|Coordinate||111,315,567 bp (GRCm38)|
|Base Change||A ⇒ 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]|
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 benign|
|Predicted Effect||probably benign|
|Meta Mutation Damage Score||0.0898|
|Is this an essential gene?||Probably nonessential (E-score: 0.074)|
|Candidate Explorer Status||CE: excellent candidate; Verification probability: 0.587; ML prob: 0.558; human score: 3.5|
Linkage Analysis Data
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Recessive|
|Last Updated||2019-09-04 9:44 PM by Diantha La Vine|
|Record Created||2015-12-01 1:04 AM by Ying Wang|
The secro phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R3745, some of which exhibited resistance to lipopolysaccharide (LPS)-induced necroptosis (Figure 1).
|Nature of Mutation|
Whole exome HiSeq sequencing of the G1 grandsire identified 48 mutations. The necroptosis phenotype was linked by continuous variable mapping to a mutation in Mlkl: a T to C transition at base pair 111,315,567 (v38) on chromosome 8, or base pair 22,890 in the GenBank genomic region NC_000074. Linkage was found with a recessive model of inheritance, wherein five variant homozygotes departed phenotypically from nine homozygous reference mice and 13 heterozygous mice with a P value of 1.245 x 10-20 (Figure 2). A substantial semidominant effect was observed, but the mutation is preponderantly recessive.
The secro mutation is within intron eight, 25 base pairs away from exon 9 (out of 11 total exons).
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.
The effect of the secro mutation on the mRNA sequence and MLKLsecro protein expression is unknown. In the event of exon 9 skipping, the aberrant transcript would have a deletion of the 50 base pair exon 9, leading to a frame-shift occurring after amino acid 383 of the protein (isoform 1), and termination after the inclusion of two aberrant amino acids.
Genomic numbering corresponds to NC_000074. The mutated nucleotide is indicated in red, the splice donor site is in green, the splice acceptor site is in blue, the aberrant amino acid in exon 8 is denoted by italics.
|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).
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 secro mice exhibit resistance to LPS-induced necroptosis; necroptosis downstream of TNF has not been examined in the secro mice. The phenotype of the secro mice indicates loss of MLKLsecro function.
1) 94°C 2:00
The following sequence of 753 nucleotides is amplified (chromosome 8, - strand):
1 aatcattgca gggacacggg aaaagaaatg tgctgagtgc ttggtttctt ggttttattt
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, Hexin Shi, Zhao Zhang, Lei Sun, Doan Dao, and Bruce Beutler|