|Coordinate||4,339,552 bp (GRCm38)|
|Base Change||A ⇒ C (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 (-)|
|MGI 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.|
|Amino Acid Change||Leucine changed to Arginine|
|Institutional Source||Beutler Lab|
|Gene Model||predicted gene model for protein(s): [ENSMUSP00000025078]|
AA Change: L273R
|Predicted Effect||probably damaging
PolyPhen 2 Score 0.989 (Sensitivity: 0.72; Specificity: 0.96)
|Phenotypic Category||TLR signaling defect: TNF production by macrophages|
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Recessive|
|Last Updated||09/11/2017 5:23 PM by Diantha La Vine|
|Record Created||08/25/2013 9:52 AM by Ying Wang|
The juicy phenotype was identified among G3 mice of the pedigree R0304, some of which showed diminished LPS-induced TNFα secretion (i.e., defective TLR4 signaling) (Figure 1).
|Nature of Mutation|
Whole exome HiSeq sequencing of the G1 grandsire identified 94 mutations. The defective TLR4 signaling phenotype was linked by continuous variable mapping to a mutation in Map3k8: a T to G transversion at base pair 4,339,552 (v38) on chromosome 18, or base pair 13,930 in the GenBank genomic region NC_000084. Linkage was found with a recessive model of linkage (P = 4.919 x 10-5), wherein 5 variant homozygotes departed phenotypically from 7 homozygous reference mice and 6 heterozygous mice (Figure 2). The mutation corresponds to residue 942 in the NM_007746 mRNA sequence in exon 5 of 8 total exons.
The mutated nucleotide is indicated in red. The mutation results in a leucine (L) to arginine (R) substitution at amino acid residue 273 in the TPL2 protein, and is strongly predicted by Polyphen-2 to cause loss of function (probably damaging; score = 0.989).
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 (Figure 3). The Juicy mutation (L273R) is within the kinase domain of TPL2 (amino acids 133-388).
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 (1). 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 (2-4). Activated TPL2 phosphorylates MEK1/2 (MAP kinase 1 and 2), which then activates ERK1/2 (5-7). The juicy phenotype is similar to that of Map3k8Sluggish/Sluggish (8) and TPL2-deficient mice in that TNFα production by juicy macrophages is abnormal in response to TLR agonists. In TPL2-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 (6).
juicy(F):5'- TGTTCTGGTGACAACGATCATCCAATG -3'
juicy(R):5'- GAAGCCAAAACCGTGAGCTTTCC -3'
juicy_seq(F):5'- AATGTTGCCATTTTGTCCCC -3'
juicy_seq(R):5'- ACACTAGGTGGGTTCCTGAC -3'
Juicy genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide transversion.
Juicy(F): 5’- TGTTCTGGTGACAACGATCATCCAATG-3’
Juicy(R): 5’- GAAGCCAAAACCGTGAGCTTTCC-3’
Juicy_seq(F): 5’- AATGTTGCCATTTTGTCCCC-3’
Juicy_seq(R): 5’- ACACTAGGTGGGTTCCTGAC-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 ∞
The following sequence of 664 nucleotides is amplified (Chr.18: 4339397-4339843, GRCm38; NC_000084):
tgttctggtg acaacgatca tccaatgttg ccattttgtc cccttcattg ctgattgtca
cagcatttcc agaccatcac caatacaatc aatcaattac ctctgttccc cggaggtcct
tgggaagata gacatcttca gtcatcttaa cactcaggcc aaaatctacc aaaacagctt
ttgtagacat gaatacaatg ttgctagctg caacgagagg ggtttttaat gtgagttaat
gcatcctaac ccaacaggaa aatcatctag aacagcaagt tccaagcaga gcagaaggaa
acgcttctga taatgactca ggtggttcac cccagaagag agaagtaggg gagcagtcag
gtgaaaacac ctgaggcctg tcaggaaccc acctagtgtg ggcagctagg agctacagcc
agcaggaaag ctcacggttt tggcttc
Chr. + strand shown. Primer binding sites are underlined and the sequencing primer is highlighted; the mutated nucleotide is shown in red text (Chr. + strand, T>G; sense strand, A>C).
1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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||Ying Wang, Hexin Shi and Bruce Beutler|