CRCK3 Antibody

What is CRCK3 and why is it significant in plant immunity research?

CRCK3 (CALMODULIN-BINDING RECEPTOR-LIKE CYTOPLASMIC KINASE 3) is encoded by the gene At2g11520 in Arabidopsis thaliana. It features an N-terminal domain with undetermined function and a C-terminal serine/threonine kinase domain. CRCK3's protein structure includes a putative signal peptide and a transmembrane motif in the N-terminal domain, with five predicted MAP kinase phosphorylation sites positioned between the transmembrane motif and the kinase domain . The significance of CRCK3 lies in its role within the SUMM2-mediated immune pathway, where it functions as a sensor for disruptions in the MEKK1-MKK1/MKK2-MPK4 kinase cascade. Unlike many NB-LRR proteins that are species-specific, CRCK3 is conserved across higher plants, suggesting broader biological functions beyond immunity .

What detection methods are commonly used for CRCK3 in experimental settings?

The most prevalent detection method for CRCK3 in research settings is immunoblotting (Western blot) using epitope tag antibodies. Researchers frequently express CRCK3 as a fusion protein with tags such as FLAG or HA to facilitate detection. FLAG-tagged CRCK3 proteins can be immunoprecipitated with anti-FLAG conjugated beads (such as those from Sigma) and subsequently detected via Western blot using anti-FLAG antibodies . For analyzing phosphorylation states, Phos-tag™ polyacrylamide gel electrophoresis followed by Western blot provides effective visualization of mobility shifts between phosphorylated and non-phosphorylated forms of CRCK3 . These techniques are essential for both expression analysis and protein-protein interaction studies involving CRCK3.

How can researchers differentiate between phosphorylated and non-phosphorylated forms of CRCK3 in experimental systems?

Distinguishing between phosphorylation states of CRCK3 requires specialized techniques beyond standard Western blotting. Phos-tag™ polyacrylamide gel electrophoresis represents the optimal approach, as demonstrated in studies comparing CRCK3-FLAG proteins from wild-type and mpk4-3 mutant plants . In this technique, phosphorylated proteins exhibit a clear mobility shift compared to their non-phosphorylated counterparts. Researchers should include phosphatase-treated CRCK3 samples as controls to establish the migration pattern of completely dephosphorylated protein. The efficacy of this method was validated in studies showing that wild-type plants contained predominantly phosphorylated CRCK3, while mpk4-3 mutants exhibited significant amounts of non-phosphorylated CRCK3 . For site-specific phosphorylation analysis, researchers should consider directed mutagenesis of predicted phosphorylation sites (such as the S172, S181, S188, S195, and S202 residues in CRCK3) to alanine, followed by mobility shift analysis. The CRCK3^5S–5A mutant protein, with all five predicted MAP kinase phosphorylation sites replaced by alanine residues, showed almost complete elimination of the mobility shift seen in wild-type CRCK3, confirming that these sites are indeed phosphorylated in planta .

What experimental approaches can validate MPK4 as a kinase for CRCK3, and how should researchers interpret conflicting phosphorylation data?

Validating MPK4 as a kinase for CRCK3 requires a multi-pronged approach combining both in vitro and in vivo methods. For in vitro validation, researchers should purify both the kinase (MPK4) and substrate (CRCK3) proteins and conduct kinase assays under controlled conditions. As demonstrated in published work, CRCK3^G390R was expressed in E. coli, purified, and used in kinase assays with MPK4-FLAG isolated from transgenic plants . Critical controls include comparing MPK4 from flg22-treated plants (activated) versus untreated plants. When conflicting phosphorylation data arises, researchers should:

• Compare phosphorylation in different genetic backgrounds (e.g., wild-type vs. mpk4 mutants)

• Utilize Phos-tag™ gel electrophoresis to visualize mobility shifts

• Perform site-directed mutagenesis of predicted phosphorylation sites

• Consider the activation state of the kinase in different experimental conditions

The interpretation should account for potential redundancy in kinase activity, as other kinases may compensate for MPK4 absence. Additionally, researchers should consider that partial phosphorylation may occur at different sites, resulting in complex mobility patterns. The finding that a "considerable amount of CRCK3-FLAG protein in mpk4-3 has similar mobility as the phosphatase-treated protein" suggests that MPK4 is responsible for a significant portion, but perhaps not all, of CRCK3 phosphorylation in vivo .

How can site-directed mutagenesis be used to investigate the functional significance of CRCK3 phosphorylation sites?

Site-directed mutagenesis provides a powerful approach to investigate the functional significance of CRCK3 phosphorylation sites through the creation of phospho-null and phospho-mimetic variants. Researchers should begin by identifying potential phosphorylation sites using bioinformatic tools; for CRCK3, five predicted MAP kinase phosphorylation sites (S172, S181, S188, S195, and S202) have been identified . To generate phospho-null variants, these serine residues should be replaced with alanine (as in the CRCK3^5S–5A mutant), which cannot be phosphorylated. Complementarily, phospho-mimetic variants can be created by substituting serine residues with aspartic or glutamic acid to simulate constitutive phosphorylation.

These variants should then be expressed in appropriate genetic backgrounds (wild-type, crck3 mutant, or mpk4 mutant plants) as epitope-tagged fusion proteins to facilitate detection. Functional analysis should include:

• Protein-protein interaction assays to determine if phosphorylation affects CRCK3-SUMM2 association

• Plant immunity assays to assess whether phosphorylation is required for activation of defense responses

• Subcellular localization studies to determine if phosphorylation affects CRCK3 trafficking

• In vitro kinase activity assays to determine if phosphorylation regulates CRCK3's intrinsic kinase activity

The observation that the phospho-null CRCK3^5S–5A variant loses the mobility shift seen in wild-type CRCK3 confirms that these sites are indeed phosphorylated in planta, providing a foundation for further functional characterization .

What are the optimal conditions for immunoprecipitation of CRCK3 for protein-protein interaction studies?

The optimal conditions for CRCK3 immunoprecipitation in protein-protein interaction studies require careful consideration of expression systems, tags, and experimental conditions. Based on successful protocols in the literature, researchers should:

• Expression system selection: Utilize either transient expression in Nicotiana benthamiana via agroinfiltration or stable expression in Arabidopsis thaliana. Transient expression provides rapid results but may yield non-physiological protein levels, while stable expression more accurately reflects natural conditions but requires more time to establish transgenic lines .

• Epitope tag selection: Express CRCK3 as a FLAG-tagged fusion protein and potential interactors (e.g., SUMM2) with alternative tags such as HA. This strategy enables sequential immunoprecipitation and detection without antibody cross-reactivity .

• Immunoprecipitation procedure:

  • Harvest and homogenize plant tissue in appropriate extraction buffer

  • Clarify lysates by centrifugation (typically 14,000g for 15 minutes at 4°C)

  • Incubate clarified lysates with anti-FLAG conjugated agarose beads (such as Sigma-Aldrich; 087K6001)

  • Wash extensively to remove non-specific binding

  • Elute bound proteins with either FLAG peptide competition or SDS-PAGE sample buffer

• Detection strategy: Analyze immunoprecipitates by SDS-PAGE followed by Western blotting with appropriate antibodies (anti-FLAG for CRCK3 and anti-HA for interactors like SUMM2) .

For domain-specific interaction studies, researchers should generate truncated versions of CRCK3 (such as the kinase domain alone) to map interaction regions. This approach has successfully demonstrated that the kinase domain of CRCK3 is sufficient for SUMM2-CRCK3 association .

How should researchers design experiments to investigate CRCK3 phosphorylation by MPK4 in vivo?

Designing robust experiments to investigate CRCK3 phosphorylation by MPK4 in vivo requires multiple complementary approaches:

• Genetic manipulation strategy:

  • Generate transgenic Arabidopsis lines expressing epitope-tagged CRCK3 (e.g., CRCK3-FLAG) under its native promoter in wild-type background

  • Cross these lines with mpk4 mutants to introduce the transgene into the mpk4 background

  • Compare phosphorylation status between wild-type and mpk4 genetic backgrounds

• Phosphorylation detection techniques:

  • Utilize Phos-tag™ polyacrylamide gel electrophoresis followed by Western blotting to visualize mobility shifts indicative of phosphorylation

  • Include lambda phosphatase-treated samples as controls for complete dephosphorylation

  • For enhanced sensitivity, consider phospho-specific antibodies against predicted phosphorylation sites, though this requires custom antibody production

• MAPK activation strategies:

  • Include treatments that activate the MAPK cascade, such as flg22 (a bacterial flagellin-derived peptide)

  • Monitor MPK4 activation status using phospho-specific antibodies against activated MAPKs

  • Include appropriate time courses to capture transient phosphorylation events

• Mutagenesis approaches:

  • Generate phospho-null mutants by replacing predicted phosphorylation sites with alanine residues

  • Create phospho-mimetic variants by substituting serine residues with aspartic acid

  • Express these variants in both wild-type and mpk4 backgrounds

This experimental strategy has successfully demonstrated that CRCK3 is predominantly phosphorylated in wild-type plants but significantly less phosphorylated in mpk4-3 mutants, confirming MPK4's role in CRCK3 phosphorylation in vivo .

What techniques can be applied to investigate the role of CRCK3 in specific plant immune pathways?

Investigating CRCK3's role in specific plant immune pathways requires a comprehensive experimental toolkit:

• Genetic analysis approaches:

  • Utilize CRISPR/Cas9 or T-DNA insertion mutants to generate crck3 knockout lines

  • Create double or triple mutants with other immune pathway components (e.g., summ2, mpk4, mkk1/mkk2)

  • Develop complementation lines expressing native or mutated CRCK3 variants in crck3 backgrounds

  • Perform epistasis analysis to position CRCK3 within signaling cascades

• Pathogen challenge assays:

  • Challenge plants with bacterial pathogens like Pseudomonas syringae (e.g., P.s.t DC3000 strains carrying avirulence genes)

  • Quantify bacterial growth in planta to assess immunity strength

  • Compare wild-type and crck3 mutant responses to various pathogen strains

  • Include appropriate controls such as known immune-compromised mutants

• Immune response marker analysis:

  • Monitor expression of defense-related genes (e.g., PR1, PR5) via qRT-PCR

  • Assess reactive oxygen species (ROS) production using luminol-based assays

  • Evaluate callose deposition through aniline blue staining

  • Quantify accumulation of defense hormones like salicylic acid

• Specificity determination:

  • Compare responses to different immune elicitors (PAMPs, effectors)

  • Assess involvement in different immune pathways (PTI vs. ETI)

  • Test requirement for immunity mediated by different NB-LRR proteins

This multi-faceted approach has revealed that CRCK3 is specifically required for SUMM2-mediated immunity but dispensable for RPS2- and RPS5-mediated immunity, demonstrating pathway specificity rather than a general role in CC-NB-LRR-mediated immunity .

How can researchers distinguish between direct and indirect effects when analyzing CRCK3 mutant phenotypes?

Distinguishing between direct and indirect effects in CRCK3 mutant phenotypes requires systematic analytical approaches:

• Genetic complementation analysis:

  • Generate multiple independent transgenic lines expressing wild-type CRCK3 in crck3 mutant backgrounds

  • Confirm that complementation restores all phenotypes to wild-type levels

  • Introduce domain-specific or phosphorylation site mutants to identify critical functional regions

• Temporal resolution of molecular events:

  • Perform time-course experiments following immune elicitation

  • Determine the sequence of molecular events (phosphorylation, protein-protein interactions, defense gene expression)

  • Compare timing in wild-type versus mutant backgrounds

• Biochemical validation of direct interactions:

  • Conduct in vitro kinase assays with purified components to confirm direct enzymatic relationships

  • Perform direct binding assays (e.g., yeast two-hybrid, pull-down assays with recombinant proteins)

  • Use proximity labeling techniques to identify proteins in close physical proximity to CRCK3 in vivo

• Genetic epistasis analysis:

  • Create double mutants between crck3 and other pathway components

  • Analyze whether phenotypes are additive, synergistic, or epistatic

  • Position CRCK3 within signaling hierarchies based on genetic interactions

This approach has been successfully applied to demonstrate that CRCK3 functions together with SUMM2 in sensing disruption of the MEKK1-MKK1/MKK2-MPK4 kinase cascade. The suppression of mkk1 mkk2 phenotypes by summ3-1 (a crck3 mutant) at both morphological and molecular levels indicates a direct role for CRCK3 in this specific immune pathway rather than a general effect on plant development or physiology .

What statistical approaches are appropriate for analyzing variable phosphorylation patterns of CRCK3 across experimental conditions?

Analyzing variable phosphorylation patterns of CRCK3 requires robust statistical approaches tailored to quantitative proteomics and biochemical data:

• Quantification methodology:

  • Densitometric analysis of Western blot bands from Phos-tag™ gels

  • Calculate the ratio of phosphorylated to non-phosphorylated forms

  • Normalize to total protein loading controls

  • Consider multiple technical and biological replicates (minimum n=3)

• Statistical tests for comparisons:

  • For comparing two conditions (e.g., wild-type vs. mpk4): Two-tailed Student's t-test or Mann-Whitney U test depending on normality

  • For multiple conditions: One-way ANOVA followed by appropriate post-hoc tests (Tukey's HSD, Dunnett's test)

  • For time-course experiments: Repeated measures ANOVA or mixed-effects models

• Visualization approaches:

  • Display both representative images and quantification data

  • Use box plots or violin plots to show distribution of phosphorylation ratios

  • Consider heat maps for visualizing phosphorylation patterns across multiple sites and conditions

• Advanced analytical considerations:

  • Account for potential non-linear relationships between signal intensity and protein quantity

  • Consider phosphorylation at multiple sites using multivariate statistics

  • Implement bootstrap or permutation tests for small sample sizes

What approaches should researchers use to develop specific antibodies against native CRCK3 protein?

Developing specific antibodies against native CRCK3 requires a strategic approach:

• Antigen design considerations:

  • Select unique regions with high antigenicity and surface exposure

  • Avoid transmembrane domains and regions with high sequence conservation across related kinases

  • Consider both full-length recombinant protein and synthetic peptide approaches

  • For peptide antigens, select sequences of 15-20 amino acids from unique regions, preferably from the variable N-terminal domain rather than the more conserved kinase domain

• Expression and purification strategies:

  • Express recombinant CRCK3 in E. coli systems using established protocols similar to those used for CRCK3^G390R in kinase assays

  • Purify using affinity chromatography under native or denaturing conditions

  • Verify purity by SDS-PAGE and protein identity by mass spectrometry

  • For challenging expression, consider fusion partners that enhance solubility (MBP, SUMO)

• Immunization and antibody production protocols:

  • Immunize rabbits or other suitable hosts following standard protocols

  • Consider multiple animals to increase chances of obtaining high-affinity antibodies

  • Collect pre-immune serum as a critical negative control

  • Perform affinity purification of antibodies against the immunizing antigen

• Validation requirements:

  • Test antibody specificity using wild-type and crck3 knockout plant extracts

  • Verify recognition of both native and denatured CRCK3 for different applications

  • Determine cross-reactivity with related plant receptor-like cytoplasmic kinases

  • Validate for specific applications (Western blot, immunoprecipitation, immunolocalization)

This comprehensive approach ensures development of CRCK3 antibodies suitable for detecting endogenous protein without relying on epitope tags, enabling studies of native CRCK3 in various plant species and conditions.

How can researchers optimize protocols for detecting CRCK3-antibody complexes in immunoprecipitation experiments?

Optimizing immunoprecipitation protocols for CRCK3-antibody complexes requires careful consideration of multiple experimental variables:

• Lysis buffer optimization:

  • Test different detergent compositions (NP-40, Triton X-100, digitonin) at varying concentrations

  • Adjust salt concentration (150-500 mM NaCl) to minimize non-specific interactions

  • Include protease inhibitors (e.g., PMSF, protease inhibitor cocktail) and phosphatase inhibitors (NaF, Na₃VO₄) when studying phosphorylated forms

  • Consider mild detergents for maintaining protein-protein interactions or stronger conditions for reducing background

• Antibody coupling strategies:

  • Compare direct immunoprecipitation vs. pre-coupling antibodies to solid supports

  • For tagged CRCK3, test commercial anti-FLAG conjugated beads (as used in published studies)

  • For native CRCK3, consider coupling custom antibodies to Protein A/G beads or NHS-activated resins

  • Optimize antibody:bead ratios to maximize capture efficiency

• Incubation parameters:

  • Test different incubation times (2 hours vs. overnight)

  • Compare incubation temperatures (4°C vs. room temperature)

  • Optimize lysate:antibody ratios based on CRCK3 expression levels

  • Consider sequential immunoprecipitation for studying multi-protein complexes

• Washing and elution conditions:

  • Determine optimal wash stringency (detergent concentration, salt concentration)

  • Compare different elution methods:

    • Denaturing (SDS sample buffer) for maximum recovery

    • Native (peptide competition) for downstream functional assays

    • Acidic glycine for antibody reuse

The optimization process should include appropriate controls (IgG control, lysate from crck3 mutants) and quantitative analysis of CRCK3 recovery efficiency and purity of immunoprecipitates using Western blotting techniques similar to those employed in published CRCK3-SUMM2 interaction studies .

How can antibody-based techniques be combined with CDR walking strategies to develop improved detection reagents for CRCK3?

Combining antibody-based techniques with CDR walking strategies offers a powerful approach for developing next-generation CRCK3 detection reagents:

• Initial antibody selection process:

  • Start with existing anti-CRCK3 antibodies or develop phage display libraries

  • Screen for antibodies with moderate affinity and specificity for CRCK3

  • Select candidates with favorable expression characteristics and stability

  • Sequence the variable domains, with particular focus on the CDR regions

• CDR walking optimization strategy:

  • Systematically mutate the CDRs (particularly CDRH3) in a stepwise manner

  • After each round of mutation, select variants with improved binding characteristics

  • Use the best mutant as template for subsequent rounds of mutagenesis

  • Continue iterations until desired affinity and specificity are achieved

• Implementation of established CDR walking success principles:

  • Apply lessons from documented successes in other antibody optimization projects

  • Follow patterns similar to Yang et al.'s development of high-affinity anti-HIV gp120 Fab (420-fold increase to Kd=1.5×10⁻¹¹ M)

  • Implement approaches like those used by Schier et al. for anti-c-erbB-2 scFv with picomolar affinity (Kd=1.3×10⁻¹¹M)

• Advanced computational approaches:

  • Implement machine learning algorithms for CDR3 optimization

  • Conduct computational mutagenesis to predict beneficial modifications

  • Apply hot-spot grafting by transferring binding site motifs from existing protein-protein complexes

  • Consider re-epitoping approaches to test existing antibodies for binding to target epitopes

This integrated approach combines the specificity of antibody-based detection with the affinity enhancement of CDR walking, potentially yielding CRCK3 detection reagents with substantially improved sensitivity and specificity for research applications.

What computational approaches can be used to analyze CRCK3 antibody binding properties and optimize detection protocols?

Computational approaches offer powerful tools for analyzing CRCK3 antibody binding properties and optimizing detection protocols:

• Structural modeling and epitope prediction:

  • Generate homology models of CRCK3 using crystal structures of related kinases

  • Predict surface-exposed epitopes using algorithms that consider accessibility, hydrophilicity, and mobility

  • Model antibody-antigen complexes using molecular docking approaches

  • Identify critical binding residues through computational alanine scanning

• Sequence-based repertoire analysis:

  • Apply error correction algorithms like those used in IgReC to accurately reconstruct antibody repertoires from sequencing data

  • Implement V(D)J classification to understand the diversity of anti-CRCK3 antibodies

  • Analyze CDR3 sequences using specialized classification tools to identify binding motifs

  • Construct full-length antibody repertoires that account for hypermutations

• Machine learning optimization:

  • Train algorithms on experimental binding data to predict optimal conditions

  • Develop models that correlate buffer compositions with antibody performance

  • Optimize protocol parameters (incubation time, temperature, pH) through predictive modeling

  • Generate virtual libraries of antibody variants for in silico screening

• Statistical approaches for protocol optimization:

  • Implement Design of Experiments (DoE) methodology to efficiently test multiple parameters

  • Use response surface methodology to identify optimal conditions with minimal experiments

  • Apply Bayesian optimization approaches for sequential experimental design

  • Develop robust statistical models for analyzing variable antibody performance across conditions

These computational approaches should complement experimental data, particularly from techniques like Phos-tag™ gel electrophoresis that have successfully distinguished phosphorylated and non-phosphorylated CRCK3 forms . By integrating computational and experimental approaches, researchers can develop highly optimized detection protocols specific to CRCK3's unique structural and biochemical properties.

How might techniques from antibody repertoire analysis be applied to develop panels of CRCK3-specific detection reagents?

Applying antibody repertoire analysis techniques to CRCK3-specific detection reagent development offers innovative approaches for creating comprehensive detection panels:

• Error-corrected repertoire construction:

  • Implement sophisticated error correction algorithms like those in IgReC to accurately reconstruct antibody repertoires from immunized animals

  • Apply barcode-based methods to minimize sequencing errors when analyzing anti-CRCK3 antibody libraries

  • Address barcode errors and collisions using computational approaches to maintain true diversity

  • Filter constructed repertoires appropriately to balance diversity preservation and error minimization

• Diversity analysis strategies:

  • Apply CDR3 classification to identify families of antibodies with similar binding properties

  • Group antibodies based on V(D)J segment usage to understand genetic origins of CRCK3 binding

  • Analyze hypermutation patterns outside CDR3 to identify affinity maturation trajectories

  • Compare diversity metrics between different immunization strategies to optimize antibody generation

• Complementarity-determining region (CDR) analysis:

  • Identify conserved motifs in CDRH3 regions of CRCK3-binding antibodies

  • Apply lessons from broadly neutralizing antibody development, where shared CDRH3 motifs indicate common binding solutions to challenging epitopes

  • Develop antibody panels targeting different epitopes based on CDR sequence clustering

• Panel development considerations:

  • Select antibodies recognizing distinct epitopes on CRCK3 for comprehensive detection

  • Include reagents specific for different phosphorylation states

  • Develop antibodies suitable for different applications (Western blot, IP, IHC)

  • Create paired antibodies for sandwich assays to quantify CRCK3 in complex samples

This integrated approach applies cutting-edge repertoire analysis techniques to develop not just single antibodies but coherent panels of detection reagents that collectively provide comprehensive analytical capabilities for CRCK3 research across multiple experimental contexts and applications.

What are common pitfalls in CRCK3 detection experiments and how can researchers overcome them?

Researchers frequently encounter several challenges when detecting CRCK3 in experimental systems:

• Post-translational modification heterogeneity:

  • Problem: Variable phosphorylation creates multiple bands or smears on Western blots

  • Solution: Include phosphatase-treated controls to identify the migration pattern of unmodified CRCK3

  • Solution: Use Phos-tag™ gel electrophoresis to clearly separate phosphorylated from non-phosphorylated forms

  • Solution: Consider phosphorylation-state specific antibodies for precise detection

• Low expression levels:

  • Problem: Endogenous CRCK3 may be expressed at levels below detection limits of standard methods

  • Solution: Use signal amplification methods such as enhanced chemiluminescence

  • Solution: Consider enrichment via immunoprecipitation before Western blotting

  • Solution: Optimize protein extraction protocols specifically for membrane-associated proteins

• Cross-reactivity with related kinases:

  • Problem: Antibodies may detect related receptor-like cytoplasmic kinases

  • Solution: Include appropriate negative controls (crck3 knockout extracts)

  • Solution: Perform peptide competition assays to confirm specificity

  • Solution: Use epitope-tagged versions in parallel with antibody detection for validation

• Protein-protein interactions masking epitopes:

  • Problem: CRCK3-SUMM2 or other protein interactions may obscure antibody binding sites

  • Solution: Test different extraction conditions that may disrupt protein complexes

  • Solution: Develop antibodies against multiple regions of CRCK3

  • Solution: Consider native vs. denaturing conditions depending on experimental goals

Implementing these solutions can significantly improve experimental outcomes, as demonstrated in studies that successfully detected CRCK3-FLAG in different phosphorylation states and identified its interactions with SUMM2 .

How should researchers structure quantitative comparisons of CRCK3 phosphorylation across different experimental conditions?

Researchers should structure quantitative phosphorylation comparisons using standardized experimental designs and clear data presentation:

Table 1: Quantitative Analysis of CRCK3 Phosphorylation Status Across Genetic Backgrounds

Data represents relative proportions of phosphorylated and non-phosphorylated CRCK3-FLAG detected by Phos-tag™ gel electrophoresis followed by Western blotting. Values shown as mean ± standard deviation. Phosphorylation ratio calculated as (phosphorylated%)/(non-phosphorylated%).

Table 2: Effect of MAP Kinase Phosphorylation Site Mutations on CRCK3 Phosphorylation Status

Data represents analysis of FLAG-tagged CRCK3 variants expressed in wild-type plants and analyzed by Phos-tag™ gel electrophoresis. Values shown as mean ± standard deviation (n=3). The CRCK3^5S-5A variant shows almost complete elimination of the mobility shift, confirming these sites as the primary MPK4 phosphorylation targets .