MAPK11 Human

What is the molecular structure and basic function of MAPK11?

MAPK11 is a 364 amino acid protein with an observed molecular weight of 42 kDa (calculated weight: 41 kDa) . It belongs to the MAP kinase subfamily and functions as one of the four p38 MAPKs that mediate cellular responses to extracellular stimuli . MAPK11 operates through the phosphorylation of substrate proteins, participating in a Ser/Thr kinase protein cascade that controls fundamental cellular processes including viability, differentiation, proliferation, and apoptosis . The primary active form of MAPK11 is its phosphorylated state (P-MAPK11), which is essential for its kinase activity and downstream signaling effects .

How is MAPK11 expression regulated in normal human tissues?

MAPK11 is expressed in multiple human tissues, with notable expression confirmed in brain, heart, and skeletal muscle . The regulation of MAPK11 involves upstream activators, particularly MAP2K6, which serves as a primary kinase responsible for MAPK11 phosphorylation and activation . While the total protein levels of MAPK11 may remain relatively stable across some tissue types, the activation state (phosphorylation) can vary significantly in response to cellular stress signals and pathological conditions . Western blot analysis has confirmed MAPK11 expression in Jurkat cells, human heart tissue, and mouse skeletal muscle tissue, indicating conservation across species .

What is the difference between MAPK11 and other p38 MAPK family members?

MAPK11 (also known as p38β) is one of four p38 MAPK family members that responds to extracellular stimuli . While sharing structural similarities with other family members, MAPK11 displays distinct substrate preferences and tissue expression patterns. Unlike some MAPK family members that show ubiquitous expression, MAPK11 demonstrates tissue specificity as confirmed by immunohistochemistry and Western blot analysis . Additionally, genetic knockout studies in mice have shown that MAPK11 deletion results in viable and fertile animals with no obvious health problems under standard conditions, suggesting potential functional redundancy with other family members or context-specific critical functions .

What role does phosphorylated MAPK11 play in clear cell renal cell carcinoma (ccRCC)?

Phosphorylated MAPK11 (P-MAPK11) demonstrates significantly upregulated expression in clear cell renal cell carcinoma (ccRCC) tissues compared to adjacent normal tissues . Analysis of the Cancer Genome Atlas (TCGA) database revealed that MAPK11 transcription levels are elevated in ccRCC . Importantly, while total MAPK11 protein expression shows no considerable difference between cancerous and healthy tissues, P-MAPK11 is significantly overexpressed in ccRCC tissues, suggesting that activation rather than expression is the critical oncogenic mechanism .

P-MAPK11 expression in ccRCC positively correlates with Fuhrman grade (p=0.0033), indicating its potential role in tumor progression and aggressiveness . Furthermore, P-MAPK11 shows a positive correlation with RUNX2 protein expression in ccRCC samples, suggesting a potential interaction pathway . Functional studies demonstrated that MAPK11 knockdown significantly impeded cellular migration, proliferation, and colony formation abilities in ccRCC cell lines (786-O and ACHN), confirming its role in promoting tumor cell malignant behavior .

How does MAPK11 contribute to Huntington's disease pathology?

MAPK11 plays a significant role in Huntington's disease (HD) pathology by regulating mutant Huntingtin (mHTT) protein levels . In HD, the accumulation of mutant Huntingtin protein is a primary pathogenic mechanism, and MAPK11 has been identified as a modulator of this process. Research has demonstrated that genetic knockout or knockdown of MAPK11 significantly lowers mHTT levels both in vitro and in vivo .

Studies using HD mouse models expressing endogenous mHtt proteins with a 140Q repeat showed that heterozygous knockout of MAPK11 significantly reduced full-length Htt levels in the striatum . Mechanistically, mHTT appears to enhance MAPK11 kinase activity, as expression of mHTT (HTTexon1 (Q72)) increased the phosphorylation of MAPK11, while wild-type HTT (HTTexon1 (Q25)) did not produce this effect . The activation of endogenous MAPK11 can be assessed through its ability to phosphorylate downstream targets such as Atf2, with Atf2 phosphorylation significantly reduced in MAPK11 knockout mice striata and increased in HD mice striata .

What is the relationship between MAPK11 and RUNX2 in cancer progression?

MAPK11, particularly in its phosphorylated form (P-MAPK11), demonstrates a significant positive correlation with RUNX2 expression in ccRCC samples, suggesting a functional relationship between these proteins in cancer progression . RUNX2, a transcription factor primarily known for its role in osteoblast differentiation and chondrocyte maturation, is highly expressed in multiple tumors, including ccRCC .

Western blotting analysis revealed that knockdown of MAPK11 significantly reduced RUNX2 protein expression in ccRCC cell lines, while the mRNA expression of RUNX2 remained unchanged . This suggests that P-MAPK11 regulates RUNX2 at the post-transcriptional level. Conversely, overexpression of RUNX2 did not alter the protein or mRNA levels of MAPK11 or P-MAPK11 . Co-immunoprecipitation assays further support the interaction between these proteins, as antibodies against P-MAPK11 and RUNX2 were used together to study their association . This unidirectional regulatory relationship suggests that MAPK11 acts upstream of RUNX2 in the oncogenic pathway in ccRCC.

What are the most effective antibodies and protocols for detecting MAPK11 in various experimental contexts?

For MAPK11 detection, polyclonal antibodies such as 17376-1-AP have been validated for multiple applications including Western Blot (WB), Immunohistochemistry (IHC), and Immunofluorescence (IF)/ICC . The recommended dilutions vary by application: 1:500-1:1000 for WB, 1:20-1:200 for IHC, and 1:50-1:500 for IF/ICC . These antibodies have demonstrated reactivity with human, mouse, and rat samples, making them versatile for cross-species studies .

For Western blotting, positive detection has been confirmed in Jurkat cells, human heart tissue, and mouse skeletal muscle tissue . For IHC applications, human brain tissue has shown positive results, with recommended antigen retrieval using TE buffer pH 9.0 or alternatively with citrate buffer pH 6.0 . For IF/ICC applications, HEK-293 cells have been validated for positive detection . When handling MAPK11 antibodies, proper storage at -20°C in PBS with 0.02% sodium azide and 50% glycerol pH 7.3 is recommended, with stability for one year after shipment .

How can researchers effectively study the phosphorylation state of MAPK11?

Studying the phosphorylation state of MAPK11 requires specific approaches to distinguish between total MAPK11 and its active phosphorylated form (P-MAPK11). Western blotting with phospho-specific antibodies is the most commonly employed technique . When performing Western blotting, researchers should include appropriate controls: positive controls (such as tissues known to express P-MAPK11) and negative controls (such as phosphatase-treated samples) .

Co-immunoprecipitation (Co-IP) assays provide another valuable method for studying MAPK11 phosphorylation and its interactions with other proteins. The protocol involves lysing cells with RIPA buffer combined with protease inhibitor cocktail, followed by centrifugation at 12,000 g for 20 minutes at 4°C . The cell lysate is then incubated with antibodies (anti-P-MAPK11 and any potential interacting protein antibodies) overnight at 4°C with rotation, followed by incubation with magnetic beads for 4 hours .

For functional validation of MAPK11 activity, researchers can assess the phosphorylation of known downstream targets such as Atf2 . Increased phosphorylation of Atf2 indicates enhanced MAPK11 activity, while decreased phosphorylation suggests reduced activity. This approach provides an indirect but functional readout of MAPK11 activation state .

What are the most effective approaches for genetic manipulation of MAPK11 in experimental models?

For genetic manipulation of MAPK11, several validated approaches have been employed in the literature:

• shRNA Knockdown: Stable cell lines with MAPK11 knockdown can be generated using shRNA targeting MAPK11. This approach has been successfully applied in ccRCC cell lines (786-O and ACHN), resulting in significantly diminished protein expression levels of both MAPK11 and P-MAPK11 .

• CRISPR/Cas9 or TALEN Technology: Complete knockout models of MAPK11 have been generated using TALEN technology in mice . These knockout models were viable and fertile with no obvious health problems under standard specific-pathogen-free conditions, consistent with previous reports . MAPK11 knockout mice have been successfully crossed with disease models, such as HD knock-in mice expressing endogenous mHtt proteins with a 140Q repeat, to study the effects of MAPK11 deletion on disease progression .

• Overexpression Systems: For gain-of-function studies, overexpression of MAPK11 can be achieved through transfection with expression vectors containing the MAPK11 gene. This approach is particularly useful when studying MAPK11 activation, as demonstrated in studies where MAPK11 overexpression was combined with detection of its phosphorylation state to assess activation by mutant Huntingtin protein .

• Epistasis Studies: To determine the positioning of MAPK11 in signaling pathways, epistasis approaches can be employed, such as the study where MAPK11 knockdown diminished the MAP2K6-mediated effect on mHTT levels, confirming that MAPK11 functions downstream of MAP2K6 .

How can researchers quantify MAPK11 activity in different experimental systems?

Quantification of MAPK11 activity requires distinguishing between protein expression and activation state. The following approaches are validated for accurate assessment:

• Western Blotting with Phospho-specific Antibodies: Using antibodies that specifically recognize the phosphorylated form of MAPK11 (P-MAPK11) allows researchers to quantify the active fraction of the protein . The ratio of P-MAPK11 to total MAPK11 provides a measure of activation state, which can be quantified through densitometry analysis of Western blot bands .

• Substrate Phosphorylation Assays: Measuring the phosphorylation of known MAPK11 substrates, such as Atf2, provides a functional readout of MAPK11 activity . Atf2 phosphorylation has been validated as a reliable indicator of MAPK11 activity, with significantly reduced phosphorylation observed in the striata of MAPK11 knockout mice .

• Kinase Activity Assays: In vitro kinase assays using recombinant MAPK11 protein and known substrates can provide direct measurement of enzymatic activity. Quantification of substrate phosphorylation through radioactive labeling or phospho-specific antibodies allows for precise activity determination.

• Active Site Sequence Representation Models: Computational models based on active site sequences have been developed to predict MAPK11 activity. These models achieve AUC scores of approximately 0.518 and enrichment scores of 1.16, indicating moderate predictive power for identifying active site residues compared to random classification .

What controls are essential when studying MAPK11 in disease contexts?

When studying MAPK11 in disease contexts, several critical controls must be incorporated:

• Matched Normal-Tumor Tissue Pairs: For cancer studies, matched pairs of tumor and adjacent normal tissues provide the most reliable control. In ccRCC research, 32 pairs of ccRCC tissues and neighboring normal tissues were analyzed to investigate the clinical role of MAPK11 .

• Cell Line Controls: Non-cancer cell lines such as HK2 (normal kidney epithelial cells) should be included as controls when studying cancer cell lines like 786-O, ACHN, 769-P, OSRC-2, and CAKI-1 .

• Genetic Controls: For knockout or knockdown studies, proper genetic controls include wild-type (+/+), heterozygous (+/-), and homozygous knockout (-/-) animals or cells to establish dose-dependent effects . For instance, heterozygous knockout of MAPK11 was sufficient to significantly lower mutant Huntingtin levels in HD mice .

• Expression Controls: When studying protein-protein interactions (such as MAPK11 and RUNX2), bidirectional manipulation experiments should be performed. This includes assessing the effect of MAPK11 knockdown on RUNX2 expression and vice versa to establish the directionality of regulation .

• Activation Controls: When studying the effects of disease-associated proteins (like mHTT) on MAPK11 activation, both mutant and wild-type versions of the protein should be tested. For example, overexpression of mHTT (HTTexon1 (Q72)) but not wtHTT (HTTexon1 (Q25)) increased MAPK11 phosphorylation .

How do expression patterns of MAPK11 correlate with clinical parameters in disease?

The expression patterns of MAPK11, particularly its phosphorylated form (P-MAPK11), show significant correlations with clinical parameters in certain diseases:

In Clear Cell Renal Cell Carcinoma (ccRCC):

In Huntington's Disease:
MAPK11 activity, as measured by Atf2 phosphorylation, is significantly increased in the striata of HD mice (Hdh Q140/Q7) compared to wild-type mice (Hdh Q7/Q7) . This increased activation correlates with the presence of the mutant Huntingtin protein, suggesting a direct relationship between disease state and MAPK11 activity .