|Coordinate||75,649,550 bp (GRCm38)|
|Base Change||A ⇒ T (forward strand)|
|Gene Name||kit oncogene|
|Synonym(s)||SCO5, Dominant white spotting, Tr-kit, belly-spot, CD117, Gsfsow3, Gsfsco5, SOW3, SCO1, Steel Factor Receptor, c-KIT, Gsfsco1|
|Chromosomal Location||75,574,916-75,656,722 bp (+)|
FUNCTION: The c-Kit proto-oncogene is the cellular homolog of the transforming gene of a feline retrovirus (v-Kit). The c-kit protein includes characteristics of a protein kinase transmembrane receptor. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
PHENOTYPE: Mutations at this locus affect migration of embryonic stem cell populations, resulting in mild to severe impairments in hematopoiesis, and pigmentation. Some alleles are homozygous lethal, sterile, or result in the formation of gastrointestinal tumors. [provided by MGI curators]
|Amino Acid Change||Isoleucine changed to Phenylalanine|
|Institutional Source||Beutler Lab|
|Gene Model||not available|
|Predicted Effect||probably damaging
PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
|Phenotypic Category||Autosomal Dominant|
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Dominant|
|Last Updated||2018-04-16 3:52 PM by Diantha La Vine|
Pretty2 mice were identified by their distinct coat color pattern. Heterozygotes have a diluted coat color, light ears and tail, and a white belly spot; in some cases white dorsal spots are observed. On the C57BL/6:C3H/HeN hybrid background, animals often have a white belly and a white spindle-shaped spot on the forehead. Heterozygote intercrosses produce black-eyed offspring with an entirely white coat. Heterozygote Pretty2 mice strongly resemble WV/+ heterozygotes that bear a mutation at the c-kit locus (1).
A very similar mutation, known as Pretty, was the first visible phenodeviant to be identified in the course of our mutagenesis, and was lost because of marked infertility of the F1 founder male.
|Nature of Mutation|
The Pretty2 mutation corresponds to an A to T transversion at position 2387 of the Kit transcript on Chromosome 5. The mutation exists in exon 17 of 21 total exons.
The mutated nucleotide is indicated in red lettering, and results in the amino acid substitution I787F.
The kinase domains of class III RTKs are divided into two regions separated by an inserted sequence that varies in length between members and is thought to contribute to substrate specificity. As in the FMS and PDGF receptors, the KIT tyrosine kinase domain contains an insert of 77 hydrophilic amino acids bisecting the kinase domain (4).
Stem cell factor (SCF), the ligand for KIT, found as both membrane-anchored and soluble forms, functions as a noncovalent homodimer to activate KIT (5). Binding of SCF to KIT leads to receptor dimerization and activation of KIT kinase activity. The crystal structure of the ectodomain of KIT, with and without bound stem cell factor (SCF), reveals that SCF interacts with the first, second and third Ig domains of KIT (6) (Figure 2; PDB ID 2E9W), in agreement with earlier data based on the structure of SCF alone (5). The main region for SCF binding resides in the second KIT Ig domain, and binding is mediated by complementary electrostatic interactions between SCF and KIT (6). Upon ligand binding, the fourth and fifth Ig domains of two neighboring KIT ectodomains are brought closer together, stabilizing the interaction between two receptor molecules (6). Based on these data, it was proposed that KIT receptor dimerization is driven by binding of the SCF homodimer, whose exclusive function is to bring two KIT molecules together (6). Dimerization induces reorientation of the fourth and fifth Ig domains, enabling their lateral interaction and stabilization of the dimer.
The KIT tyrosine kinase domain has the characteristic bi-lobed architecture of all protein kinases (Figure 3; PDB ID 1PKG). Residues 582-671 comprise the small N-terminal lobe (N-lobe), and residues 678-953 comprise the large C-terminal lobe (C-lobe) [(9;10) and reviewed in (11)]. The cleft created between the two lobes contains the catalytic site. The Pretty2 mutation results in the substitution of isoleucine 787 by phenylalanine. I787 resides in the C-lobe of the kinase, and immediately precedes the catalytic loop of the kinase (consisting of the sequence HRDLAARN), which surrounds the actual site of phosphate transfer.
KIT is expressed in most stem cell types. Its expression is lost during cell differentiation with the exceptions of mature mast cells, melanocytes, and the intestinal interstitial cells of Cajal (12). KIT is expressed in hematopoietic stem cells, dendritic, erythroid, megakaryotic, and myeloid progenitor cells, and pro-B and pro-T cells (12). It is localized at the cell membrane.
The W (dominant white spotting) locus in mice, reported in the early 1900s, was identified when normally pigmented mice developed a white spot on their bellies (13;14). In addition to this coat color phenotype, W mice were infertile due to defects in germ cell development, developed macrocytic anemia and exhibited drastically reduced numbers of mast cells due to defects in hematopoiesis, leading to perinatal death (13;15). W mice also exhibited deafness (16;17). These various phenotypes could be attributed to the failure of stem cell populations to migrate and/or proliferate effectively during development [see (18) for a discussion of findings described here]. Defects in W mutants are intrinsic to the stem cells of the affected tissues, which fail to proliferate and survive during embryogenesis. Thus, melanoblasts and primordial germ cells in W mutants fail to proliferate as they migrate from their origins in the neural crest and yolk sac, respectively. The hematopoietic defects (anemia, mast cell deficiency) of W mice are similarly stem cell-intrinsic. The blood-forming tissues of anemic W mice contain reduced numbers of stem cells, as measured by the number of colony-forming units generated from splenic tissue. Mast cells, derived from hematopoietic stem cells, are decreased to less than 1% of their normal level in W skin, and none are detected in other W tissues (15). Transplant of wild type bone marrow into W mutants completely rescues erythroid and non-erythroid cell populations, indicating that the mutant hematopoietic environment is capable of supporting normal hematopoiesis. The loss of hearing due to a lack of endocochlear potential in W mice is correlated with the lack of pigmentation in the stria vascularis in the inner ear (17). In addition, cochlear and outer hair cell morphology is abnormal in W mice (16). This is likely due to abnormal neural crest cell migration and/or proliferation. Injection of wild type neural crest cells into 9.5-day-old W mutant embryos partially rescues stria pigmentation (19).
v-kit was first identified in 1986 as the viral oncogene of the Hardy-Zuckerman 4 feline sarcoma virus, captured by the retrovirus in a truncated and activated form (20). The corresponding cellular gene, designated c-kit, was found to encode a receptor tyrosine kinase (3), and two years later in 1988, mutations in KIT were identified as the causative lesion in W alleles (18;21). More than 90 white spotting mice have been reported since the first W mutants were described. The complete absence of KIT kinase activity leads to death in utero or perinatally (13), but mice with loss-of-function mutations in KIT [for example in the Kit viable dominant spotting allele, Wv (1)] are viable, having variable defects in hematopoiesis, fertility and pigmentation (22). The severity of the mutant phenotype generally correlates with the level of KIT tyrosine kinase activity.
Stem cell factor (SCF) or Kit ligand (KL), encoded by the Steel locus (Sl), is the ligand for KIT (23-25), and mice with loss-of-function mutations in the Sl locus have a phenotype that is virtually identical to that of W mice (26). However, defects in Sl mutants are not cell intrinsic, and Sl melanoblasts or bone marrow cells transplanted into a wild type environment develop normally. SCF is widely expressed during embryogenesis; it is detected in brain, endothelium, gametes, heart, kidney, lung, melanocytes, skin, and the stromal cells of the bone marrow, liver, and thymus (12).
Src family kinases and the protein tyrosine phosphatases SHP-1 and SHP-2 are also reported to associate with KIT (12). The significance of the KIT/SHP-1 interaction was demonstrated in vivo using double mutant KitW-v/W-v Shp1me/me mice (29;30). The spontaneously occurring motheaten (me) mutant expresses a null allele of Shp1 (see the record for spin), and develops autoimmune disease and systemic inflammation resulting in alopecia, skin inflammation of the paws and interstitial pneumonitis (31;32). Homozygosity for both the Wv and me alleles rescues aspects of both mutant phenotypes, including the pneumonitis associated with me and the embryonic lethality and mast cell deficiency associated with Wv (29;30). However, anemia associated with Wv mutation is not affected by the me allele. The juxtamembrane tyrosines 567 and 569, which form the binding site for Src kinases and SHP-1 and SHP-2, have also been examined in knock-in mice with Y to F mutations (KitFF) (33). Most KitFF mice die by 3 months of age. They display extensive white spotting, reduced numbers of mast cells, and splenomegaly due to increased numbers of B cells, but fertility is completely normal in both males and females (33). Although the precise signaling defects in KitFF mice are unknown (Y719 phosphorylation is also reduced by the FF mutation), this type of knock-in experiment clearly reveals that distinct signaling pathways mediated by different KIT binding partners can regulate separate biological processes.
In humans, loss-of-function mutations in KIT result in piebaldism (34) (OMIM #172800), an autosomal dominant disease characterized by a white forelock and large, non-pigmented patches on the forehead, eyebrows, chin, chest, abdomen and extremities. Interestingly, no hematopoietic defects have been reported in humans with KIT mutations. Activating mutations in KIT are implicated in several cancers, including gastrointestinal stromal tumors (35), mastocytosis (36), and germ cell tumors (37).
The Pretty2 mutation (I787F) likely prevents proper activation of KIT kinase activity. As mentioned above (Protein Prediction), I787 resides in the C-lobe of the kinase, and immediately precedes the catalytic loop of the kinase (consisting of the sequence HRDLAARN), which surrounds the actual site of phosphate transfer. In addition, as seen in the crystal structure of activated KIT, I787 forms hydrogen bonds with R815 of the C-lobe, stabilizing the activation loop in an extended conformation required for kinase activation (10). Thus, mutation of I787 may both disrupt catalytic loop structure or orientation, as well as prevent the activation loop from adopting an open conformation induced by ligand binding.
|Primers||Primers cannot be located by automatic search.|
Pretty2 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. This protocol has not been tested.
Pretty2 (F): 5’- CCTCTTCCTTGTGTCCTTGGGAGAA -3’
Pretty2 (R): 5’- AAATGGATTGCTAGTTTCAGCCTATCGT -3’
1) 95°C 2:00
2) 95°C 0:30
3) 56°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 29X
6) 72°C 7:00
7) 4°C ∞
Primers for sequencing
Pretty2_seq(F): 5’- GCAATTATAGTCATTAGAGCCCCG -3’
Pretty2_seq(R): 5’- GGTACTAACATGTGACATTACAAGG -3’
The following sequence of 758 nucleotides (from Genbank genomic region NC_000071 for linear genomic sequence of Kit) is amplified:
74117 cctc ttccttgtgt ccttgggaga agacgtcaag ttgaagctgt
74161 gaaacttttt tttttttttt ttttttggag aaaacgttca aagagatgca tacaaaatga
74221 actttcattt tagaaatggg atttgactat ttataatgca ttttcctgtg aatggaagga
74281 agggagaaag acgtttatta aaattgggtt ggaaagcaat tatagtcatt agagccccga
74341 tcctgtgaaa cacaaaacgg gaatatcact tgcaccataa tttttatttt cggtgtgcta
74401 aatactttaa aacgaaagtt tctttttttt tttcatgtaa acaccattgt agtattaaaa
74461 tcatcttctc tcggagagct gaaatgaatg gctgttgctg tctttccttt tctcccccaa
74521 cagtgtattc acagagattt ggcagccagg aatatcctcc tcactcacgg gcggatcaca
74581 aagatttgcg atttcgggct agccagagac atcaggaatg attcgaatta cgtggtcaaa
74641 ggaaatgtga gtacctttct ccatctcatg agtctaccca gggtgctttg gtatccagtc
74701 ttgattctaa attgttttct atgatcatta caactcctac cttgtaatgt cacatgttag
74761 taccactaag gcttgttaat agaattttta gctataattg tataattggg ggtgtgcgaa
74821 ataaacaaaa atagccttaa tttttcacga taggctgaaa ctagcaatcc attt
Primer binding sites are underlined; sequencing primer binding sites are highlighted in gray; the mutated A is indicated in red.
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24. Martin, F. H., Suggs, S. V., Langley, K. E., Lu, H. S., Ting, J., Okino, K. H., Morris, C. F., McNiece, I. K., Jacobsen, F. W., Mendiaz, E. A., and . (1990) Primary structure and functional expression of rat and human stem cell factor DNAs, Cell 63, 203-211.
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33. Kimura, Y., Jones, N., Kluppel, M., Hirashima, M., Tachibana, K., Cohn, J. B., Wrana, J. L., Pawson, T., and Bernstein, A. (2004) Targeted mutations of the juxtamembrane tyrosines in the Kit receptor tyrosine kinase selectively affect multiple cell lineages, Proc. Natl. Acad. Sci. U. S. A 101, 6015-6020.
34. Giebel, L. B. and Spritz, R. A. (1991) Mutation of the KIT (mast/stem cell growth factor receptor) protooncogene in human piebaldism, Proc. Natl. Acad. Sci. U. S. A 88, 8696-8699.
35. Hirota, S., Isozaki, K., Moriyama, Y., Hashimoto, K., Nishida, T., Ishiguro, S., Kawano, K., Hanada, M., Kurata, A., Takeda, M., Muhammad, T. G., Matsuzawa, Y., Kanakura, Y., Shinomura, Y., and Kitamura, Y. (1998) Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors, Science 279, 577-580.
36. Longley, B. J., Tyrrell, L., Lu, S. Z., Ma, Y. S., Langley, K., Ding, T. G., Duffy, T., Jacobs, P., Tang, L. H., and Modlin, I. (1996) Somatic c-KIT activating mutation in urticaria pigmentosa and aggressive mastocytosis: establishment of clonality in a human mast cell neoplasm, Nat. Genet. 12, 312-314.
|Science Writers||Eva Marie Y. Moresco|
|Illustrators||Diantha La Vine|
|Authors||Xin Du, Bruce Beutler|