MAPK15 Antibody

What is MAPK15 and what are its key functions in cellular processes?

MAPK15, also known as ERK7 or ERK8, is an atypical member of the MAP kinase family with a molecular weight of 59.8 kilodaltons and 544 amino acid residues. It is primarily localized in the cytoplasm and belongs to the CMGC Ser/Thr protein kinase family .

Key cellular functions include:

• Autophagy regulation: MAPK15 stimulates AMPK-dependent ULK1 activity, directly interacts with the ULK1 complex, and mediates ULK1 activation during starvation-induced autophagy

• DNA damage protection: It efficiently protects cells from DNA damage and prevents p53-dependent cell cycle arrest mechanisms

• Cell proliferation: MAPK15 enhances tumorigenicity in vivo and promotes cell proliferation in vitro in multiple cancer types

• Early autophagosome biogenesis: It increases ATG12-ATG5 complex formation and ATG12 puncta, indicating involvement in early stages of autophagosome formation

• Radioresistance: MAPK15 regulates radioresistance by attenuating ROS accumulation and promoting DNA damage repair after irradiation exposure

How can researchers validate MAPK15 antibody specificity for their experimental systems?

Ensuring antibody specificity is critical for generating reliable MAPK15 data. Multiple validation approaches should be employed:

For positive validation:

• Overexpression systems: Create stable cell lines expressing MAPK15 (e.g., NTera2/D1_MAPK15) or use transient transfection of MAPK15 constructs

• Cell/tissue type controls: Utilize tissues with known high MAPK15 expression (lung and kidney show maximal expression) as positive controls

• Western blot: Verify antibody detects the expected 59.8 kDa band that increases with overexpression

For negative validation:

• siRNA knockdown: Transfect cells with MAPK15-specific siRNA (which has been shown to result in ~50% decrease in cell counts within 72 hours in NTera2/D1 cells)

• Species-specific controls: Some antibodies recognize only human MAPK15 but not mouse orthologs, making mouse samples valuable negative controls for human-specific antibodies

For specificity validation:

• Multiple antibody confirmation: Use different antibodies targeting distinct MAPK15 epitopes to confirm consistent detection patterns

• Mutant controls: Compare wild-type MAPK15 with kinase-dead (KD) mutants or the MAPK15 AXXA mutant (which specifically affects autophagy induction without disturbing kinase activity)

What role does MAPK15 play in autophagy regulation, and how can this be experimentally investigated?

MAPK15 serves as a key modulator of autophagy through several mechanisms:

• It stimulates 5′-AMP-activated protein kinase-dependent activity of ULK1, the only protein kinase among ATG-related proteins

• MAPK15 directly interacts with the ULK1 complex and mediates ULK1 activation induced by starvation

• It induces an increase in ULK1-dependent ATG13-Ser318 phosphorylation

• MAPK15 promotes early autophagosome biogenesis by increasing ATG12-ATG5 complex levels

Experimental approaches to investigate MAPK15's role in autophagy:

How does MAPK15 contribute to DNA damage response, and which assays are most suitable for studying this relationship?

MAPK15 plays a protective role against DNA damage through multiple mechanisms:

• It prevents DNA damage accumulation even in the absence of extrinsic genotoxic stress

• MAPK15 limits p53 activation and prevents triggering of p53-dependent mechanisms resulting in cell cycle arrest

• Its control of the autophagic process is necessary for basal management of DNA damage

• MAPK15 regulates radioresistance by attenuating ROS accumulation and promoting DNA damage repair after irradiation

• Its expression level correlates with cisplatin sensitivity by affecting DNA repair capacity

Recommended assays for studying MAPK15's role in DNA damage response:

• DNA damage markers:

  • Measure nuclear phospho-Ser139 H2AX (γH2A.X) signal intensity by immunofluorescence or flow cytometry

  • Quantify 53BP1 foci formation

• Cell cycle analysis:

  • Flow cytometry to assess cell cycle distribution after MAPK15 knockdown

  • BrdU incorporation analysis to measure proliferation rates

• p53 pathway assessment:

  • Monitor p53 phosphorylation and activation

  • Measure expression of p53 target genes involved in cell cycle arrest

• ROS detection:

  • Use dichlorofluorescin diacetate fluorescent probe to measure intracellular ROS levels

• Clonogenic survival assays:

  • Evaluate cell survival after exposure to irradiation or cisplatin with MAPK15 knockdown/overexpression

• Cell viability measurements:

  • CCK-8 viability assay following DNA-damaging treatments

• Apoptosis detection:

  • Caspase-3 colorimetric assay

  • Annexin V staining to differentiate between apoptotic and necrotic cells

What are the signaling pathways involving MAPK15 and how can they be studied?

MAPK15 participates in several key signaling pathways:

Autophagy signaling pathway

• MAPK15 stimulates AMPK-dependent ULK1 activity

• It directly interacts with the ULK1 complex components (ULK1, ATG13, FIP200, and ATG101)

• MAPK15 mediates ULK1 activation induced by starvation

Study approaches: Monitor ULK1 substrate phosphorylation (e.g., ATG13-Ser318); assess autophagosome formation using LC3B-II Western blotting; examine ATG12-ATG5 complex formation

DNA damage response pathway

• MAPK15 protects cells from DNA damage

• It limits p53 activation and prevents cell cycle arrest

Study approaches: Measure γH2A.X and 53BP1 as DNA damage markers; monitor p53 activation and downstream effectors

NF-κB signaling pathway

• MAPK15 is transcriptionally regulated by TNF-α-activated NF-κB signaling

• TNF-α synergizes with cisplatin in a MAPK15-dependent manner

Study approaches: Treat cells with TNF-α and measure MAPK15 expression; use NF-κB inhibitors to block this pathway and assess effects on MAPK15 levels

Cell cycle regulation pathway

• MAPK15 knockdown leads to cell cycle arrest at G1-S phase

• It sustains cell cycle progression by limiting p53 activation

Study approaches: Flow cytometry for cell cycle analysis; BrdU incorporation to measure proliferation; analysis of cell cycle regulator expression

How do researchers effectively knockdown or knockout MAPK15 to study its function?

Several approaches have been successfully employed to modulate MAPK15 expression:

siRNA-mediated knockdown:

• MAPK15-specific siRNA has been used in multiple studies with significant functional effects

• In NTera2/D1 cells, MAPK15-specific siRNA resulted in ~50% decrease in cell counts within 72 hours

• Important considerations:

  • Validate knockdown efficiency through Western blot or qPCR

  • Include appropriate negative controls (scrambled siRNA)

  • Optimal transfection conditions may vary by cell type

Dominant-negative approaches:

• MAPK15 kinase-dead (KD) mutant demonstrates a dominant-negative effect, reducing basal ULK1-dependent phosphorylation

• This approach can distinguish between kinase-dependent and kinase-independent functions

• The dominant-negative effect is likely based on direct protein-protein interactions

CRISPR/Cas9 knockout models:

• Mapk15-/- mouse models have been developed and used in studies of metabolic-associated steatotic liver disease

• Can be applied to generate stable knockout cell lines for long-term studies

Function-specific mutants:

• MAPK15 AXXA mutant specifically affects autophagy induction without disturbing kinase activity

• Useful for dissecting specific functional domains and pathways

Overexpression approaches:

• Complementary to knockdown studies

• Stable cell lines expressing MAPK15 (e.g., NTera2/D1_MAPK15) provide consistent expression levels

• Transient transfection can be used for shorter-term studies

What experimental controls should be included when studying MAPK15 in cancer models?

MAPK15 has been implicated in various cancers including germ cell tumors , gastric cancer , nasopharyngeal cancer , and lung cancer . Appropriate controls are essential:

Expression controls:

• Positive tissue controls: Use tissues with known high MAPK15 expression (lung and kidney)

• Negative controls: Include MAPK15 knockdown samples using validated siRNA approaches

• Copy number validation: For gastric cancer studies, validate copy number gains which correlate with MAPK15 overexpression (found in 17% of gastric tumors)

Functional controls:

• MAPK15 wild-type (WT): Standard overexpression construct

• MAPK15 kinase-dead (KD) mutant: Distinguishes between kinase-dependent and kinase-independent functions

• MAPK15 AXXA mutant: Specifically affects autophagy induction without disturbing kinase activity

Pathway-specific controls:

• Autophagy pathway: Include ULK1/2 knockdown or inhibition with SBI-0206965

• DNA damage pathway: Monitor p53 status and activation

• NF-κB pathway: Include TNF-α treatment and/or NF-κB inhibitors when studying MAPK15 regulation

Cell and tissue model controls:

• Matched normal vs. tumor tissue: Essential for expression studies

• Paired cell lines: Use matched models with different MAPK15 expression (e.g., radioresistant CNE2-IR vs. parental CNE2 cells)

• Xenograft models: Include vector control stable cell lines (e.g., NTera2/D1_pCEFL vs. NTera2/D1_MAPK15)

Treatment response controls:

• Starvation conditions: To activate autophagy when studying MAPK15's role in this process

• DNA-damaging agents: Include appropriate dose-response curves for irradiation or cisplatin

• ROS measurements: Include positive controls for ROS induction

How does MAPK15 expression vary across different cancer types, and what implications does this have for research?

MAPK15 expression patterns show specific associations with different cancer types:

Germ cell tumors (GCT):

• Positive correlation between MAPK15 expression and specific GCT subtypes

• Highest expression levels found in aggressive embryonal carcinomas (EC)

• NTera2/D1 cells (derived from EC) are commonly used to study MAPK15 in this context

Gastric cancer:

• Copy number gains of MAPK15 found in 15 (17%) of 88 tumor tissues

• Higher MAPK15 mRNA levels in gastric cancer tissues and cell lines with copy number gains

• Knockdown of MAPK15 in gastric cancer cells suppresses cell proliferation and induces G1-S phase arrest

Nasopharyngeal cancer (NPC):

• MAPK15 identified as a potential regulator of radioresistance in NPC

• Depletion of MAPK15 expression decreased clonogenic survival following radiation exposure

• MAPK15 might regulate radioresistance through attenuating ROS accumulation and promoting DNA damage repair

Lung cancer:

• MAPK15 expression correlates with cisplatin sensitivity in human lung cancer cells

• TNF-α-activated NF-κB pathway regulates MAPK15 expression transcriptionally

• TNF-α synergizes with cisplatin in a MAPK15-dependent manner

Research implications:

• Cell line selection: Choose models with appropriate MAPK15 expression patterns for the cancer type being studied

• Treatment resistance studies: Consider MAPK15's role in radioresistance and chemosensitivity

• Molecular targeting: Potential for developing MAPK15-specific inhibitors to enhance therapeutic efficacy in resistant tumors

• Biomarker development: MAPK15 expression or copy number could serve as biomarkers for treatment response prediction

• Pathway analysis: Different cancer types may utilize distinct MAPK15-regulated pathways (autophagy, DNA damage response, cell cycle regulation)

What approaches can be used to study the relationship between MAPK15 and ULK1 in autophagy regulation?

The MAPK15-ULK1 interaction represents a critical node in autophagy regulation. Several experimental approaches can illuminate this relationship:

Protein-protein interaction studies:

• Co-immunoprecipitation: Immunoprecipitate endogenous MAPK15 from human 293T cells and blot for ULK1 to demonstrate coimmunoprecipitation

• Epitope-tagged pulldowns: ULK1 protein can coimmunoprecipitate both wild-type MAPK15 and MAPK15 KD mutant

• Complex component analysis: Co-transfect MAPK15 with FLAG-tagged ULK1 complex components (ULK1, ATG13, FIP200, ATG101) and immunoprecipitate using FLAG antibody

Colocalization analysis:

• Immunofluorescence: Examine cellular colocalization of MAPK15 and ULK1 to autophagosomal compartments (GABARAP-positive vesicles)

• Confocal microscopy: High-resolution imaging to confirm spatial proximity on autophagosomal structures

Functional analysis:

• ULK1 kinase activity: Monitor ULK1-dependent phosphorylation of ATG13 at Ser318 as a readout of ULK1 activity

• MAPK15 mutants: Compare effects of wild-type MAPK15 vs. kinase-dead mutant on ULK1 activity

• Pharmacological inhibition: Use ULK1/2 inhibitor SBI-0206965 (IC50 of 108 and 711 nM for ULK1 and ULK2, respectively) to assess contribution of these kinases to MAPK15-induced autophagy

Autophagosome biogenesis assessment:

• ATG12-ATG5 complex formation: Measure levels via Western blot as an indicator of early-stage autophagosome formation

• ATG12 puncta formation: Monitor via immunofluorescence to assess early autophagosomal membrane formation

• Double knockdown experiments: Compare effects of MAPK15 knockdown alone versus combined MAPK15/ULK1/ULK2 knockdown

How can researchers investigate MAPK15's role in DNA damage response and cancer therapy resistance?

MAPK15 has emerged as an important regulator of DNA damage response and therapy resistance, particularly in cancer contexts:

DNA damage assessment:

• γH2A.X quantification: Measure intensity of nuclear phospho-Ser139 H2AX signal by immunofluorescence to assess DNA damage levels

• 53BP1 foci: Quantify 53BP1 recruitment as a marker of DNA double-strand breaks

• Time course studies: Determine if MAPK15 affects initial DNA damage or repair kinetics following treatment

Therapy resistance models:

• Radioresistance: Compare radioresistant cell lines (e.g., CNE2-IR) with parental lines (CNE2) for MAPK15 expression and function

• Chemoresistance: Correlate MAPK15 expression with cisplatin sensitivity in lung cancer cells

• Clonogenic survival: Quantify colony formation after radiation or chemotherapy with MAPK15 modulation

Mechanistic studies:

• ROS measurement: Use dichlorofluorescin diacetate to assess if MAPK15 reduces therapy-induced oxidative stress

• p53 pathway: Monitor p53 activation and downstream effectors to determine if MAPK15 limits this tumor suppressor pathway

• Autophagy connection: Determine if MAPK15's role in autophagy contributes to therapy resistance through enhanced cellular stress management

TNF-α/NF-κB signaling:

• Pathway activation: Stimulate cells with TNF-α and assess MAPK15 expression changes

• Combination therapy: Test synergistic effects of TNF-α with cisplatin in MAPK15-expressing vs. knockdown cells

• Transcriptional regulation: Investigate NF-κB-dependent regulation of MAPK15 gene expression

Translational applications:

• Sensitization strategies: Determine if MAPK15 inhibition could enhance radiation or chemotherapy efficacy

• Biomarker development: Assess if MAPK15 expression levels predict therapy response in patient samples

• Pharmacological targeting: Explore potential for developing MAPK15-specific inhibitors as chemotherapy/radiotherapy sensitizers