Phospho-LIMK2 (S283) Antibody

What is LIMK2 and why is phosphorylation at Ser283 significant?

LIMK2 (LIM domain kinase 2) is a serine/threonine kinase containing two LIM domains, a PDZ domain, and a C-terminal kinase domain. It plays crucial roles in regulating actin cytoskeletal reorganization primarily through phosphorylation of cofilin at Ser3.

The phosphorylation of LIMK2 at Ser283 has specific biological significance:

• It is mediated by PKC (Protein Kinase C) and regulates nucleocytoplasmic shuttling of LIMK2

• This phosphorylation site affects LIMK2's subcellular localization, particularly inhibiting nuclear import

• In endothelial cells, PKC-mediated phosphorylation at Ser283 was confirmed using phospho-specific antibodies

• Unlike phosphorylation at Thr505 (which activates LIMK2's kinase activity), Ser283 phosphorylation primarily regulates localization rather than enzymatic activity

This distinct regulation mechanism makes phospho-Ser283 detection particularly valuable for studying LIMK2 trafficking between cellular compartments.

What experimental applications are suitable for Phospho-LIMK2 (S283) Antibody?

Phospho-LIMK2 (S283) Antibody has been validated for multiple research applications:

The antibody specifically detects endogenous levels of LIMK2 protein only when phosphorylated at Ser283, making it ideal for:

• Studying PKC signaling pathways

• Examining LIMK2 nucleocytoplasmic shuttling dynamics

• Investigating LIMK2 regulation in cancer, neurological conditions, and vascular biology

How should samples be prepared for optimal Phospho-LIMK2 (S283) detection?

For reliable phospho-LIMK2 (S283) detection, sample preparation is critical:

• Cell/Tissue Lysis Protocol:

  • Use buffer containing phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate)

  • Include protease inhibitor cocktail

  • Maintain cold temperature (4°C) throughout processing

  • For brain tissue samples, rapid extraction is essential as postmortem dephosphorylation occurs quickly

• Western Blot Sample Handling:

  • Avoid multiple freeze-thaw cycles of lysates

  • Heat samples at 95°C for 5 minutes in Laemmli buffer

  • Load 20-40μg total protein per lane for cell lysates

  • Use freshly prepared samples when possible

• Immunohistochemistry Preparation:

  • Formalin-fixed paraffin-embedded (FFPE) sections require antigen retrieval

  • Sodium citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) can be used for retrieval

  • Block endogenous peroxidase activity with hydrogen peroxide

  • Use BSA or serum for blocking non-specific binding

For subcellular localization studies of phospho-LIMK2, preserving phosphorylation status during fixation is critical, as studies have shown that LIMK2 shuttles between nucleus and cytoplasm, with phosphorylation at Ser283 inhibiting nuclear import .

How does Ser283 phosphorylation affect LIMK2 function compared to other phosphorylation sites?

LIMK2 activity and localization are regulated through multiple phosphorylation sites with distinct functions:

Research has revealed that:

• Phosphorylation at Thr505 is reduced following seizure episodes, while LIMK2 protein expression is increased

• Combined phosphorylation at Ser283 and Thr494 completely blocks LIMK2 nucleocytoplasmic shuttling

• Aurora A can phosphorylate LIMK2 at S283, T494, and T505, affecting its kinase activity, subcellular localization, and protein levels

• PKC-mediated phosphorylation at Ser283 does not activate LIMK2-mediated cofilin phosphorylation in endothelial cells

This complex phosphorylation pattern suggests that LIMK2 functions as an integration point for multiple signaling pathways, with Ser283 phosphorylation specifically regulating its nuclear accessibility.

What controls and validation steps are recommended for Phospho-LIMK2 (S283) Antibody experiments?

Rigorous validation is essential for phospho-specific antibody experiments:

• Positive Controls:

  • PMA-stimulated endothelial cells (triggers PKC activation and Ser283 phosphorylation)

  • Cells expressing constitutively active PKC

  • Brain tissue samples (shows endogenous expression)

• Negative Controls:

  • Blocking peptide competition (pre-incubate antibody with phospho-Ser283 peptide)

  • Phosphatase-treated samples

  • LIMK2 knockout or knockdown cells/tissues

• Specificity Validation:

  • Demonstrate lack of reactivity with non-phosphorylated LIMK2

  • Show reduced signal after treatment with Ser/Thr phosphatase

  • Compare with total LIMK2 antibody staining pattern

  • Test cross-reactivity with phosphorylated LIMK1 (which shares structural similarity)

• Technical Validation:

  • For IHC, include control tissues with known expression patterns

  • For Western blotting, confirm single band at expected molecular weight (~72kDa)

  • For phosphorylation studies, include relevant kinase inhibitors (PKC inhibitors)

Research by Goyal et al. demonstrated that Ser283 in LIMK2 is phosphorylated in PMA-stimulated endothelial cells, providing a reliable positive control system for antibody validation .

How can researchers integrate Phospho-LIMK2 (S283) detection into studies of disease mechanisms?

Phospho-LIMK2 (S283) detection can provide valuable insights into several pathological contexts:

• Cancer Research Applications:

  • Aurora A regulates LIMK2 through phosphorylation at multiple sites including S283

  • LIMK2 ablation completely abrogates Aurora A-mediated tumorigenesis in animal models

  • Phospho-LIMK2 detection can monitor Aurora A-LIMK2 pathway activation

  • Potential biomarker for Aurora A-driven cancers (breast, pancreatic, etc.)

• Neurological Disorder Studies:

  • LIMK2 is upregulated in seizure models and contributes to programmed necrotic neuronal death

  • Phospho-LIMK2 status may serve as an indicator of neuronal stress

  • Monitoring phosphorylation patterns during neuropathological events

  • Potential therapeutic target in neurodegenerative conditions

• Vascular Biology Research:

  • In endothelial cells, LIMK2 regulates actin cytoskeletal reorganization via Rho/Rho-kinase pathway

  • PKC-mediated phosphorylation at Ser283 affects LIMK2 localization

  • Implications for vascular permeability, angiogenesis, and endothelial dysfunction

• Methodological Approach:

  • Combine phospho-LIMK2 (S283) detection with other phospho-sites (T505, T494)

  • Correlate phosphorylation status with subcellular localization

  • Study the dynamics of phosphorylation in response to stimuli

  • Investigate phosphatase regulation of LIMK2

Researchers can implement temporal analysis of LIMK2 phosphorylation at different sites to understand the sequential regulation during disease progression or cellular responses to stimuli.

What are the technical challenges in detecting phospho-LIMK2 (S283) and how can they be overcome?

Researchers face several challenges when working with phospho-specific antibodies:

• Low Signal Intensity:

  • Challenge: Phospho-epitopes often represent a small fraction of total protein

  • Solution: Enrich phosphorylated proteins using phospho-protein enrichment kits before Western blotting

  • Alternative: Use signal amplification systems like TSA (Tyramide Signal Amplification)

• Specificity Issues:

  • Challenge: Cross-reactivity with other phosphorylated epitopes

  • Solution: Always include peptide competition controls

  • Method: Pre-incubate antibody with phosphorylated and non-phosphorylated peptides separately

• Preserving Phosphorylation Status:

  • Challenge: Rapid dephosphorylation during sample preparation

  • Solution: Use phosphatase inhibitor cocktails immediately during lysis

  • Protocol: Process samples quickly at 4°C and avoid phosphatase-activating conditions

• Fixation-Induced Epitope Masking:

  • Challenge: Some fixatives can mask phospho-epitopes

  • Solution: Compare different fixation methods (PFA vs. methanol)

  • Approach: Optimize antigen retrieval conditions specifically for phospho-LIMK2

• Quantification Challenges:

  • Challenge: Accurately determining phosphorylation levels

  • Solution: Always normalize to total LIMK2 expression

  • Method: Use quantitative techniques like ELISA or phospho-flow cytometry when possible

Studies have shown that PMA stimulation of endothelial cells provides a reliable system for detecting LIMK2 Ser283 phosphorylation , making this an excellent positive control model for method optimization.

How should researchers design experiments to study the functional consequences of LIMK2 Ser283 phosphorylation?

To investigate the specific role of LIMK2 Ser283 phosphorylation:

• Site-Specific Mutagenesis Approach:

  • Generate LIMK2 S283A (phospho-deficient) and S283D/E (phospho-mimetic) mutants

  • Express in LIMK2-depleted cells to avoid interference from endogenous protein

  • Compare subcellular localization patterns using immunofluorescence

  • Assess impact on LIMK2 function (cofilin phosphorylation, actin dynamics)

• Kinase Manipulation Studies:

  • Use PKC inhibitors or activators to modulate Ser283 phosphorylation

  • Implement genetic approaches (siRNA, CRISPR) to target specific PKC isoforms

  • Monitor subsequent effects on LIMK2 localization and function

  • Test in multiple cell types to identify context-dependent regulation

• Live-Cell Imaging Experiments:

  • Create fluorescent protein-tagged LIMK2 constructs (wild-type and mutants)

  • Use FRAP (Fluorescence Recovery After Photobleaching) to measure nucleocytoplasmic shuttling dynamics

  • Apply stimuli that activate PKC to observe real-time changes in localization

  • Combine with biosensors for downstream targets

• Experimental Controls:

  • Include LIMK2 T505A/E mutants for comparison with canonical activation site

  • Test double mutants (S283A/T505A, S283D/T505E) to understand pathway integration

  • Use Leptomycin B to inhibit nuclear export and assess nuclear accumulation

Research has demonstrated that PMA stimulation induces PKC-mediated phosphorylation of LIMK2 at Ser283, inhibiting its nuclear import and affecting its subcellular distribution , providing a foundation for these functional studies.

What are the recommended methods for analyzing LIMK2 phosphorylation in complex tissue samples?

Analyzing phospho-LIMK2 in tissues requires specialized approaches:

• Tissue Preparation Methods:

  • Rapid fixation is critical to preserve phosphorylation status

  • For frozen sections, use acetone or methanol fixation to maintain phospho-epitopes

  • For FFPE sections, optimize antigen retrieval (heat-induced in citrate buffer)

  • Consider phosphatase inhibitor perfusion for animal models before tissue collection

• Detection Strategies:

  • Multiplexed Immunofluorescence:

    • Co-stain with phospho-LIMK2 (S283) and total LIMK2 antibodies

    • Include cell type-specific markers to identify responsive populations

    • Use spectral imaging to resolve multiple fluorophores

  • Advanced IHC Techniques:

    • Implement tyramide signal amplification for low-abundance phospho-proteins

    • Use automated staining platforms for consistency

    • Consider chromogenic multiplex IHC for co-localization studies

• Quantification Methods:

  • Digital image analysis with automated tissue segmentation

  • Measure nuclear/cytoplasmic signal ratios across tissue regions

  • Population-level analysis with single-cell resolution when possible

• Validation in Tissue Context:

  • Compare phospho-LIMK2 (S283) patterns in normal versus pathological tissues

  • Correlate with upstream regulators (PKC activity) and downstream effects

  • Use genetic models (conditional knockout) to confirm specificity

Immunohistochemistry analysis of human brain tissue has successfully demonstrated specificity of phospho-LIMK2 (S283) antibody, with signal effectively blocked by phosphopeptide competition , establishing this as a viable approach for tissue studies.

How can researchers resolve contradictory results when studying LIMK2 phosphorylation at multiple sites?

When faced with apparently contradictory data regarding LIMK2 phosphorylation:

• Sequential Phosphorylation Analysis:

  • Implement time-course experiments to determine phosphorylation order

  • Use kinase inhibitors to block specific pathways sequentially

  • Analyze interdependence of phosphorylation events using phospho-site mutants

  • Employ mass spectrometry to identify all phosphorylation sites simultaneously

• Context-Dependent Regulation Assessment:

  • Compare results across different cell types (neurons vs. endothelial cells vs. cancer cells)

  • Test effects of multiple stimuli (thrombin, PMA, growth factors)

  • Examine phosphorylation patterns under physiological vs. pathological conditions

  • Consider microenvironmental factors (hypoxia, inflammation)

• Methodological Reconciliation:

  • Antibody Validation:

    • Test multiple phospho-specific antibodies from different sources

    • Validate with phosphatase treatment and phospho-deficient mutants

    • Consider epitope accessibility issues in different applications

  • Technological Approaches:

    • Implement phospho-proteomics for unbiased site identification

    • Use proximity ligation assays to confirm specific phosphorylation in situ

    • Apply CRISPR-based tagging to track endogenous LIMK2 modifications

• Integrative Data Analysis:

  • Construct mathematical models of LIMK2 regulation incorporating all phosphorylation sites

  • Apply systems biology approaches to understand network-level effects

  • Consider combinatorial effects of multiple modifications

Research has revealed seemingly contradictory findings: Aurora A phosphorylates LIMK2 at S283, T494, and T505 , while PKC primarily targets S283 and T494 . These differences likely reflect context-dependent regulation and highlight the complexity of LIMK2 phosphorylation.

How can Phospho-LIMK2 (S283) Antibody be integrated into high-throughput screening approaches?

For researchers developing drug discovery or large-scale screening programs:

• High-Content Imaging Applications:

  • Automated microscopy platforms can quantify phospho-LIMK2 (S283) nuclear/cytoplasmic ratios

  • Screen for compounds affecting PKC-mediated phosphorylation

  • Multiplex with actin cytoskeleton markers to link phosphorylation to functional outcomes

  • Use machine learning algorithms for complex phenotype recognition

• Phospho-Flow Cytometry Implementation:

  • Optimize fixation and permeabilization for intracellular phospho-LIMK2 detection

  • Develop multi-parameter panels to correlate with cell cycle or activation markers

  • Apply to heterogeneous primary cell populations or cancer samples

  • Enable single-cell analysis of phosphorylation status

• ELISA-Based Screening:

  • Develop sandwich ELISA using capture and phospho-specific detection antibodies

  • Adapt to 384 or 1536-well format for high-throughput applications

  • Implement parallel measurement of total LIMK2 for normalization

  • Use for kinase inhibitor library screening

• Methodological Considerations:

  • Standardize positive controls (PMA treatment for PKC activation)

  • Include plate-based controls for phosphorylation state

  • Optimize signal-to-noise ratio for automation compatibility

  • Develop robust statistical analysis methods for hit identification

Modern phospho-specific antibody-based assays, like those described for phospho-Tau , provide methodological templates that can be adapted for high-throughput phospho-LIMK2 detection in drug discovery applications.

What are the most promising therapeutic implications of targeting LIMK2 phosphorylation at Ser283?

The therapeutic potential of modulating LIMK2 Ser283 phosphorylation spans multiple disease areas:

• Cancer Therapy Applications:

  • LIMK2 has been identified as a crucial regulator and effector of Aurora A-kinase-mediated tumorigenesis

  • LIMK2 ablation fully abrogates Aurora A-mediated tumor formation in nude mice

  • Targeting Ser283 phosphorylation could disrupt the positive feedback loop between Aurora A and LIMK2

  • Combined inhibition approaches:

    • Aurora A inhibitors + LIMK2 inhibitors show synergistic cell death induction

    • Disrupting specific phosphorylation events may provide selective targeting

• Neurological Applications:

  • LIMK2 contributes to programmed necrotic neuronal death following seizures

  • Phosphorylation status affects LIMK2 subcellular localization and function

  • Potential neuroprotective strategies by modulating specific phosphorylation events

  • Targeting context-specific phosphorylation may offer selective neuroprotection

• Vascular Disease Relevance:

  • LIMK2 regulates endothelial cell cytoskeletal dynamics

  • PKC-mediated phosphorylation affects LIMK2 function in endothelial cells

  • Potential applications in vascular permeability disorders

  • Therapeutic implications for ischemia-reperfusion injury

• Rational Drug Design Considerations:

  • Develop compounds that specifically block Ser283 phosphorylation

  • Target the structural interface between PKC and LIMK2

  • Create conformation-specific inhibitors that recognize phosphorylated states

  • Design dual-specificity compounds affecting multiple regulatory phosphorylation sites

Research has established that LIMK2 inhibition acts synergistically with Aurora A inhibition in promoting cancer cell death , suggesting that targeting specific phosphorylation events may enhance therapeutic efficacy while potentially reducing off-target effects.

What emerging technologies can enhance the study of site-specific LIMK2 phosphorylation?

Recent technological advances offer new opportunities for phosphorylation research:

• AI-Based Antibody Design:

  • AI technologies are enabling de novo generation of antibodies with enhanced specificity

  • Custom antibodies targeting specific phosphorylation states and conformations

  • Computational design of antibodies with reduced cross-reactivity to similar phospho-epitopes

  • Machine learning approaches to predict optimal immunogens for phospho-epitopes

• CRISPR-Based Technologies:

  • Genomic tagging of endogenous LIMK2 with fluorescent or epitope tags

  • Base editing to create phospho-deficient mutations in endogenous genes

  • CRISPR activation/repression systems to modulate LIMK2 expression

  • Optogenetic control of kinase activity for temporal regulation studies

• Advanced Imaging Approaches:

  • Super-resolution microscopy to visualize subcellular phospho-LIMK2 localization

  • FRET-based biosensors for real-time monitoring of phosphorylation events

  • Expansion microscopy for enhanced spatial resolution of phosphorylation patterns

  • Correlative light and electron microscopy to link phosphorylation to ultrastructural changes

• Proteomics Integration:

  • Targeted mass spectrometry approaches for absolute quantification of phosphorylation stoichiometry

  • Phospho-proteomic profiling to identify all LIMK2 phosphorylation sites simultaneously

  • Cross-linking mass spectrometry to identify phosphorylation-dependent protein interactions

  • Thermal proteome profiling to assess phosphorylation-induced conformational changes

• Single-Cell Technologies:

  • Single-cell phospho-proteomics to reveal cell-to-cell variation in LIMK2 regulation

  • Spatial transcriptomics combined with phospho-protein detection

  • Microfluidic approaches for temporal analysis of phosphorylation dynamics

  • CyTOF (mass cytometry) for high-dimensional analysis of phosphorylation networks

These emerging technologies, particularly AI-based antibody design approaches , can address current limitations in phospho-specific detection and enable more comprehensive analysis of LIMK2 regulation across different biological contexts.

What are the most promising future research directions for Phospho-LIMK2 (S283) studies?

Based on current knowledge and technological capabilities, several research directions show particular promise:

• Integrated Multi-Site Phosphorylation Analysis:

  • Develop comprehensive models of how multiple phosphorylation sites (S283, T494, T505) collectively regulate LIMK2

  • Investigate temporal sequences of phosphorylation events under different stimuli

  • Map phosphorylation-specific protein interaction networks

  • Explore the "phosphorylation code" of LIMK2 that determines localization and function

• Disease-Specific Phosphorylation Patterns:

  • Compare LIMK2 phosphorylation profiles across different cancer types

  • Investigate age-dependent changes in neurological disorders

  • Examine tissue-specific phosphorylation patterns in developmental contexts

  • Establish phosphorylation signatures as potential biomarkers

• Therapeutic Targeting Strategies:

  • Develop site-specific phosphorylation inhibitors

  • Evaluate combination approaches targeting kinase cascades regulating LIMK2

  • Investigate tissue-specific delivery of LIMK2 modulators

  • Explore the potential of degraders (PROTACs) targeting phosphorylated LIMK2 forms

• Advanced Technological Applications:

  • Create genetically encoded biosensors for specific phosphorylation events

  • Implement spatial phospho-proteomics to map subcellular phosphorylation domains

  • Develop computational models of LIMK2 regulation integrating multiple phosphorylation sites

  • Apply AI approaches to predict functional outcomes of phosphorylation patterns

The dual function of LIMK2 in cytoskeletal regulation and nuclear processes , coupled with its involvement in cancer progression and neuronal death pathways , highlights the therapeutic potential of targeting specific phosphorylation events in a context-dependent manner.