- Forkhead associated domain (FHA): 8-98 aa.
This domain is characterized as a modular phosphopeptide recognition domain with specificity to phosphothreonine containing sequences (Hammet et al., 2003; Durocher et al., 1999). The MKI67 FHA domain is shown to lie at the N-terminus of the protein. According to computer alignment, the core domain is found within 27-76 amino acids but the functional domain is believed to expand from 8 to 98 residue. So far, two proteins have been identified to interact with MKI67 FHA domain, the hNIFK (human nucleolar protein interacting with the FHA domain of pKi-67) and the Hklp2 (human kinesin-like protein 2).
- Protein phosphatase 1 binding site: 502-563 aa.
The Protein Phosphatase 1 (PPI) is a Ser/Thr phosphatase which belongs to the PPP family of phosphatases, and it is believed to dephosphorylate its substrates in large complexes consisting of regulatory as well as target proteins (Moorhead et al., 2008). In MKI67 protein there is a docking site of PPI at the N-terminal part of the protein within residues 502 to 563.
- Ki67 Repeats:
1) 1002-1113 aa, 2) 1124-1235 aa, 3) 1246-1357 aa, 4) 1368-1478 aa, 5) 1489-1599 aa, 6) 1610-1721 aa, 7) 1732-1843 aa, 8) 1854-1965 aa, 9) 1976-2087 aa, 10) 2098-2204 aa, 11) 2216-2327 aa, 12) 2337-2448 aa, 13) 2459-2570 aa, 14) 2582-2690 aa, 15) 2701-2806 aa, 16) 2820-2929 aa.
The Ki67 repeat is a cluster of 122 residues repeated 16 times within the 13th exon of the molecule with identity ranging from 43 to 62% to the consensus sequence. Within the Ki67 repeat there is a highly conserved motif of 22 amino acids named "the Ki67 motif" which has 72-100% identity to the consensus sequence and includes the F-K-E-L epitope recognized by the prototype Ki67 antibody (Schlüter et al., 1993). So far, their function remains an enigma.
- ATP/ GTP binding site motif A-P loop: 3034-3041 aa.
At the C-terminus of the protein there is a sequence resembling the predictive nucleotide binding site of Walker A motif. This well-known motif is a nucleotide binding fold recognized by Walker et al. (1982) and it is found in many nucleotide binding proteins.
- PEST sequences:
The cDNA sequences show 40 weak and 10 strong PEST sites. These sites are named as PEST due to the fact that they are rich in proline, glutamic acid (E), serine and threonine and to a lesser extent aspartic acid. They are found in several proteins with diverse functions such as key metabolic enzymes, transcription factors, protein kinases, phosphatases and cyclins. The regulation of proteins based on PEST sequences is characterized by strict expression, high susceptibility to proteolysis and short half-life, especially in those proteins participating in cell cycle and mitosis regulation. In MKI67 protein, the 10 strongest PEST sequences are located within exon 13, surrounding the conserved cysteine residues at position 8 of the Ki67 repeats (Ross and Hall, 1995). PEST sequences seem to be functional in the MKI67 protein, based on its biological behavior with susceptibility to proteases, short half-life and rapid loss of the protein after mitosis (Ross and Hall, 1995; Schlüter et al., 1993).
- Putative Monopartite Nuclear Targeting Sequences: 502-505 and 687-690 aa.
The MKI67 protein has two putative monopartite nuclear targeting sequences at the N-terminal part of the protein, which could function as nuclear localization signals (NLS) through the classical importin a/b nuclear import mechanism.
- Putative Bipartite Nuclear Targeting Sequences: 1) 536-550 aa, 2) 1516-1530 aa, 3) 2244-2258 aa, 4) 2365-2379 aa, 5) 2651-2665 aa, 6) 2890-2904 aa, 7) 2997-3011 aa, 8) 3141-3155 aa.
There are other classical nuclear targeting sequences within this molecule, which could mediate nuclear localization with importin a/b pathway as well. These bipartite sequences are characterized by two clusters of basic residues separated by a 10 to 12 aa linker that is tolerant to residue substitution (Kosugi et al., 2009).
- Post-translational modifications:
The predicted post-translational modifications of MKI67 protein comprise 19 N-myristoylation, 3 amidation and over 200 phosphorylation sites (143 PKC, 89 casein kinase II, 2 tyrosine kinase sites and 8 consensus sites for Cdc2 kinase) (Schlüter et al., 1993).
- MKI67 protein interactions:
The MKI67 protein has been found to interact directly with four proteins. The HP1 proteins, which are small non-histone chromosomal proteins found in several chromatin complexes, interact with its C-terminal region (Kametaka et al., 2002). The Hklp2, a kinesin-like motor, interacts with the N-terminal FHA domain of MKI67 protein (Sueishi et al., 2000). Another protein shown to interact with the N-terminal FHA domain of MKI67 protein is a putative RNA-binding protein with length of 293 residues, named hNIFK (Takagi et al., 2001). MKI67 protein was found to be a candidate protein for interacting with the HiNF-P transcription factor in a yeast two-hybrid screen (Miele et al., 2007).
Finally, several studies have shown that MKI67 protein interacts with DNA and more specifically with the heterochromatin (Kreitz et al., 2000; Bridger et al., 1998).
a. Early G1 phase: Accumulates in several foci within the nucleoplasm (Gerdes et al., 1983; Kill, 1996).
b. Late G1, S, G2 phase: Localizes predominantly within nucleoli at the dense fibrillar component but also there is diffuse nucleoplasmic staining (Gerdes et al., 1983; Kill, 1996).
2. Mitosis: Coats chromosome surface (Gerdes et al., 1983).
a. Metaphase: a reticulate but uniform network of fibrils around chromosomes, at the perichromosomal layer (Ross and Hall, 1995).
b. Anaphase: a reticulate more granular network of fibrils around chromosomes with the highest density of staining (Ross and Hall, 1995).
c. Telophase: the staining moves from the perichromosomal layer and becomes speckled at the nucleoplasm and subsequently concentrates at the newly reformed nucleoli (Ross and Hall, 1995).
1. Nucleolar localization of MKI67 protein is believed to be related to:
a. Structural modulation of the nucleolus in order to enhance the high rates of ribosomal synthesis during cell proliferation, since its expression is related to high rates of protein synthesis (Plaat et al., 1999; Scholzen et al., 2002).
b. Sequestration of MKI67 protein within nucleoli until the mitosis starts again. During mitosis the nucleolar structures are dispersed and the MKI67 protein is free to interact with nuclear components (Scholzen et al., 2002).
2. Localization of MKI67 to the surfaces of chromosomes during mitosis (prometaphase to anaphase) is believed to be related to:
a. Protection of chromosomal surface during mitosis (Verheijen et al., 1989; Yasuda and Maul, 1990).
3. DNA interaction:
a. It was believed that the central part of the MKI67 protein and especially the "Ki67 repeat" region has the ability to bind DNA through its Thr-Pro-X-X motif (Schlüter et al., 1993).
b. The LR domain has the ability to bind DNA in order to compact chromatin (Kametaka et al., 2002).
1. In Phyllodes tymor the expression of MKI67 and TP53 mRNA is associated with the grade of this tumor and could help in distinguishing the benign from the malignant form (Kucuk et al., 2013).
2. The expression profile of the estrogen receptor, ERBB2 (c-erbB2) and MKI67 mRNA could help in distinguishing between Toker cells, which are normal components of nipple epidermis, and cells of Pagets disease (Park and Suh, 2009).
Konstantinos Ntzeros ; Philip Stanier ; Dimitrios Mazis ; Neoklis Kritikos ; Meletios Rozis ; Eustathios Anesidis ; Chrisoula Antoniou ; Michael Stamatakos
MKI67 (marker of proliferation Ki-67)
Atlas Genet Cytogenet Oncol Haematol. 2014-05-01
Online version: http://atlasgeneticsoncology.org/gene/41369/mki67-(marker-of-proliferation-ki-67)