|Mutation Type||critical splice donor site|
|Coordinate||111,319,716 bp (GRCm38)|
|Base Change||C ⇒ G (forward strand)|
|Gene Name||mixed lineage kinase domain-like|
|Chromosomal Location||111,311,797-111,338,177 bp (-)|
FUNCTION: This gene belongs to the protein kinase superfamily. The encoded protein contains a protein kinase-like domain; however, is thought to lack protein kinase activity. This protein plays a critical role in tumor necrosis factor (TNF)-induced necroptosis, a programmed cell death process, via interaction with receptor-interacting protein 3 (Rip3), which is a key signaling molecule in necroptosis pathway. Knockout of this gene in mice showed that it is essential for necroptosis. Alternatively spliced transcript variants have been found for this gene. [provided by RefSeq, Sep 2015]
PHENOTYPE: Mice homozygous for a knock-out allele exhibit imapired macrophage and mouse embryonic fibroblast necroptosis. [provided by MGI curators]
|Amino Acid Change|
|Institutional Source||Beutler Lab|
|Gene Model||predicted gene model for protein(s): [ENSMUSP00000055521 †] [ENSMUSP00000113718 †] † probably from a misspliced transcript|
Structure of MLKL [X-RAY DIFFRACTION]
Crystal structure of the mouse MLKL kinase-like domain [X-RAY DIFFRACTION]
Crystal structure of the mouse RIP3-MLKL complex [X-RAY DIFFRACTION]
|Predicted Effect||probably null|
|Predicted Effect||probably null|
|Meta Mutation Damage Score||0.9487|
|Is this an essential gene?||Probably nonessential (E-score: 0.077)|
|Candidate Explorer Status||CE: excellent candidate; Verification probability: 0.905; ML prob: 0.86; human score: 4|
Linkage Analysis Data
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Recessive|
|Last Updated||2019-09-04 9:42 PM by Diantha La Vine|
|Record Created||2016-08-15 9:58 AM by Ying Wang|
The mecro phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R4760, some of which exhibited resistance to lipopolysaccharide (LPS)-induced necroptosis (Figure 1).
|Nature of Mutation|
Whole exome HiSeq sequencing of the G1 grandsire identified 89 mutations. The necroptosis phenotype was linked by continuous variable mapping to a mutation in Mlkl: a G to C transversion at base pair 111,319,716 (v38) on chromosome 8, or base pair 18,741 in the GenBank genomic region NC_000074. Linkage was found with a recessive model of inheritance, wherein two variant homozygotes departed phenotypically from four homozygous reference mice and six heterozygous mice with a P value of 3.982 x 10-11 (Figure 2). A substantial semidominant effect was observed, but the mutation is preponderantly recessive.
The mecro mutation is within the splice donor site of intron 6, one base pair away from exon 6 (out of 11 total exons). The effect of the mecro mutation on the mRNA sequence and MLKLmecro protein expression is unknown. In the event of exon 6 skipping, the aberrant transcript would have a deletion of the 136 base pair exon 6 (corresponding to amino acids 263-308), a frame-shift, and coding of 29 aberrant amino acids followed by a premature stop codon (within exon 8) after amino acid 291.
Genomic numbering corresponds to NC_000074. The mutated nucleotide is indicated in red, the splice donor site is in green, and the splice acceptor site is in blue.
|Illustration of Mutations in
Gene & Protein
Mlkl encodes mixed lineage kinase domain-like (MLKL), a pseudokinase and member of the protein kinase superfamily. MLKL has a pseudokinase domain (amino acids 171-464), but does not exhibit kinase activity (1) (Figure 3). Similar to kinases, MLKL has a (Val-Ala-Ile-Lys) motif, which positions the α- and β-phosphates of ATP during phosphoryl transfer, but does not have a catalytic loop or an activation loop. The pseudokinase domain has a kinase fold with N- and C-lobes (1-3). The N-lobe has an antiparallel, five-stranded β-sheet and an α-helix (helix αC), whereas the C-lobe contains seven α-helices and a pair of β-strands (4). MLKL has an N-terminal four-helical bundle (amino acids 1-130) followed by a two-helix linker (termed brace; amino acids 131-170) that tethers the N-terminus to a pseudokinase domain (1).
The mecro mutation is predicted to result in skipping of exon 6, resulting in deletion of amino acids 263-308 encoded by exon 6, a frame-shift, and coding of 29 aberrant amino acids and a premature stop codon after amino acid 291. The mecro mutation would affect the pseudokinase domain.
For more information about Mlkl, please see the record for necro.
Necroptosis is a pro-inflammatory form of cell death regulated by the kinases RIP1 and RIP3. Necroptosis occurs after stimulation of the DNA receptor, DNA-dependent activator of interferon regulatory factors (DAI), or activation of death receptors [e.g., TNF receptor 1 (TNFR1; see the record PanR1 for information about TNF) and Fas (see the record for cherry)], Toll-like receptors [TLRs; e.g., TLR3 and TLR4 (see the record for lps3)], T-cell antigen receptor (TCR), or interferon receptor [IFNAR1 (see the record for macro-1) and IFNAR2 (see the record for macro-2)] signaling. During necroptosis, RIP3 binds RIP1 through their respective RIP homotypic interaction motif domains, forming the necroptosome. RIP1 and RIP3 phosphorylation in the necroptosome leads to the recruitment of MLKL (5;6). MLKL is essential for necroptosis (2;4-6). Inhibition of MLKL function using necrosulfonamide prevents necrosome formation and subsequent necropototic signaling (5;6). Mouse dermal fibroblasts (MDFs), mouse embryonic fibroblasts (MEFs), and bone-marrow-derived macrophages (BMDMs) derived from the Mlkl-/- mice were resistant to TNF-induced necroptotic cell death (1).
The mecro mice exhibit resistance to LPS-induced necroptosis; necroptosis downstream of TNF has not been examined in the mecro mice. The phenotype of the mecro mice indicates loss of MLKLmecro function.
1) 94°C 2:00
The following sequence of 401 nucleotides is amplified (chromosome 8, - strand):
1 tggggctgga tctttacaat accttgcttc ttttccacta gtgaagcccc ctgagttctc
Primer binding sites are underlined and the sequencing primers are highlighted; the mutated nucleotide is shown in red.
1. Murphy, J. M., Czabotar, P. E., Hildebrand, J. M., Lucet, I. S., Zhang, J. G., Alvarez-Diaz, S., Lewis, R., Lalaoui, N., Metcalf, D., Webb, A. I., Young, S. N., Varghese, L. N., Tannahill, G. M., Hatchell, E. C., Majewski, I. J., Okamoto, T., Dobson, R. C., Hilton, D. J., Babon, J. J., Nicola, N. A., Strasser, A., Silke, J., and Alexander, W. S. (2013) The Pseudokinase MLKL Mediates Necroptosis Via a Molecular Switch Mechanism. Immunity. 39, 443-453.
2. Chen, W., Zhou, Z., Li, L., Zhong, C. Q., Zheng, X., Wu, X., Zhang, Y., Ma, H., Huang, D., Li, W., Xia, Z., and Han, J. (2013) Diverse Sequence Determinants Control Human and Mouse Receptor Interacting Protein 3 (RIP3) and Mixed Lineage Kinase Domain-Like (MLKL) Interaction in Necroptotic Signaling. J Biol Chem. 288, 16247-16261.
3. Murphy, J. M., Lucet, I. S., Hildebrand, J. M., Tanzer, M. C., Young, S. N., Sharma, P., Lessene, G., Alexander, W. S., Babon, J. J., Silke, J., and Czabotar, P. E. (2014) Insights into the Evolution of Divergent Nucleotide-Binding Mechanisms among Pseudokinases Revealed by Crystal Structures of Human and Mouse MLKL. Biochem J. 457, 369-377.
4. Xie, T., Peng, W., Yan, C., Wu, J., Gong, X., and Shi, Y. (2013) Structural Insights into RIP3-Mediated Necroptotic Signaling. Cell Rep. 5, 70-78.
5. Sun, L., Wang, H., Wang, Z., He, S., Chen, S., Liao, D., Wang, L., Yan, J., Liu, W., Lei, X., and Wang, X. (2012) Mixed Lineage Kinase Domain-Like Protein Mediates Necrosis Signaling Downstream of RIP3 Kinase. Cell. 148, 213-227.
|Science Writers||Anne Murray|
|Authors||Ying Wang and Bruce Beutler|