|Coordinate||4,333,965 bp (GRCm38)|
|Base Change||G ⇒ A (forward strand)|
|Gene Name||mitogen-activated protein kinase kinase kinase 8|
|Synonym(s)||Tpl2, Tpl-2, c-COT, Cot, Cot/Tpl2|
|Chromosomal Location||4,331,327-4,353,015 bp (-)|
FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] This gene is an oncogene that encodes a member of the serine/threonine protein kinase family. The encoded protein localizes to the cytoplasm and can activate both the MAP kinase and JNK kinase pathways. This protein was shown to activate IkappaB kinases, and thus induce the nuclear production of NF-kappaB. This protein was also found to promote the production of TNF-alpha and IL-2 during T lymphocyte activation. This gene may also utilize a downstream in-frame translation start codon, and thus produce an isoform containing a shorter N-terminus. The shorter isoform has been shown to display weaker transforming activity. Alternate splicing results in multiple transcript variants that encode the same protein. [provided by RefSeq, Sep 2011]
PHENOTYPE: Mutant mice resist endotoxic shock. Their MHC II expression is enhanced. Macrophages' TNF-alpha response to viruses and to all TLR ligands is impaired. Macrophage and T-cell secretion of other cytokines in response to various TLR ligands or OVA is aberrant. Anti-OVA Ig classes are abnormally skewed. [provided by MGI curators]
|Amino Acid Change||Arginine changed to Cysteine|
|Institutional Source||Beutler Lab|
|Gene Model||predicted gene model for protein(s): [ENSMUSP00000025078] [ENSMUSP00000133469]|
AA Change: R376C
|Predicted Effect||probably damaging
PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
|Predicted Effect||probably benign|
|Meta Mutation Damage Score||0.9621|
|Is this an essential gene?||Non Essential (E-score: 0.000)|
|Candidate Explorer Status||CE: excellent candidate; human score: 1; ML prob: 0.742|
Linkage Analysis Data
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Recessive|
|Last Updated||2017-09-11 5:26 PM by Diantha La Vine|
|Record Created||2015-09-04 9:50 PM by Bruce Beutler|
The gnostic_gospel phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R3625, some of which showed reduced TNFα (see the record for PanR1) secretion in response to the Toll-like receptor ligands CpG oligodeoxynucleotide (TLR9; Figure 1), lipopolysaccharide (TLR4; Figure 2), Pam3CSK4 (TLR1/2; Figure 3), and poly(I:C) (TLR3; Figure 4).
|Nature of Mutation|
Whole exome HiSeq sequencing of the G1 grandsire identified 40 mutations. All of the above anomalies were linked by continuous variable mapping to a mutation in Map3k8: a C to T transition at base pair 4,333,965 (v38) on chromosome 18, or base pair 19,517 in the NC_000084 GenBank genomic region. The strongest association was found with a recessive model of linkage to the normalized level of TNFα secretion to LPS, wherein five variant homozygotes departed phenotypically from eight homozygous reference mice and 17 heterozygous mice with a P value of 1.067 x 10-5 (Figure 5). A substantial semidominant effect was observed in most of the assays but the mutation is preponderantly recessive, and in no assay was a purely dominant effect observed.
The mutation corresponds to residue 1,250 in the mRNA sequence NM_008713 within exon 7 of 8 total exons.
The mutated nucleotide is indicated in red. The mutation results in an arginine (R) to cysteine (C) substitution at position 376 (R376C) in the TPL2 protein, and is strongly predicted by PolyPhen-2 to be damaging (score = 1.000) (1).
Map3k8 encodes TPL2 (tumor progression locus 2)/COT (cancer Osaka thryoid)/MAP3K8, a serine/threonine kinase member of the mitogen-activated protein kinase kinase kinase (MAP3K) family of proteins. Full-length TPL2 contains three domains: the N-terminal domain (amino acids 1-132), the kinase domain (amino acids 133-388), and a C-terminal region (Figure 6). The N-terminal domain may have a role in regulating protein stability (2). The kinase domain of TPL2 shows sequence homology to the kinase domains of other MAP3 kinases (2-4). The C-terminus of TPL2 appears both to inhibit TPL2 kinase activity (5-7), and to target the protein for degradation (7;8). The gnostic_gospel mutation results in an arginine (R) to cysteine (C) substitution at position 376 (R376C) within the kinase domain of TPL2.
Please see the record Sluggish for information about Map3k8.
TPL2 activates the MEK/ERK pathway downstream of most TLRs. Upon TLR stimulation, both p105 and TPL2 are phosphorylated by the IKK complex, resulting in degradation of p105 and the release and activation of TPL2 (9). Phosphorylation of TPL2 by the IKK complex occurs at T290, and is necessary for both the dissociation of TPL2 from p105, as well as kinase activity (10-12). Activated TPL2 phosphorylates MEK1/2 (MAP kinase 1 and 2), which then activates ERK1/2 (13-15). The gnostic_gospel phenotype is similar to that of Sluggish (16), juicy and Mapk38-deficient mice in that TNFα production by gnostic_gospel macrophages is abnormal in response to TLR agonists. In Mapk38-deficient macrophages, the levels of TNF-α are normal, but the transport of TNF-α mRNA to the cytoplasm in response to LPS is defective, suggesting that TPL2 regulates TNF-α mRNA transport, but not stability (14).
NOTE: These primers have not been validated.
Genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide transition.
R36250037_Map3k8_PCR_F: 5’- CATCAGAGCTTTGCTGTAGACAG-3’
R36250037_Map3k8_PCR_R: 5’- AATAGGTCTCTCCCCTAGCC-3’
R36250037_Map3k8_SEQ_F: 5’- TGGCTCCACACCCACAGG-3’
R36250037_Map3k8_SEQ_R: 5’- TAGCCTTTCCTGCAGAAGATATACC-3’
1) 94°C 2:00
2) 94°C 0:30
3) 55°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 40X
6) 72°C 10:00
7) 4°C hold
The following sequence of 414 nucleotides is amplified (NCBI RefSeq: NC_000084, chromosome 18):
catcagagct ttgctgtaga cagcaggagg gaggccagtg gctccacacc cacaggcata
cacccacaca cccagcatgg tgtcctacca gcaatgttct caggaagttg tagttccttc
ctgctcagca gcctcttccg ttcaaagagg gcagagtcca gactctgaca tcgtggctgg
tcctctcttg ggggattcag ggcttcatgt ttcagtaggt ctgctgcttt ggggcggtgg
ttggggttcc tctccagggc agcttctatc agctccctca tgcctggact gcagtcacca
gcgatgtctt ccaggggagg tgcctgcttg tggatctgcc aacaaaccag ctctgtaagc
atggatggag tagaggtata tcttctgcag gaaaggctag gggagagacc tatt
Primer binding sites are underlined and the sequencing primer is highlighted; the mutated G is shown in red text (Chr. + strand, G>A).
1. Adzhubei, I. A., Schmidt, S., Peshkin, L., Ramensky, V. E., Gerasimova, A., Bork, P., Kondrashov, A. S., and Sunyaev, S. R. (2010) A Method and Server for Predicting Damaging Missense Mutations. Nat Methods. 7, 248-249.
2. Aoki, M., Hamada, F., Sugimoto, T., Sumida, S., Akiyama, T., and Toyoshima, K. (1993) The human cot proto-oncogene encodes two protein serine/threonine kinases with different transforming activities by alternative initiation of translation, J Biol. Chem. 268, 22723-22732.
3. Ohara, R., Miyoshi, J., Aoki, M., and Toyoshima, K. (1993) The murine cot proto-oncogene: genome structure and tissue-specific expression, Jpn. J Cancer Res. 84, 518-525.
4. Patriotis, C., Makris, A., Bear, S. E., and Tsichlis, P. N. (1993) Tumor progression locus 2 (Tpl-2) encodes a protein kinase involved in the progression of rodent T-cell lymphomas and in T-cell activation, Proc. Natl. Acad. Sci. U. S. A 90, 2251-2255.
5. Ceci, J. D., Patriotis, C. P., Tsatsanis, C., Makris, A. M., Kovatch, R., Swing, D. A., Jenkins, N. A., Tsichlis, P. N., and Copeland, N. G. (1997) Tpl-2 is an oncogenic kinase that is activated by carboxy-terminal truncation, Genes Dev. 11, 688-700.
6. Robinson, M. J., Beinke, S., Kouroumalis, A., Tsichlis, P. N., and Ley, S. C. (2007) Phosphorylation of TPL-2 on serine 400 is essential for lipopolysaccharide activation of extracellular signal-regulated kinase in macrophages, Mol. Cell Biol. 27, 7355-7364.
7. Gandara, M. L., Lopez, P., Hernando, R., Castano, J. G., and Alemany, S. (2003) The COOH-terminal domain of wild-type Cot regulates its stability and kinase specific activity, Mol. Cell Biol. 23, 7377-7390.
8. Wu, B. and Wilmouth, R. C. (2008) Proteomics analysis of immunoprecipitated proteins associated with the oncogenic kinase cot, Mol. Cells 25, 43-49.
9. Waterfield, M., Jin, W., Reiley, W., Zhang, M., and Sun, S. C. (2004) IkappaB Kinase is an Essential Component of the Tpl2 Signaling Pathway. Mol Cell Biol. 24, 6040-6048.
10. Luciano, B. S., Hsu, S., Channavajhala, P. L., Lin, L. L., and Cuozzo, J. W. (2004) Phosphorylation of Threonine 290 in the Activation Loop of Tpl2/Cot is Necessary but Not Sufficient for Kinase Activity. J Biol Chem. 279, 52117-52123.
11. Cho, J., and Tsichlis, P. N. (2005) Phosphorylation at Thr-290 Regulates Tpl2 Binding to NF-kappaB1/p105 and Tpl2 Activation and Degradation by Lipopolysaccharide. Proc Natl Acad Sci U S A. 102, 2350-2355.
12. Cho, J., Melnick, M., Solidakis, G. P., and Tsichlis, P. N. (2005) Tpl2 (Tumor Progression Locus 2) Phosphorylation at Thr290 is Induced by Lipopolysaccharide Via an Ikappa-B Kinase-Beta-Dependent Pathway and is Required for Tpl2 Activation by External Signals. J Biol Chem. 280, 20442-20448..
13. Waterfield, M. R., Zhang, M., Norman, L. P., and Sun, S. C. (2003) NF-kappaB1/p105 Regulates Lipopolysaccharide-Stimulated MAP Kinase Signaling by Governing the Stability and Function of the Tpl2 Kinase. Mol Cell. 11, 685-694.
14. Dumitru, C. D., Ceci, J. D., Tsatsanis, C., Kontoyiannis, D., Stamatakis, K., Lin, J. H., Patriotis, C., Jenkins, N. A., Copeland, N. G., Kollias, G., and Tsichlis, P. N. (2000) TNF-Alpha Induction by LPS is Regulated Posttranscriptionally Via a Tpl2/ERK-Dependent Pathway. Cell. 103, 1071-1083.
15. Beinke, S., Deka, J., Lang, V., Belich, M. P., Walker, P. A., Howell, S., Smerdon, S. J., Gamblin, S. J., and Ley, S. C. (2003) NF-kappaB1 p105 Negatively Regulates TPL-2 MEK Kinase Activity. Mol Cell Biol. 23, 4739-4752.
|Science Writers||Anne Murray|
|Authors||Lei Sun, Zhao Zhang, Hexin Shi, Jeff SoRelle, Takuma Misawa, and Bruce Beutler|