Phospho-EIF2AK2 (T446) Recombinant Monoclonal Antibody

What is Phospho-EIF2AK2 (T446) and why is it significant in cellular research?

EIF2AK2, commonly known as Protein Kinase R (PKR), is a serine-threonine and tyrosine kinase that plays a critical role in the innate immune response to viral infection and cellular stress. PKR is activated through homodimerization and subsequent autophosphorylation on threonine residues 446 and 451. Phosphorylation at T446 represents a key activation marker for PKR function . This phosphorylation event is particularly important because it serves as a definitive indicator of PKR activation status in experimental systems.

The significance of T446 phosphorylation lies in its position as a critical regulatory modification that enables PKR to phosphorylate eukaryotic initiation factor-2α (eIF2α), which subsequently leads to inhibition of mRNA translation, stress granule formation, and ultimately contributes to antiviral defense mechanisms . Antibodies targeting this specific phosphorylation site allow researchers to precisely monitor PKR activation in various experimental contexts.

How does PKR activation occur at the molecular level?

PKR activation follows a well-characterized sequence of events. Upon binding to double-stranded RNA (dsRNA), which often occurs during viral infection, PKR undergoes a conformational change that facilitates homodimerization . This dimerization enables autophosphorylation, particularly at threonine residues 446 and 451, which are located in the activation loop of the kinase domain . Research has demonstrated that this phosphorylation is essential for full catalytic activity, enabling PKR to phosphorylate downstream substrates like eIF2α.

Interestingly, research has identified additional regulatory phosphorylation sites, including Ser6 and Ser97, which are positioned 3 amino acids upstream of the double-stranded RNA binding motifs (DRBMs) and appear to modulate PKR activity in a complex manner . When these serine residues are mutated to alanine to prevent phosphorylation, PKR exhibits enhanced spontaneous activation, suggesting these sites provide inhibitory regulation under normal conditions .

What is the role of Phospho-EIF2AK2 (T446) in the integrated stress response?

PKR represents one of four eIF2α kinases that constitute the integrated stress response (ISR), alongside HRI (EIF2AK1), PERK (EIF2AK3), and GCN2 (EIF2AK4) . Each kinase responds to distinct cellular stressors but converges on the phosphorylation of eIF2α at serine 51. In the case of PKR, activation occurs primarily in response to viral infection and double-stranded RNA, although additional activators have been identified .

Recent research has highlighted the critical role of eIF2α phosphorylation in autophagy induction, with evidence suggesting that this phosphorylation event is centrally involved in the response to various autophagy-inducing compounds . The ability to specifically monitor PKR activation through phospho-T446 detection provides researchers with a powerful tool to distinguish PKR-mediated stress responses from those initiated by other eIF2α kinases.

What applications are validated for Phospho-EIF2AK2 (T446) antibodies?

Based on multiple sources, Phospho-EIF2AK2 (T446) antibodies have been rigorously validated for several key applications in molecular and cellular biology research:

For Western blot applications, optimal results are typically achieved at antibody dilutions around 1:3000, with incubation times of approximately 1 hour at room temperature . Specific positive controls include HeLa cell lysates treated with Calyculin A and TNF-alpha, which effectively induce PKR phosphorylation .

How should researchers prepare samples for optimal detection of Phospho-EIF2AK2 (T446)?

Proper sample preparation is critical for successful detection of phosphorylated PKR. The phosphorylation status of proteins can be highly labile, and care must be taken to preserve phosphoepitopes during sample handling. Based on validated protocols, the following recommendations can be made:

• Include phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, and β-glycerophosphate) in all lysis buffers to prevent dephosphorylation .

• Perform cell lysis using cold buffers and maintain samples on ice throughout processing to minimize enzymatic activity that could alter phosphorylation status.

• For Western blotting applications, freshly prepared samples typically yield better results than frozen samples due to potential phosphoepitope degradation during freeze-thaw cycles .

• For immunohistochemistry applications with human colon tissue samples, standard formalin fixation and paraffin embedding protocols have been validated to preserve the phospho-T446 epitope .

• When using cell culture models, consider treatments that enhance PKR phosphorylation as positive controls, such as Calyculin A (100 nM) or TNF-alpha, which have been demonstrated to enhance detection of phospho-T446 PKR in HeLa cells .

What controls are essential when working with Phospho-EIF2AK2 (T446) antibodies?

Inclusion of appropriate experimental controls is crucial for reliable interpretation of results when working with phospho-specific antibodies. Based on established research protocols, the following controls should be considered:

• Positive control samples: Human colon tissue is a validated positive control for immunohistochemistry applications . For Western blot analysis, HeLa cell lysates treated with Calyculin A (a phosphatase inhibitor) and TNF-alpha can serve as reliable positive controls .

• Negative controls: Include samples where PKR phosphorylation is expected to be absent or reduced. This could involve unstimulated cells or the use of PKR inhibitors.

• Phosphatase treatment controls: Treating duplicate samples with lambda phosphatase prior to immunoblotting can confirm antibody phospho-specificity by demonstrating signal loss after dephosphorylation.

• Loading controls: When performing Western blot analysis, include detection of total PKR protein to normalize phosphorylation levels and account for variations in total protein content.

• Genetic controls: When available, PKR-knockout cell lines (PKR-KO) can serve as valuable negative controls to validate antibody specificity .

How do phosphorylation events at T446 coordinate with other PKR phosphorylation sites?

PKR activation involves a complex interplay of multiple phosphorylation events. While T446 and T451 are the primary autophosphorylation sites associated with kinase activation, research has identified additional regulatory phosphorylation sites that modulate PKR function in more nuanced ways .

Recent studies have identified novel phosphorylation sites at Ser6 and Ser97, which are positioned 3 amino acids upstream of the first and second double-stranded RNA binding motifs (DRBM1 and DRBM2), respectively . Intriguingly, mutation of these serine residues to alanine (phosphoinhibiting mutations) resulted in spontaneous PKR activation, while phosphomimetic mutations (serine to aspartate) inhibited PKR activation following either poly(I:C) transfection or virus infection .

These findings suggest a regulatory model where phosphorylation at different sites has opposing effects on PKR activity:

• Phosphorylation at T446/T451: Required for kinase domain activation

• Phosphorylation at Ser6/Ser97: Appears to have inhibitory effects on PKR activation

The ability to specifically monitor T446 phosphorylation allows researchers to dissect these complex regulatory mechanisms and understand how different phosphorylation events are coordinated in response to various cellular stressors.

How can Phospho-EIF2AK2 (T446) antibodies be used to study autophagy mechanisms?

Emerging research has established connections between PKR activation and the regulation of autophagy. The phosphorylation of eIF2α, a primary downstream target of activated PKR, appears to be a central event in the stimulation of autophagy in response to various pharmacological agents .

Researchers investigating autophagy mechanisms can utilize Phospho-EIF2AK2 (T446) antibodies to:

• Monitor PKR activation status during autophagy induction by different compounds or stressors.

• Distinguish between PKR-mediated autophagy and autophagy induced by other eIF2α kinases (HRI, PERK, GCN2).

• Investigate temporal relationships between PKR activation, eIF2α phosphorylation, and the formation of autophagic puncta.

• Assess potential crosstalk between PKR signaling and other autophagy-regulatory pathways such as mTOR or AMPK.

The quantification of cytoplasmic fluorescence intensity after immunostaining with phospho-specific antibodies has been validated as an effective approach for evaluating eIF2α phosphorylation in autophagy research . This methodological approach can be adapted for phospho-PKR detection in similar experimental contexts.

What techniques are most effective for studying PKR activation dynamics during viral infection?

Understanding the temporal dynamics of PKR activation during viral infection requires sophisticated experimental approaches. Several methodologies have been validated for monitoring phospho-T446 PKR in this context:

• Time-course Western blot analysis: Sequential sampling following viral infection allows researchers to track the kinetics of PKR phosphorylation at T446. This approach requires careful experimental design with appropriate time points and synchronized infection.

• Live-cell imaging with phospho-specific antibodies: Though technically challenging, immunofluorescence approaches using membrane-permeable antibody delivery systems can enable real-time visualization of PKR phosphorylation dynamics.

• Phosphoproteomics: Mass spectrometry-based approaches can provide comprehensive profiling of PKR phosphorylation states and identify potential novel phosphorylation sites.

• Genetic approaches using phosphomutants: The introduction of PKR variants with mutations at Ser6 and Ser97 (either phosphoinhibiting or phosphomimetic) can provide insights into how these modifications modulate PKR response to viral infection .

Research has demonstrated that phosphomimetic mutations at Ser6 and Ser97 (S6D, S97D, S6D-S97D) inhibited PKR activation following viral infection, suggesting these sites participate in a regulatory feedback mechanism to control PKR activity during infection .

What are common challenges in Western blot detection of Phospho-EIF2AK2 (T446)?

Researchers working with Phospho-EIF2AK2 (T446) antibodies may encounter several technical challenges that can affect experimental outcomes:

• Antibody specificity issues: Ensure the antibody specifically recognizes phosphorylated T446 and not other phosphorylated residues. Phosphatase treatment controls can help confirm specificity.

• Rapid dephosphorylation during sample processing: Phosphorylation at T446 may be labile, necessitating rapid sample processing and inclusion of phosphatase inhibitors in all buffers.

• Storage considerations: Multiple sources recommend storing the antibody at -20°C for long-term stability, with short-term storage at 4°C for up to one month . Repeated freeze-thaw cycles should be avoided as they can compromise antibody performance.

• Inconsistent signal intensity: This may result from variations in the efficiency of protein transfer during Western blotting or inconsistent PKR activation across experiments. Standardizing experimental conditions and including positive controls (such as HeLa cells treated with Calyculin A and TNF-alpha) can help address this issue .

How can researchers validate the specificity of Phospho-EIF2AK2 (T446) antibodies?

Validation of antibody specificity is critical for generating reliable research data. Several approaches are recommended:

• Phosphatase treatment: Treating duplicate samples with lambda phosphatase before immunoblotting should abolish or significantly reduce the signal if the antibody is truly phospho-specific.

• Peptide competition assays: Pre-incubating the antibody with phosphorylated and non-phosphorylated peptides corresponding to the T446 region can help confirm epitope specificity.

• Genetic approaches: Utilizing PKR-knockout cell lines provides a definitive negative control . Additionally, cells expressing PKR with T446A mutations (preventing phosphorylation at this site) should show no signal.

• Induction controls: Using treatments known to induce PKR phosphorylation, such as Calyculin A (100 nM) or TNF-alpha in HeLa cells, can verify antibody functionality .

• Cross-validation with multiple antibodies: When possible, confirm results using phospho-T446 antibodies from different sources or generated using different immunogens.

How is Phospho-EIF2AK2 (T446) detection being applied to neurodegenerative disease research?

PKR activation has been implicated in various neurodegenerative conditions, with multiple publications connecting Phospho-EIF2AK2 (T446) to brain diseases and nervous system disorders . The ability to specifically detect activated PKR through T446 phosphorylation has provided researchers with a valuable tool to investigate these connections:

• Alzheimer's disease: Research suggests PKR activation may contribute to neuronal loss through translational control mechanisms and interactions with tau protein. Phospho-T446 detection allows for precise quantification of PKR activation in disease models and patient samples.

• Parkinson's disease: Studies have implicated PKR in the cellular stress responses associated with protein aggregation and mitochondrial dysfunction characteristic of Parkinson's.

• Amyotrophic lateral sclerosis (ALS): PKR activation and subsequent eIF2α phosphorylation have been linked to the stress responses observed in motor neurons affected by ALS.

The methodological approach typically involves immunohistochemical analysis of brain tissue samples using Phospho-EIF2AK2 (T446) antibodies, allowing researchers to identify specific brain regions and cell types where PKR activation occurs in the context of neurodegenerative conditions .

What role does PKR phosphorylation play in antiviral research?

PKR represents a critical component of the innate immune response to viral infection, and its phosphorylation at T446 serves as a key activation marker in antiviral research . Multiple applications for Phospho-EIF2AK2 (T446) antibodies in this field include:

• Viral evasion mechanism studies: Many viruses have evolved strategies to inhibit PKR activation. Phospho-T446 detection enables researchers to evaluate the effectiveness of these viral evasion mechanisms.

• Antiviral compound screening: Compounds that enhance PKR phosphorylation at T446 may have potential as broad-spectrum antiviral agents.

• Host-virus interaction studies: Examining the kinetics of PKR phosphorylation during infection with different viruses provides insights into host response dynamics.

Research has demonstrated that PKR exerts antiviral activity against a wide range of DNA and RNA viruses, including hepatitis C virus (HCV), hepatitis B virus (HBV), measles virus (MV), and herpes simplex virus 1 (HHV-1) . The specific detection of phosphorylated PKR at T446 allows researchers to monitor activation status during infection with these diverse viral pathogens.

How can Phospho-EIF2AK2 (T446) analysis contribute to cancer research?

PKR activation has been implicated in various aspects of cancer biology, and Phospho-EIF2AK2 (T446) antibodies provide valuable tools for investigating these connections . Key applications include:

• Tumor suppressor functions: PKR has been reported to have tumor suppressor properties in some contexts, with activation leading to apoptosis in cancer cells. Monitoring T446 phosphorylation allows researchers to assess PKR activation status in different cancer types.

• Stress response in cancer cells: Cancer cells often experience various forms of stress, including ER stress and oxidative stress, which may activate PKR. Phospho-T446 detection enables researchers to examine how cancer cells respond to these stressors.

• Therapeutic response monitoring: Some cancer therapies may induce PKR activation as part of their mechanism of action. Phospho-T446 detection can serve as a pharmacodynamic marker for treatment response.

• Inflammation and cancer: Given PKR's role in inflammatory signaling, Phospho-T446 detection can help elucidate connections between inflammation and cancer progression.

Publications have specifically linked PKR activation to melanoma and other neoplasms, highlighting the relevance of phospho-specific detection methods in oncology research .