KNL1 (cancer susceptibility candidate 5)

2016-10-01   Masato Takimoto , Jean-Loup Huret 

Institute for Genetic Medicine, Hokkaido University, Sapporo, Hokkaido, Japan

Identity

HGNC
LOCATION
15q15.1
LOCUSID
ALIAS
AF15Q14,CASC5,CT29,D40,MCPH4,PPP1R55,Spc7,hKNL-1,hSpc105
FUSION GENES

Abstract

Review on KNL1, with data on DNA, on the protein encoded, and where the gene is implicated.

DNA/RNA

Note

Whole genomic size is about 70 kbp, but consists of 27 exons.

Transcription

KNL1 mRNA expression is dominant in normal human testis and slight expression are observed in other organs, such as placenta. Analysis on cancer cell lines, such as HeLa, gave single band with 8.5 kb. There is another alternative splicing site at the 5 side of this gene that generates a short exon with 78 bp in cDNA. There are potential other alternative splicing at cancer cell lines.
Analysis on testis mRNA shows two bands with size of approximately 6 and 8,5 kb which are probably derived from the two isoforms.

Proteins

Atlas Image
Figure 1. Domain structure of KNL1 protein. Brown arrows indicate protein interactions, and blue arrows and line with T-shape indicate phosphorylation and dephosphorylation, respectively. MT :microtubules. KT : kinetochore.

Description

Encodes 1833 amino acids and 2342 amino acids.
The KLN1 protein contains: conserved motifs, which are the following: a (S/G)ILK motif (aa 25-28), a RRVSF motif (aa 57-61), and, for BUB3 recognition, MELT repeats (aa 140-161, 308-329, 474-494, 562-582, 750-769, 859-882, 902-924, 940-962, 1025-1044, 1073-1096, 1114-1136, 1152-1174). The Bubs recognition KI motifs KI(D/N)XXXF(L/I)XXLK, are KIDTTSFLANLK (aa 202-213) for BUB1, and KIDFNDFIKRLK (aa 238-249) for BUB1B: (BUBR1); a nuclear localization signal (aa 1789-1803); a coiled coil region (aa 1942-2133) and the ZWINT (Zwint-1) binding region (aa 1834 or 19811 for a smaller region -2108); and RWD repeats (aa 2109- 2353) With the NSL1 (hMis14)-binding region (aa 2109-2316), according to Kiyomitsu et al., 2011, VEGA checking, and SwissProt.

Expression

KNL1 protein expressions with molecular weight of approximately 250 kDa and 300 kDa are observed in human testicular germ cells and cancer cell lines.

Localisation

In germ cell of testis, significant high expressions of KNL1 protein are observed in nucleus of spermatocytes and slightly in spermatogonia. It is noteworthy that round spermatids express significantly high KNL1 protein in their pre-acrosome. As KNL1 protein has no hydrophobic signal peptide in its amino terminus, it probably localizes outer surface of pre-acrosome membrane of spermatids inside of the cells..
In mitosis KNL1 protein is localized in kinetochore in a human cancer cell line.

Function

KNL1 is a large of kinetochore protein, constituting KMN (KNL1/ MIS12 complex/ NDC80 complex) network. KMN network is the central hub of outer kinetochore, not only connecting mitotic chromosomes and spindles but also coordinating microtubule-binding, chromosome congression and spindle assembly checkpoint (SAC) signaling. There are two microtubule binding activity in KMN network, one in NDC80 and the other in KNL1. KNL1 protein binds directly microtubule through its far N-terminal region, consisting of 68 amino acids, in vitro, and indirectly through the interactions with MIS12 and NDC80 complexes at its C-terminus. KNL1 depletion affects metaphase chromosome congression. In C. elegans,
The depletion preclude metaphase chromosomal congression, while in yeast, Drosophila and human cells, the depletions show partial alignment phenotypes, in which some chromosomes congress to the equator but many chromosomes remain stranded near the spindle poles. In the N-terminus of KNL1 protein, AURKB (Aurora kinase B) phosphorylates SILK and RVSF motifs, and then disrupts the interaction between KNL1 and protein phosphatase I (PP1), which also binds the motifs. KNL1 augments Aurora B kinase activity that phosphorylates outer kinetochore protein, such as NDC80, resulting in reduction its microtubule-binding activity. Protein phosphatase 2A ( PP2A) is recruited by BUB1B (BubR1), one of SAC protein. Both PP1 and PPA2 are suggested to counteract Aurora B kinase activity. Especially, PP1 was shown to stabilize microtubule attachments to kinetochores probably through KNL1, while Aurora B kinase destabilizes microtubule-binding by NDC80 phosphorylation as described. The destabilized binding of KNL1 to microtubules is presumably important for correcting and eliminating erroneous kinetochore-microtubule attachment during SAC.
It is suggested that the bindings with microtubule and with PP1 also play roles in SAC silencing. Although their binding sites are in close proximity, one of their bindings do not affect the other, and they contribute independently to the silencing of SAC. Two KI motifs, KI1 and KI2, localized in the N-terminus, bind SAC protein, BUB1 and BUB1B, respectively, through tetratricopeptide repeats (TPRs) of the proteins, resulting into folding the motifs into short alpha-helices. Although BUB1 fragment with mutation in KI-binding domain was not able to bind to KNL1 in vitro, BUB1 and BUB1B mutants with KI-binding sites were able to attach to kinetochore. Mutations in the BUB3-binding domain (BUB3-BD) in BUB1 and BUB1B prevent kinetochore localization of the SAC protein. BUB1 fragment consisting only of N-terminus with TPRs does not localize to kinetochore and longer fragments that accommodate BUB3-BD did. Those results suggest that BUB3-BD of BUB1 and BUB1B, rather than TPRs, is critical for their recruitment to kinetochore and that the interaction between TPRs of Bub proteins and KI-motif of KNL1 might play a subsidiary role in the localization of BUB1 and BUB1B to kinetochore. In response to SAC signal, the first step of this response is phosphorylation of MELT motifs of KNL1, located in the N-terminal and central region, by Mps1 kinase, and then the phosphorylated MELT motifs bind BUB3/BUB1complex, mediating SAC signaling. KNL1- BUB3-BUB1(KBB) complex binds MXD1 (MAD1)/ MAD2L1 (MAD2) complex, and then, together with MAD1 phosphorylation by Msp1, the binding leads to CDC20/MAD2 formation, an essential part of Mitotic Checkpoint Complex (MCC) that inhibits Anaphase Promoting Complex/Cyclosome (APC/C).
Recently, it suggested that there are two pathways for recruiting MAD1-MAD2 that results in SAC activation. One is the pathway through KBB, as described above, the other is KNTC1 (ROD)-RW10- ZWILCH, (RZZ) complex, which interacts with KNL1 through ZWINT (Zwint-1 protein). The former is required for SAC activation when kinetochores are misaligned but is not essential when kinetochores are unattached from microtubules. The latter binds SPDL1 (Spindly protein) and MAD1-MAD2, and causes the anaphase-onset delay in response to unattached kinetochore independently of the former.
It was suggested that the binding of KNL1 with microtubules and with PP1 contribute to silencing of SAC, in which motor protein dynein, moving along on microtubules, is suggested to work to strip MAD1-MAD2. The C-terminal region of KNL1 interacts with MIS12 and Zwint-1 protein, through RWD motif and coiled-coiled region, respectively. The binding with the former plays role in connecting inner kinetochore with KNL1 and the latter mediates the interaction of KNL1 with RZZ complex which works in SAC regulation as described above.

Implicated in

Top note
KNL1 expression was observed in many human cancer cell lines and primary cancers derived from different organs and tissues. In one study, KNL1 mRNA expression was detected in all the cancer cell lines examined and positive in 31 out of 103 primary human cancers (30%). As KNL1 is overexpressed in normal testis, KNL1 is a member of Cancer/Testis gene family. Among the primary cancers examined lung cancer showed the highest positive rate, more than 40%, and its clincopathological characteristics are significantly correlated with KNL1 mRNA expression as described below.
Entity name
Leukemia
Note
A small subset of leukemia with a t(11;15)(q23;q14) has been described for long and has often be referred as: t(11;15)(q23;q15) MLL/AF15q14. KMT2A, (previous symbol: mixed leukemia gene (MLL)) is translocated with KNL1 (previous symbol CASC5, originally described as AF15q14), which makes research of published cases often arduous.
  • t(11;15)(q23;q15) and/or KMT2A/KNL1
    t(11;15)(q23;q14-15)
    Data on 16 cases with a t(11;15)(q23;q14-15), according to (Yang et al., 2014) are the following: there was 2 of myelodysplastic syndrome (MDS) cases, 10 acute myeloid leukemia (AML) cases (2 M1, 4 M2, 3 M4, and 1 NOS), and 4 acute lymphoblastic leukemia (ALL) cases. Mean age of the patients was 20.6 years (range 1-54); there were 11 males and 5 females. Abnormalities of chromosome 3 were seen in 10 out of 16 cases. Out of 8 patients for whom clinical data were available, only 3 are in complete remission, whereas 5 patients died with a mean survival period of 10.4 months.
    t(11;15)(q23;q15) and KMT2A/KNL1
    Of 7 cases with a t(11;15)(q23;q15) and KMT2A/KNL1 hybrid gene and fusion protein ((Chinwalla et al., 2003; Kuefer et al., 2003; Meyer et al., 2006; Yang et al., 2014). Diagnosis was: therapy related MDS (t-MDS) in 2 cases, AML in 4 cases (1 M2, 2 AML-M4, 1 AML-NOS), and 1 de novo T-ALL. Sex ratio was 5M:1F; There was 3 children and 4 adult patients. Of three cases with data on survival, patients died at: 8 mths, 8 mths, and 22 mths. Of four cases with documented karyotypes: the karyotype was a complex karyotype with markers in two cases, abnormalities of chromosome 3 were seen in three cases, +21 in two cases. KMT2A (MLL) exon 8, 9, or 10 were fused to exon 10, 11 or 12 of KNL1, the fusion protein contains the 1362 or 1418 first aa from MLL with aa 1796, 1818 or 1819 from KNL1 (according to authors and/or VEGA).
  • A t(3;15;p14;q15) KNL1/ ADAMTS9-AS2 is mentioned, without further data in the ChiTARS database (Gorohovski et al., 2016) as a chimeric EST (dbEST Id:12413828; accession BQ375909).
  • Entity name
    Lung cancer
    Note
    In one study on primary lung cancer, KNL1 mRNA expression was observed in more than 40% of the cases, which is the highest among all the different types of cancers examined. The study also revealed that clinicopathological findings correlates with KNL1 expression. KNL1 mRNA expression is more frequent in the tumors with low differentiation than the ones with moderate and high differentiation. Further, the tumors derived from smoker express higher incidence of KNL1 mRNA than the ones from non-smoker.
    Entity name
    Spermatogenesis
    Note
    KNL1 mRNA was highly expressed in normal testis. As KNL1 protein expressions were observed in spermatogonia and spermatocytes in seminiferous tube of human testes, this protein may also play a role in cell division as a kinetochore protein in meiotic cells. It is noteworthy that KNL1 protein is also significantly expressed in pre-acrosome of spermatids, especially from its early stage, suggesting that KNL1 might be playing important role in the formation of acrosome, an essential organelle for fertilization. KNL1 expressions in testes of the patients with infertility were significantly lower than normal ones.
    Entity name
    Microcephaly
    Disease
    Autosomal recessive primary microcephaly (MCPH) is a very rare neuro-developmental disorder with brain size reduction, no structural malformation of the brain at birth, mild-to moderate mental retardation and absence of other neurological or somatic disease. There are 12 genetic loci responsible for MCPH, and it was suggested that one of the responsible genetic locus, MCPH4, resides on chromosome 15. Subsequently, the study on the patients with MCPH4 of consanguineous families in Morocco revealed that a homozygous missense mutation was observed in exon 18 of KNL1 gene. This point mutation caused skipping this exon in splicing reaction in the mRNA maturation of KNL1, suggesting that the affected nucleotide is a part of Exonic splicing enhancers. The mutation resulted in frame-shift and truncation of KNL1 protein. As RWD repeats at near its carboxy terminus were deleted by this mutation, the truncated protein is no longer able to bind to Mis12, leading to the defected recruitment of KNL1 protein to kinetochore. One of the patients in the Morroco families has cryptorchidism in addition to microcephaly. The results of studies on MCH4 is a direct demonstration that KNL1 is essential to cell division in vivo.

    Bibliography

    Pubmed IDLast YearTitleAuthors
    106948772000Exonic splicing enhancers: mechanism of action, diversity and role in human genetic diseases.Blencowe BJ et al
    244569772014Kinetochore signalling: the KIss that MELTs Knl1.Bollen M et al
    243106192014KNL1: bringing order to the kinetochore.Caldas GV et al
    126187662003A t(11;15) fuses MLL to two different genes, AF15q14 and a novel gene MPFYVE on chromosome 15.Chinwalla V et al
    257476562015The (phospho) needle in the (MELT) Haystack.Faesen AC et al
    229839542012Kinetochore KMN network gene CASC5 mutated in primary microcephaly.Genin A et al
    278995962017ChiTaRS-3.1-the enhanced chimeric transcripts and RNA-seq database matched with protein-protein interactions.Gorohovski A et al
    105213161999Primary autosomal recessive microcephaly: homozygosity mapping of MCPH4 to chromosome 15.Jamieson CR et al
    202322242010Finding the middle ground: how kinetochores power chromosome congression.Kops GJ et al
    126187682003Characterization of the MLL partner gene AF15q14 involved in t(11;15)(q23;q14).Kuefer MU et al
    165115152006The MLL recombinome of acute leukemias.Meyer C et al
    266512942015KNL1-Bubs and RZZ Provide Two Separable Pathways for Checkpoint Activation at Human Kinetochores.Silió V et al
    117928042001Kinetochore localisation and phosphorylation of the mitotic checkpoint components Bub1 and BubR1 are differentially regulated by spindle events in human cells.Taylor SS et al
    244256912014Molecular characterization and clinical impact of t(11;15)(q23;q14-15) MLL-CASC5 rearrangement.Yang JJ et al

    Other Information

    Locus ID:

    NCBI: 57082
    MIM: 609173
    HGNC: 24054
    Ensembl: ENSG00000137812

    Variants:

    dbSNP: 57082
    ClinVar: 57082
    TCGA: ENSG00000137812
    COSMIC: KNL1

    RNA/Proteins

    Gene IDTranscript IDUniprot
    ENSG00000137812ENST00000346991Q8NG31
    ENSG00000137812ENST00000399668Q8NG31
    ENSG00000137812ENST00000526913H0YCZ2
    ENSG00000137812ENST00000527044E9PPJ1
    ENSG00000137812ENST00000528975A0A1Y8EIW7
    ENSG00000137812ENST00000532056A0A0G2JL96
    ENSG00000137812ENST00000532406H0YEY7
    ENSG00000137812ENST00000534204H0YN41

    Expression (GTEx)

    0
    5
    10
    15
    20
    25

    Pathways

    PathwaySourceExternal ID
    Signal TransductionREACTOMER-HSA-162582
    Signaling by Rho GTPasesREACTOMER-HSA-194315
    RHO GTPase EffectorsREACTOMER-HSA-195258
    RHO GTPases Activate ForminsREACTOMER-HSA-5663220
    Cell CycleREACTOMER-HSA-1640170
    Cell Cycle, MitoticREACTOMER-HSA-69278
    M PhaseREACTOMER-HSA-68886
    Mitotic PrometaphaseREACTOMER-HSA-68877
    Resolution of Sister Chromatid CohesionREACTOMER-HSA-2500257
    Mitotic Metaphase and AnaphaseREACTOMER-HSA-2555396
    Mitotic AnaphaseREACTOMER-HSA-68882
    Separation of Sister ChromatidsREACTOMER-HSA-2467813
    Chromosome MaintenanceREACTOMER-HSA-73886
    Nucleosome assemblyREACTOMER-HSA-774815
    Deposition of new CENPA-containing nucleosomes at the centromereREACTOMER-HSA-606279

    Protein levels (Protein atlas)

    Not detected
    Low
    Medium
    High

    References

    Pubmed IDYearTitleCitations
    202313802010Regulated targeting of protein phosphatase 1 to the outer kinetochore by KNL1 opposes Aurora B kinase.153
    179811352007Human Blinkin/AF15q14 is required for chromosome alignment and the mitotic checkpoint through direct interaction with Bub1 and BubR1.132
    180459862008KNL1 and the CENP-H/I/K complex coordinately direct kinetochore assembly in vertebrates.81
    252466132014PP2A-B56 opposes Mps1 phosphorylation of Knl1 and thereby promotes spindle assembly checkpoint silencing.50
    243441832013Arrayed BUB recruitment modules in the kinetochore scaffold KNL1 promote accurate chromosome segregation.47
    280723882017A sequential multi-target Mps1 phosphorylation cascade promotes spindle checkpoint signaling.43
    211999192011Protein interaction domain mapping of human kinetochore protein Blinkin reveals a consensus motif for binding of spindle assembly checkpoint proteins Bub1 and BubR1.42
    243634482014A minimal number of MELT repeats supports all the functions of KNL1 in chromosome segregation.42
    256014042015Multiple assembly mechanisms anchor the KMN spindle checkpoint platform at human mitotic kinetochores.41
    256614892015Sequential multisite phospho-regulation of KNL1-BUB3 interfaces at mitotic kinetochores.39

    Citation

    Masato Takimoto ; Jean-Loup Huret

    KNL1 (cancer susceptibility candidate 5)

    Atlas Genet Cytogenet Oncol Haematol. 2016-10-01

    Online version: http://atlasgeneticsoncology.org/gene/318/knl1

    Historical Card

    2012-06-01 KNL1 (cancer susceptibility candidate 5) by  Masato Takimoto 

    Institute for Genetic Medicine, Hokkaido University, Sapporo, Hokkaido, Japan

    2006-09-01 KNL1 (cancer susceptibility candidate 5) by  Masato Takimoto 

    Institute for Genetic Medicine, Hokkaido University, Sapporo, Hokkaido, Japan

    2000-03-01 KNL1 (cancer susceptibility candidate 5) by  Jean-Loup Huret,Christiane Charrin 

    Genetics, Dept Medical Information, University of Poitiers, CHU Poitiers Hospital, F-86021 Poitiers, France (JLH); Service dHematologie, Hopital Edouard Herriot, Lyon, France (CC).