Phospho-MAP2K3 (Ser189) Antibody

What is MAP2K3 and why is the Ser189 phosphorylation site significant?

MAP2K3 (Mitogen-Activated Protein Kinase Kinase 3) is a dual specificity kinase that plays a crucial role in cellular responses to cytokines and environmental stressors. The Ser189 site is particularly significant because:

• It is located in the activation loop of MAP2K3, making it essential for kinase activity

• Phosphorylation at Ser189 is required for the subsequent activation of downstream targets, particularly p38 MAPK

• MAP2K3 catalyzes the concomitant phosphorylation of threonine and tyrosine residues in MAP kinase p38 after its own activation

The sequence context surrounding the phosphorylation site is: V-D-S(p)-V-A , which is important for recognition by specific antibodies.

Proper storage and handling are critical for maintaining antibody performance:

• Store at -20°C for long-term preservation

• Aliquot to avoid repeated freeze/thaw cycles, which can degrade antibody performance

• For short-term storage (up to 6 months), some antibodies can be kept at 4°C

• Most commercial preparations are supplied in phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, 150mM NaCl, with 0.02% sodium azide and 50% glycerol

• Concentration typically ranges from 1.0mg/mL

What cross-reactivity should I expect with Phospho-MAP2K3 (Ser189) antibodies?

When selecting an antibody for your experiments, consider the following reactivity information:

• Most commercial antibodies react with human, mouse, and rat MAP2K3

• Some antibodies may cross-react with MAP2K6 (MKK6) phosphorylated at Thr193, due to sequence similarity in the phosphorylation motif

• Some antibodies are available as part of dual recognition sets, with separate antibodies against total MAP2K3 protein and phospho-Ser189 MAP2K3

• Predicted cross-reactivity with other species (e.g., pig, bovine, horse, sheep, rabbit, dog, chicken, Xenopus) exists for some antibodies but should be experimentally validated

How is MAP2K3 Ser189 phosphorylation regulated in cells?

Multiple cellular conditions and pathways regulate MAP2K3 Ser189 phosphorylation:

• Activated by cytokines and environmental stress in vivo

• Lipopolysaccharide (LPS) stimulation induces phosphorylation as part of the Toll-like receptor (TLR) signaling pathway

• TRAF6, a key adaptor molecule for the TLR pathway, regulates this phosphorylation

• Various cellular stressors activate the phosphorylation cascade leading to MAP2K3 activation

• COPI subunit depletion can induce phosphorylation of MAP2K3 and subsequent signaling events

What are the recommended positive controls for Phospho-MAP2K3 (Ser189) antibodies?

When validating your experimental system, consider these established positive controls:

• For Western blot: HeLa cells, especially after stress induction

• For IHC: Human breast carcinoma tissue sections

• For ICC/IF: HeLa cells

• Cell lysates from cells treated with LPS, cytokines, or stress-inducing agents can serve as positive controls

How does MAP2K3 Ser189 phosphorylation compare to similar phosphorylation sites in related kinases?

The phosphorylation of MAP2K3 at Ser189 is part of a conserved regulatory mechanism across related kinases:

• Ser189 in MAP2K3 is equivalent to Ser207 in MAP2K6/MKK6

• The phosphorylation site is comparable to Ser526 in MEKK3 and Ser519 in MEKK2

• The sequence context surrounding these phosphorylation sites is highly conserved, suggesting evolutionary importance

• Despite the similarity, different stimuli may preferentially activate specific kinases, indicating pathway specificity

A comparative analysis of key phosphorylation sites:

What novel research applications utilize Phospho-MAP2K3 (Ser189) antibodies?

Recent studies have employed these antibodies in innovative research contexts:

• Proximity Ligation Assay (PLA) for single-molecule detection of phosphorylated proteins in situ

• Integration with Phospho-seq approaches for multi-modal profiling of intracellular signaling networks

• Investigation of MAP2K3 as a prognostic biomarker in gliomas and other cancers

• Studies on the miR-19b-3p-MAP2K3-STAT3 feedback loop in tumorigenesis

• Analysis of MAP2K3 as a non-canonical kinase for YAP phosphorylation at Ser127, independent of Hippo pathway components LATS1/2

What experimental pitfalls should I be aware of when working with Phospho-MAP2K3 (Ser189) antibodies?

Researchers should consider these technical challenges:

• Antibody specificity may vary between applications (WB vs. IHC vs. IF); validation in each application is recommended

• Post-translational modifications beyond phosphorylation can affect antibody recognition; Yersinia yopJ may acetylate Ser/Thr residues, preventing phosphorylation and blocking antibody detection

• The phosphorylation can be rapidly lost during sample preparation; phosphatase inhibitors should be included in all buffers

• Basal phosphorylation levels may be low in unstimulated cells, requiring stimulus-induced activation for detection

• When using the antibody pair approach, optimization of both antibodies (anti-phospho and anti-total protein) is necessary for reliable results

How can I integrate Phospho-MAP2K3 (Ser189) detection into multi-parameter analyses?

Advanced experimental designs can incorporate Phospho-MAP2K3 (Ser189) detection alongside other measurements:

• Dual staining with other phospho-specific antibodies to map signaling networks

• Combination with transcriptional profiling to correlate phosphorylation states with gene expression patterns

• Integration with functional assays such as migration, invasion, or proliferation assays to link phosphorylation to phenotypic outcomes

• Use in genetically modified systems (CRISPR/Cas9 knockout or knockdown) to establish pathway dependencies

• Incorporation into proteome-wide phosphorylation studies using mass spectrometry to position MAP2K3 within the broader signaling landscape

What emerging roles of MAP2K3 Ser189 phosphorylation have been identified in recent research?

Recent studies have uncovered novel functions of phosphorylated MAP2K3:

• Non-canonical role in YAP phosphorylation at Ser127, independent of the core Hippo pathway kinases LATS1/2

• Involvement in the regulation of autophagy processes through the MAPKAPK2/MAPKAPK3 pathway

• Role in tumor suppression through regulation of the STAT3 pathway

• Potential prognostic biomarker in gliomas, related to immunity and tumor progression

• Participation in the miR-19b-3p-MAP2K3-STAT3 feedback loop that regulates cell proliferation and invasion in esophageal squamous cell carcinoma

What are the recommended protocols for validating a new Phospho-MAP2K3 (Ser189) antibody?

A comprehensive validation strategy should include:

• Western blot analysis comparing phosphorylated vs. non-phosphorylated MAP2K3:

  • Using samples from stimulated vs. unstimulated cells

  • Including phosphatase treatment controls

  • Testing kinase-dead mutants (e.g., MEKK2CT(KM))

• Specificity confirmation:

  • Using cells with MAP2K3 knocked out by CRISPR/Cas9 system

  • Testing with phospho-peptide competition assays

  • Comparing reactivity with the mutated form (S189A)

• Functional validation:

  • Correlation of phosphorylation with downstream target activation (e.g., p38 MAPK)

  • Integration with cellular assays to link phosphorylation to biological outcomes

How can I use Phospho-MAP2K3 (Ser189) antibodies to study signaling dynamics?

For time-course and dynamic studies:

• Stimulation protocols:

  • Treat cells with relevant stimuli (cytokines, LPS, stress inducers)

  • Collect samples at multiple time points (e.g., 0, 5, 15, 30, 60 minutes post-stimulation)

  • Include both early (seconds to minutes) and late (hours) time points

• Quantification approaches:

  • Densitometry analysis of Western blots, normalizing phospho-signal to total MAP2K3

  • Fluorescence intensity measurements in IF/ICC experiments

  • Flow cytometry for single-cell resolution of phosphorylation status

• Inhibitor studies:

  • Use specific pathway inhibitors to establish signaling hierarchies

  • Test both upstream regulators and downstream effectors

  • Include appropriate positive and negative controls

What cloning and expression systems are suitable for studying MAP2K3 phosphorylation?

For recombinant expression and genetic manipulation studies:

• Expression vectors:

  • pCDNA3 and pRc/RSV-FLAG-MAP2K3 have been successfully used

  • MAP2K3 5' regulatory region can be cloned into pGL3-Luc for promoter studies

• Mutational analysis:

  • S189A mutation to prevent phosphorylation

  • Kinase-dead mutations for control experiments

  • Phosphomimetic mutations (S189D or S189E) to mimic constitutive phosphorylation

• Selection and verification:

  • G418 (Geneticin) can be used for selection of stable transfectants

  • Western blot analysis to confirm expression and phosphorylation status

  • Functional assays to validate biological activity