MAPK1 Human, His

What is MAPK1 and what are its primary cellular functions?

MAPK1 (also known as ERK2) is a ubiquitously expressed member of the MAP kinase family that functions in several cell signaling cascades. It mediates diverse cellular processes including immune responses, proliferation, differentiation, and development. Under normal physiological conditions, MAPK1 expression is modest and persistent in most tissues, but it undergoes significant changes in several malignancies including breast, bladder, lung, and gastric cancers . MAPK1 has dual functionality: it acts as a protein kinase, phosphorylating various substrates, and also functions as a transcription factor capable of binding to G/CAAAG/C consensus sequences at gene promoters to regulate gene expression .

How does MAPK1 differ from other members of the MAPK family, particularly MAPK3 (ERK1)?

While MAPK1 (ERK2) and MAPK3 (ERK1) were initially assumed to be functionally equivalent, genetic studies have revealed distinct roles. MAPK1 deletion results in early embryonic lethality due to failures in trophoblast formation, mesodermal differentiation, and placental development, whereas MAPK3-deficient mice survive embryonic development with only minor developmental defects in thymocyte and adipocyte differentiation . Conditional knockout studies further demonstrate that MAPK1 plays unique roles in certain developmental contexts that cannot be compensated by MAPK3. For example, MAPK1-deficient mice exhibit marked abnormalities in social behaviors related to autism-spectrum disorders, and these impairments are not exacerbated by pharmacological inhibition of MAPK3 .

What experimental models are most suitable for studying human MAPK1 function?

Human gastric adenocarcinoma cell lines (such as AGS) have been successfully used to study MAPK1 functions in cancer progression . For developmental studies, conditional knockout mouse models using tissue-specific Cre drivers (such as Le-Cre for lens development) have proven valuable for understanding MAPK1's role in specific tissues without the complications of embryonic lethality associated with complete knockout . These models allow researchers to distinguish between the roles of MAPK1 and MAPK3 in various developmental contexts. For biochemical studies, various expression systems can be used to produce recombinant His-tagged MAPK1 proteins for in vitro experiments.

How does MAPK1 function as both a kinase and a transcription factor?

MAPK1 exhibits dual functionality that operates through different mechanisms. As a kinase, MAPK1 phosphorylates various protein substrates including other kinases (RAF1, RPS6KA1/RSK1, RPS6KA3/RSK2, SYK, etc.) and phosphatases (DUSP1, DUSP4, DUSP6), enabling signal propagation to additional cytosolic and nuclear targets . This kinase activity is critical for mediating cellular responses to extracellular signals.

As a transcription factor, MAPK1 directly binds to specific DNA sequences (G/CAAAG/C consensus sequence) in the promoter regions of target genes to regulate their expression . Research has demonstrated that MAPK1 can function as a bidirectional transcription factor, both upregulating and downregulating gene expression. In gastric cancer cells, MAPK1 has been shown to bind to promoter regions of target genes, upregulating seven genes (KRT13, KRT6A, KRT81, MYH15, STARD4, SYTL4, and TMEM267) while downregulating one gene (FGG) . This direct transcriptional regulation represents a mechanism distinct from its canonical kinase signaling pathway.

What mechanisms regulate MAPK1 nuclear translocation and DNA binding?

MAPK1 activation typically involves phosphorylation, which enhances its translocation to the nucleus where it can function as a transcription factor. Pathogenic variants in MAPK1 have been shown to promote increased phosphorylation of the kinase, which enhances translocation to the nucleus and boosts MAPK signaling both in vitro and in vivo .

The regulation of MAPK1 activity involves interactions with phosphatases, particularly MKP3 (a dual-specificity protein phosphatase that negatively regulates ERK function). Some pathogenic MAPK1 variants directly disrupt binding to MKP3, leading to dysregulated signaling . Importantly, even when MAPK1 signaling is dysregulated by pathogenic variants, it remains stimulus-reliant and retains dependence on MEK activity, indicating that the protein still functions within the context of the RAF-MEK-ERK pathway .

How do post-translational modifications affect His-tagged MAPK1 function in experimental systems?

When working with His-tagged MAPK1 in experimental systems, researchers must consider how the tag might affect post-translational modifications critical for MAPK1 function. Phosphorylation at specific threonine and tyrosine residues is crucial for MAPK1 activation, and the presence of a His-tag could potentially alter the kinetics or accessibility of these modifications. When conducting phosphorylation studies with His-tagged MAPK1, it's essential to verify that the tag doesn't interfere with kinase recognition sites or three-dimensional protein folding that could affect modification patterns.

What is the role of MAPK1 in cancer progression, particularly in gastric cancer?

MAPK1 plays a significant role in gastric cancer progression through both its kinase activity and transcription factor functions. Overexpression of MAPK1 has been associated with cell migration in BGC-823 gastric cancer cells, while downregulation significantly decreases proliferation of MGC-803 gastric cancer cells, leading to cell cycle progression alterations and promoting apoptosis .

Research using AGS gastric adenocarcinoma cells has demonstrated that MAPK1 promotes invasion and migration by regulating specific target genes in different directions. It upregulates several genes (KRT13, KRT6A, KRT81, MYH15, STARD4, SYTL4, and TMEM267) and downregulates FGG. Among these, five genes (FGG, MYH15, STARD4, SYTL4, and TMEM267) were found to be newly associated with cancer progression, while the others (KRT81, KRT6A, and KRT13) had previously been confirmed to relate to cancer metastasis and migration . These findings suggest that MAPK1's role as a transcription factor directly contributes to the malignant phenotype of gastric cancer cells.

How do MAPK1 mutations contribute to neurodevelopmental disorders?

De novo missense variants in MAPK1 have been identified as causing neurodevelopmental diseases within the RASopathy phenotypic spectrum, reminiscent of Noonan syndrome in some subjects . These pathogenic variants promote increased phosphorylation of the kinase, which enhances its translocation to the nucleus and boosts MAPK signaling both in vitro and in vivo.

Two classes of pathogenic variants have been identified: one class directly disrupts binding to MKP3, a dual-specificity protein phosphatase that negatively regulates ERK function . This disruption leads to increased MAPK1 activity. Importantly, the signal dysregulation driven by pathogenic MAPK1 variants is stimulus-reliant and retains dependence on MEK activity, suggesting potential therapeutic targets for treating these disorders.

What are the current approaches to target MAPK1 in disease therapies?

Understanding MAPK1's dual functionality as both a kinase and transcription factor provides multiple potential therapeutic approaches. Since pathogenic MAPK1 variants retain dependence on MEK activity , MEK inhibitors may represent a promising therapeutic strategy for disorders caused by MAPK1 dysregulation. Additionally, compounds that could enhance MKP3-MAPK1 interactions might restore normal regulation in cases where pathogenic variants disrupt this interaction.

For cancer therapies, approaches that specifically target MAPK1's transcriptional activity on genes promoting invasion and metastasis (such as KRT13, KRT6A, and KRT81) could potentially reduce malignant progression without affecting all MAPK1 functions . This selective targeting might reduce side effects compared to complete MAPK1 inhibition.

What are the optimal methods for expressing and purifying His-tagged human MAPK1?

For recombinant expression of His-tagged human MAPK1, researchers typically clone the full-length coding sequence into an appropriate expression vector. Based on protocols used for related research, the MAPK1 CDS can be obtained by reverse transcription-PCR using cDNA from human cells (such as AGS cells) as a template . Primers should be designed according to the gene annotation of the GRCh38.p13 human genome assembly in databases like Ensembl.

For expression, the CDS should be cloned into a vector containing a His-tag sequence (either N-terminal or C-terminal, though N-terminal tags may be preferable to avoid interfering with C-terminal regulatory domains). Optimal expression systems include E. coli BL21(DE3) for bacterial expression or baculovirus-infected insect cells for eukaryotic expression with more native-like post-translational modifications.

Purification typically involves immobilized metal affinity chromatography (IMAC) using Ni-NTA resin, followed by size exclusion chromatography to ensure high purity. Buffer conditions should maintain protein stability while minimizing aggregation (typically including 20-50 mM Tris-HCl pH 7.5-8.0, 100-300 mM NaCl, 5-10% glycerol, and 1-5 mM DTT or 0.5-1 mM TCEP).

How can ChIP-seq be effectively used to identify MAPK1 binding sites?

ChIP-seq is a powerful technique for identifying MAPK1 binding sites genome-wide. For effective ChIP-seq with MAPK1, researchers should:

• Use validated antibodies specific to MAPK1 (not cross-reactive with MAPK3/ERK1) or use anti-His antibodies when working with His-tagged MAPK1 in experimental systems.

• Optimize crosslinking conditions - typically 1% formaldehyde for 10 minutes at room temperature is effective for protein-DNA interactions.

• Ensure sufficient chromatin fragmentation (200-500 bp fragments) through sonication optimization.

• Include appropriate controls, such as input chromatin and IgG immunoprecipitation controls.

• Validate ChIP-seq results with ChIP-qPCR on selected targets.

Analysis of MAPK1 ChIP-seq data should focus on identifying enriched motifs, particularly the G/CAAAG/C consensus sequence previously identified for MAPK1 binding . Integration with RNA-seq data can help correlate binding events with transcriptional outcomes, as demonstrated in studies showing MAPK1's bidirectional regulation of target genes in gastric cancer cells .

What cell-based assays are most informative for studying MAPK1's dual functionality?

To study MAPK1's dual functionality as both a kinase and transcription factor, researchers should employ complementary approaches:

For kinase activity:

• Western blotting with phospho-specific antibodies to detect phosphorylation of known MAPK1 substrates

• In vitro kinase assays using purified His-tagged MAPK1 and substrate proteins

• Phosphoproteomic analysis to identify novel phosphorylation targets

For transcription factor activity:

• Luciferase reporter assays with promoters containing MAPK1 binding sites

• ChIP followed by qPCR for specific target genes

• Electrophoretic mobility shift assays (EMSA) to confirm direct DNA binding

• Gene expression analysis (qRT-PCR or RNA-seq) combined with MAPK1 overexpression or knockdown

Cell migration and invasion assays (e.g., transwell assays) can be used to assess the functional consequences of MAPK1 activity in cancer cell lines, as demonstrated in studies with gastric cancer cells . Cell proliferation assays and cell cycle analysis are also informative, particularly when comparing wild-type and mutant MAPK1 variants or when studying tissue-specific effects as observed in lens development models .

How should researchers interpret conflicting results about MAPK1 functions in different experimental systems?

When faced with conflicting results regarding MAPK1 function across different experimental systems, researchers should consider several factors:

Resolution strategies include performing experiments in multiple cell types, using both gain-of-function and loss-of-function approaches, and employing complementary techniques to study both kinase and transcription factor activities simultaneously.

What bioinformatic approaches can identify novel MAPK1 transcriptional targets?

To identify novel MAPK1 transcriptional targets, researchers should integrate multiple bioinformatic approaches:

• Motif analysis: Scan genomic regions (particularly promoters) for the G/CAAAG/C consensus sequence known to be bound by MAPK1 . Tools like MEME, HOMER, or JASPAR are useful for this purpose.

• Multi-omics integration: Combine ChIP-seq data identifying MAPK1 binding sites with RNA-seq data showing gene expression changes following MAPK1 manipulation (overexpression, knockdown, or mutation). This approach was successfully used to identify eight MAPK1 target genes in gastric cancer cells .

• Network analysis: Use protein-protein interaction databases and pathway enrichment tools to identify biological processes enriched among potential MAPK1 targets, helping to prioritize candidates for validation.

• Comparative genomics: Examine conservation of putative MAPK1 binding sites across species, as functionally important sites tend to be evolutionarily conserved.

• Machine learning approaches: Train algorithms on known MAPK1 targets to predict additional targets based on promoter features, expression patterns, and other genomic characteristics.

These computational predictions should be validated experimentally through methods such as ChIP-qPCR, reporter assays, and gene expression analysis following MAPK1 manipulation.

How can phosphoproteomics data be effectively analyzed to understand MAPK1 signaling networks?

Phosphoproteomics generates complex datasets that require sophisticated analysis approaches to extract meaningful insights about MAPK1 signaling networks:

• Quantitative analysis: Apply appropriate normalization methods and statistical tests to identify phosphosites significantly affected by MAPK1 activation or inhibition. Consider fold-change thresholds and adjust p-values for multiple testing.

• Motif analysis: Examine sequences surrounding affected phosphosites for MAPK1 consensus phosphorylation motifs (typically P-X-S/T-P) to distinguish direct from indirect targets.

• Temporal dynamics: When time-course data is available, cluster phosphosites by their temporal profiles to identify primary versus secondary effects and feedback mechanisms.

• Pathway mapping: Map phosphoproteins onto known signaling pathways using tools like KEGG, Reactome, or STRING to identify biological processes affected by MAPK1 activity.

• Integration with other data: Combine phosphoproteomics with transcriptomics, ChIP-seq, or protein-protein interaction data to build comprehensive models of MAPK1 signaling.

• Network analysis: Apply algorithms that infer signaling network structure from phosphorylation data, potentially revealing novel connections within MAPK1-dependent pathways.

These approaches can help distinguish MAPK1's direct kinase targets from downstream effects and provide insights into how MAPK1 dysregulation in diseases like cancer and neurodevelopmental disorders affects cellular signaling networks.