS6KA2 can vary significantly between different cell lines . Solution: Validate antibody performance in each new cell line before conducting experiments and consider the biological context of expression.

• Degradation during sample preparation: RPS6KA2 may be sensitive to proteolytic degradation. Solution: Use fresh samples, maintain cold temperatures throughout preparation, and include protease inhibitors in lysis buffers.

How can researchers optimize immunohistochemistry protocols for RPS6KA2 detection in different tissue types?

For optimal immunohistochemical detection of RPS6KA2 across various tissue types:

• Antigen retrieval optimization: Different tissues may require specific antigen retrieval methods. For RPS6KA2, studies suggest using TE buffer at pH 9.0 for optimal results, although citrate buffer at pH 6.0 may serve as an alternative .

• Antibody dilution titration: Test a range of antibody dilutions, typically starting with 1:50-1:500 for IHC applications . The optimal dilution may vary based on tissue type and fixation method.

• Positive and negative controls: Include known positive tissues (lung cancer tissue and placenta have been validated ) and negative controls (primary antibody omission) in each experiment.

• Signal amplification: For tissues with low RPS6KA2 expression, consider using signal amplification systems like tyramide signal amplification (TSA) or polymer-based detection systems.

• Counterstaining optimization: Adjust counterstaining intensity to provide context without obscuring RPS6KA2 staining. Hematoxylin is commonly used, but the incubation time may need adjustment.

• Tissue-specific considerations: Be aware that RPS6KA2 expression varies across tissues, with higher levels reported in lung and skeletal muscle as well as liver, kidney, pancreas, testis, prostate, and placenta . Staining patterns may differ between normal and pathological tissues, particularly in cancer where expression is often reduced .

• Quantification methods: Consider using histochemistry score (H-score) methods as demonstrated in studies comparing normal and ovarian cancer tissues for standardized assessment of staining intensity and distribution.

How can RPS6KA2 antibodies be used to investigate its role in MAPK signaling pathways?

To investigate RPS6KA2's role in MAPK signaling:

• Co-immunoprecipitation (CoIP) studies: Use RPS6KA2 antibodies for CoIP to identify interaction partners within the MAPK cascade. RPS6KA2 acts downstream of ERK signaling , and CoIP can help elucidate the specific protein-protein interactions involved.

• Pathway activation monitoring: Combine RPS6KA2 antibodies with phospho-specific antibodies against other MAPK pathway components (ERK1/2, p38) to monitor pathway activation state following various stimuli. Studies have linked RPS6KA2 to the p38/MAPK signaling pathway in cancer contexts .

• Translocation studies: Employ immunofluorescence with RPS6KA2 antibodies to track subcellular localization changes following pathway activation, as RSK family proteins often shuttle between cytoplasm and nucleus upon activation.

• Functional knockdown/knockout validation: Use RPS6KA2 antibodies to confirm protein depletion in knockdown/knockout models before assessing effects on downstream MAPK pathway targets.

• Phosphorylation profiling: Combine immunoprecipitation using RPS6KA2 antibodies with phospho-proteomic analyses to identify phosphorylation sites and dynamics under different conditions.

• Chromatin immunoprecipitation (ChIP): For investigating transcriptional regulation roles, use RPS6KA2 antibodies in ChIP experiments to identify genomic binding regions when RPS6KA2 translocates to the nucleus.

What methodological approaches can resolve contradictory findings about RPS6KA2's role as a tumor suppressor versus oncogene?

Research has yielded seemingly contradictory findings regarding RPS6KA2's role in cancer, with evidence supporting both tumor suppressor and oncogenic functions . To resolve these contradictions:

• Context-specific analysis: Use tissue-specific expression profiling with RPS6KA2 antibodies across diverse cancer types. Research suggests RPS6KA2 has a tumor suppressive role in ovarian cancer but may function differently in other contexts.

• Isoform-specific investigations: Employ antibodies that can distinguish different RPS6KA2 isoforms, as studies indicate that different variants may have opposing functions . Design experiments that specifically express or inhibit individual isoforms to assess their distinct roles.

• Signaling pathway integration: Combine RPS6KA2 antibodies with antibodies against relevant signaling partners (ERK, p38, cyclin D1, c-Myc) to map context-dependent signaling networks. Research has shown interactions between RPS6KA2, cyclin D1, and c-Myc that vary by cellular context .

• Growth condition variations: Test cells under different growth conditions (serum starvation, anchorage-dependent versus independent growth) as RPS6KA2's effects have been shown to depend on these environmental factors .

• In vivo validation: Confirm in vitro findings through in vivo models using RPS6KA2 antibodies for tissue analysis. Studies have demonstrated that in vivo tumor growth can be affected by RPS6KA2 expression levels .

• Genetic background consideration: Analyze RPS6KA2 function across cell lines with different genetic backgrounds (p53 status, PTEN status) as these factors can influence RPS6KA2 expression and function .

• Multi-omics approach: Integrate antibody-based protein studies with transcriptomics, epigenomics, and functional genomics to develop a comprehensive understanding of RPS6KA2's context-dependent roles.