# Patent

## Title
Methods and compositions for modulating tumor suppression

## Abstract

The purification of native RB (retinoblastoma) as a complex, including P107, P130, and a 600 kDa subunit, termed MTAF600 (microtubule associated factor 600) is described. MTAF600 binds to RB regardless of the phosphorylation status of RB, and binds to RB without disrupting the interaction between RB and E2F. It is further shown that E2F and DP proteins co-purified with MTAF600 and RB, such that hypophosphorylated RB may gain access to E2F as a complex with MTAF600. In addition, MTAF600 binds to microtubules and plays a role in active repression of E2F-responsive genes, cell cycle arrest, and genomic stability. The sequence of MTAF600 is described herein, along with its binding properties to proteins such as RB and microtubules, and its sequence homology. Further, methods and reagents for assaying the presence of MTAF600 or mutants thereof, pharmaceutical formulations, and methods for treating disease are also described.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application gains priority from provisional application serial No. 60/278,245 and provisional application 60/278,244 both filed on Mar. 23, 2001 and incorporated herein by reference.


TECHNICAL FIELD AND BACKGROUND ART
The present invention relates to reversing inhibition of tumor suppression.
The failure of normal function of the retinoblastoma tumor suppressor gene (RB) has been implicated as a contributing factor in a number of tumor types, including retinoblastomas and osteosarcomas, as well as lung, breast, and bladder carcinomas. (For reviews, see Goodrich et al., Biochim. Biophys. Acta., Vol. 1155, pp. 43–61, 1993; Zacksenhaus et al., Adv. Cancer. Res., Vol. 61, pp. 115–141, 1993; Sellers et al., J. Clin. Oncol., Vol. 15, pp. 3301–3312, 1997; Lohmann, D. R., Hum. Mutat., Vol. 14, pp. 283–288, 1999). A major role of RB is repression of the E2F family of DNA-binding transcriptional activators, which regulate the cell cycle through various genes required for S-phase entry. In resting cells, RB exists in the hypophosphorylated form that binds directly to E2F. (Reviewed in Weinberg, R. A., Cell, Vol. 81, pp. 323–330, 1995; Dyson, N., Genes Dev., Vol. 12, pp. 2245–2262, 1998). Importantly, mutations in E2F-recognition sequences, at least in some promoters, lead to derepression in G0/G1 cells, rather than repression in S-phase. (Neuman et al., Mol. Cell. Biol., Vol. 14, pp. 6607–6615, 1994). Although RB binds to the promoters only through E2F, RB is capable of repressing not only E2F, but also various activators that bind to E2F-responsive promoters. It has been proposed that chromatin modifiers, including histone deacetylases, (Brehm et al, Nature, Vol. 391, pp. 597–601, 1998), ATP-dependent chromatin remodeling factors (Zhang et al., Cell, Vol. 101, pp. 79, 2000), and DNA methyltransferases (Fuks et al., Nat. Genet., Vol. 24, pp. 88–91, 2000; Robertson et al., Nat. Genet., Vol. 25, pp. 338–3342, 2000) are involved in the mechanisms of this active repression. (Harbour et al., Curr. Opin. Cell Biol., Vol. 12, pp. 685–689, 2000).
Once RB becomes hyperphosphorylated, it dissociates from E2F resulting in expression of E2F-responsive genes. This hyperphosphorylation event at the time of the G1/S transition of the cell cycle, (For reviews, see Weinberg, Cell, Vol. 81, pp. 323–330, 1995; Sherr, “Cancer cell cycles”, Science, Vol. 274, pp. 1672–1677, 1996; Dyson, Genes Dev., Vol. 12, pp. 2245–2262, 1998; Mittnacht, Vol. 8, pp. 21–27, 1998) is thought to occur through the enzymatic activity of cyclin-dependent kinases (CDK). Accordingly, RB regulates S-phase entry through binding to E2F in a cell cycle-dependent manner. This cell cycle-dependent regulation is disturbed by viral transforming factors, including adenovirus E1A, simian virus 40 large-T antigen, and human papillomavirus (HPV) E7. (For reviews, see Zalvide et al., Mol. Cell. Biol., Vol. 15, pp. 5800–5810, 1995; Flint et al., Annu. Rev. Genet., Vol. 31, pp. 177–212, 1997). These transforming factors bind to the evolutionally conserved C-terminal region of RB, referred to as the pocket domain, and inhibit access of RB to E2F, leading to loss of G1 control.
In mammals, two proteins, namely p107 and p130, are structurally and functionally related to RB (For reviews, see Dyson, N., Genes Dev., Vol. 12, pp. 2245–2262, 1998; Lipinski et al., Oncogene, Vol. 18, pp. 7873–7882, 1999). All family members, namely RB, p107, and p130, bind to E2F and actively inhibit E2F-responsive transcription, leading to G0/G1 arrest. Although the RB family members are similar in these properties, they are differentially expressed during mouse development (Reviewed in Jiang et al., Oncogene, Vol. 14, pp. 1789–1797, 1997; Lipinski et al., Oncogene, Vol. 18, pp. 7873–7882, 1999). While RB nullzygous mutant embryos die at midgestation with multiple defects (Clarke et al., Nature, Vol. 359, pp. 328–330, 1992; Jacks et al., Nature, Vol. 359, pp. 295–300, 1992; Lee et al., Nature, Vol. 359, pp. 288–294, 1992), p107 and p130 nullzygous mice do not have any obvious developmental or tumor phenotype (Cobrinik et al, Genes Dev., Vol. 10, pp. 1633–1644, 1996; Lee et al., Genes Dev., Vol. 10, pp. 1621–1632, 1996). This phenotypic difference may be due to unique roles of RB and/or distinct expression profiles of RB. On the other hand, mouse embryonic fibroblasts carrying inactivating disruptions in all three RB gene family members are viable and proliferate in culture (Dannenberg et al., Genes Dev., Vol 14, pp. 3051–3064, 2000; Sage et al., Genes Dev, Vol. 14, pp. 3037–3050, 2000). Importantly, triple knockout fibroblasts have a shorter cell cycle and are insensitive to G0/G1 arrest signals following contact inhibition or serum starvation. These results support the view that the RB family members play an essential role in growth arrest.
In mammals, the E2F family has six members, namely E2F-1 to -6 (For reviews, see Dyson, N., Genes Dev., Vol. 12, pp. 2245–2262, 1998; Black et al., Gene, Vol. 237, pp. 281–302, 1999). All family members recognize the same DNA sequence as a heterodimer with either DP-1 or DP-2. E2F-6 differs from other E2F family members in that it lacks the transactivation and RB-binding domains, suggesting that it acts antagonistically to other E2F family members by occupying the binding sites on promoters (For reviews, see Cartwright et al., Oncogene, Vol. 17, pp. 611–623, 1998; Gaubatz et al., Proc. Natl. Acad. Sci. U.S.A., Vol. 95, pp. 9190–9195, 1998; Trimarchi et al., Proc. Natl. Acad. Sci. U.S.A., Vol. 95, pp. 2850–2855, 1998). On the other hand, E2F-1 to -5 all have transactivation and RB-binding domains. While their function could be partly redundant, several lines of evidence indicate specific roles for each E2F member (Reviewed in Dyson, N., Genes Dev., Vol. 12, pp. 2245–2262, 1998; Black et al., Gene, Vol. 237, pp. 281–302, 1999). First, each E2F protein preferentially binds to different RB family members: RB binds to E2F-1 to -4; p107 interacts with E2F-4; and p130 interacts with E2F-4 and -5. In addition, E2F-1 to -5 are differently regulated according to cell type and developmental stage. Furthermore, E2F-1 to -3 appear to be exclusively nuclear, whereas a significant portion of E2F-4 and E2F-5 are present in cytoplasm (Allen et al., J. Cell. Sci., Vol. 110, pp. 2819–2831, 1997; Lindeman et al., Proc. Natl. Acad. Sci. U.S.A., Vol. 94, pp. 5095–5100, 1997; Verona et al., Mol. Cell. Biol., Vol. 17, pp. 7268–7282, 1997).
Although RB was identified over a decade ago as the first tumor suppressor (Friend et al., Nature, Vol. 323, pp. 643–646, 1986; Fung et al., Science, Vol. 236, pp. 1657–1661, 1987; Lee et al., Nature, Vol. 329, pp. 642–645, 1987), to our knowledge RB has never been purified to homogeneity. Here, we report its purification in a native form, and we demonstrate that RB is present in a complex. The 600 kDa subunit, referred to as microtubule-associated factor (MTAF) 600, interacts directly with RB and microtubules and plays a role in active repression of E2F-responsive genes, cell cycle arrest, and genomic stability. These findings indicate that RB functions as a complex in vivo.
Because of the importance of RB in tumor suppression and growth arrest, and the demonstrated occurrence of tumors in subjects in which the RB gene has been mutated, there is significant clinical interest in identifying how the process of tumor suppression can be manipulated. In the future, as prognostic tests for a variety of diseases improve, it will be desirable to modulate the expression of key proteins associated with disease.
SUMMARY OF THE INVENTION
In a first embodiment of the invention there is provided a polypeptide comprising an amino acid sequence at least 90% homologous to SEQ ID No 1. In accordance with another embodiment of the invention, a peptide is provided. The peptide comprises an amino acid sequence for MTAF600 such that the amino acid sequence has at least 90% homology with amino acid sequence 3,910–4851 of SEQ ID No 1. Similarly, in accordance with a further embodiment of the invention, a peptide may comprise an amino acid sequence corresponding to amino acid 4293–4534 of SEQ ID: No. 1. In accordance with yet another embodiment of the invention, a small molecule is provided. The molecule has a molecular weight of less than 1500 D and capable of binding at least one of RB (379–928), P107, P130, E2F1–5, or microtubules.
In accordance with another embodiment of the invention, a pharmaceutical composition is provided. The pharmaceutical composition includes an effective dose for treating a hyperproliferative condition of at least one of a polypeptide comprising an amino acid sequence at least 90% homologous to SEQ ID No 1, a peptide having an amino acid sequence for MTAF600 such that the amino acid sequence has at least 90% homology with amino acid sequence 3,910–4851 of SEQ ID No 1, a peptide including an amino acid sequence corresponding to amino acid 4293–4534 of SEQ ID: No. 1, or a small molecule having a molecular weight of less than 1500 D and capable of binding at least one of RB (379–928), P107, P130, E2F1–5 or microtubules.
In accordance with another embodiment of the invention, a method for treating a hyperproliferative disease comprises administering an effective dose of a pharmaceutical composition wherein the pharmaceutical composition includes an effective dose for treating a hyperproliferative condition of at least one of a polypeptide comprising an amino acid sequence at least 90% homologous to SEQ ID No 1, a peptide having an amino acid sequence for MTAF600 such that the amino acid sequence has at least 90% homology with amino acid sequence 3,910–4851 of SEQ ID No 1, a peptide including an amino acid sequence corresponding to amino acid 4293–4534 of SEQ ID: No. 1, or a small molecule having a molecular weight of less than 1500 D and capable of binding at least one of RB (379–928), P107, P130, E2F1–5 or microtubules.
In related embodiments of the invention, the hyperproliferative condition may be a cancer. In further related embodiments, the cancer may be retinoblastoma, osteosarcoma, lung cancer, breast cancer or bladder cancer
In accordance with another embodiment of the invention, a vector comprises a nucleic acid sequence encoding SEQ ID No: 1 or a biologically active substituent thereof operably linked to a regulatory sequence for providing transcriptional activity in a host cell. In accordance with related embodiments, the nucleic acid sequence comprises at least 50% of the nucleotides 11,728–14,553 of the gene sequence encoding MTAF600 corresponding to a peptide having amino acids 3,910–4851 or nucleotide 13,611–14,767 of the gene sequence encoding MTAF600 corresponding to a peptide having amino acids 4,293–4,534. In accordance with yet another related embodiment, an in vitro cell culture includes a nucleic acid sequence encoding SEQ ID No: 1 or a biologically active substituent thereof operably linked to a regulatory sequence for providing transcriptional activity in a host cell.
In accordance with another embodiment of the invention, a method of diagnosing a susceptibility to cancer of a subject is provided. The method comprises: (a) obtaining a tissue sample from the subject and (b) screening the tissue sample for mutations in the chromosome 1 p36 relating to expression of MTAF600 protein.
In accordance with a further embodiment of the invention, a reagent for assaying for the presence of normal or mutated MTAF600 is provided. The reagent comprises at least one of a polyclonal antibody, a monoclonal antibodies or a Fab fragments having specificity for epitopes of intact MTAF600 or fragments thereof. In accordance with a related embodiment, a reagent for assaying for the presence of normal or mutated MTAF600 comprises nucleic acid probes and primers for detecting DNA or mRNA encoding MTAF600 or fragments thereof. In accordance with a further related embodiment, a method of diagnosing a susceptibility to a cancer or a type of cancer in a subject comprises obtaining a cell sample from the subject, and subjecting the cell sample to an immunoassay comprising at least one of a polyclonal antibody, a monoclonal antibodies or a Fab fragments having specificity for epitopes of intact MTAF600 or fragments thereof. In accordance with yet another related embodiment, a method of diagnosing a susceptibility to a cancer or a type of cancer in a subject comprises obtaining a cell sample from the subject, and subjecting the cell sample to an immunoassay comprising nucleic acid probes and primers for detecting DNA or mRNA encoding MTAF600 or fragments thereof
In accordance with another embodiment of the invention, an animal model is provided. The animal model includes a mouse having a deletion in its genome corresponding to the entire gene or a fragment of a gene at a locus selected from the RB gene and the MTAF600 gene.
In accordance with a further embodiment of the invention, a method of inhibiting E2F-mediated transcription in a cell is provided. The method comprises administering to the cell a polypeptide comprising an amino acid sequence at least 90% homologous to SEQ ID No 1, a peptide having an amino acid sequence for MTAF600 such that the amino acid sequence has at least 90% homology with amino acid sequence 3,910–4851 of SEQ ID No 1, a peptide including an amino acid sequence corresponding to amino acid 4293–4534 of SEQ ID: No. 1, or a small molecule having a molecular weight of less than 1500 D and capable of binding at least one of RB (379–928), P107, P130, E2F1–5 or microtubules that binds to retinoblastoma protein to inhibit the E2F-mediated transcription.
In accordance with another embodiment of the invention, a method of treating a subject that has a heterozygous or homozygous mutation in the RB gene is provided. The method comprises administering to the subject an effective amount of a polypeptide comprising an amino acid sequence at least 90% homologous to SEQ ID No 1, a peptide having an amino acid sequence for MTAF600 such that the amino acid sequence has at least 90% homology with amino acid sequence 3,910–4851 of SEQ ID No 1, a peptide including an amino acid sequence corresponding to amino acid 4293–4534 of SEQ ID: No. 1, or a small molecule having a molecular weight of less than 1500 D and capable of binding at least one of RB (379–928), P107, P130, E2F1–5 or microtubules.
In accordance with a further embodiment of the invention, a method of treating a subject that has a heterozygous or homozygous mutation in the RB gene is provided. The method comprises administering to the subject an effective amount of a peptide or small molecule that interacts with a polypeptide comprising an amino acid sequence at least 90% homologous to SEQ ID No 1, a peptide having an amino acid sequence for MTAF600 such that the amino acid sequence has at least 90% homology with amino acid sequence 3,910–4851 of SEQ ID No 1, a peptide including an amino acid sequence corresponding to amino acid 4293–4534 of SEQ ID: No. 1, or a small molecule having a molecular weight of less than 1500 D and capable of binding at least one of RB (379–928), P107, P130, E2F1–5 or microtubules.
In accordance with another embodiment of the invention, a screening assay for identifying molecules with binding affinity to RB includes (a) selecting a protein having a large pocket corresponding to amino acids 379–928, (b) subjecting the protein to a library of small molecules so as to identify small molecules capable of binding the large pocket, and (c) testing the small molecule for competitive inhibition of binding of MTAF600 with RB.



BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing features of the invention will be more readily understood by reference to the following detailed description, taken with reference to the accompanying drawings, in which:
FIG. 1a shows a characterization of the purified RB Complex from Hela cells resolved by SDS-PAGE and stained with Coomassie brilliant blue R250 showing the results obtained from Hela cells expressing RB with the FLAG epitope tag at the N-terminus (f: RB) by immunoprecipitation with anti-FLAG antibody.
FIG. 1b shows the polypeptide sequence of MTAF600 (SEQ ID No: 1).
FIG. 1c shows agarose gel electrophoresis stained with ethidium bromide where MTAF600 is widely expressed in various tissues. Expression in heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas are shown here. Messenger RNA-encoding MTAF600 was amplified by RT-PCR from poly-A+ RNA that had been isolated from various tissues. mRNA for a house-keeping gene and glycerol-3-phosphate dehydrogenase (G3PDH), was amplified as a positive control.
FIG. 2 shows the interaction of MTAF600 with RB and E2F-1.
FIG. 2a shows that MTAF600 binds to both RB and E2F-1. HeLa nuclear extracts were immunoprecipitated with rabbit anti-MTAF600 polyclonal antibody and mouse anti-E2F-1 monoclonal antibody, respectively and MTAF600, RB, and E2F-1 in the immunoprecipitates were detected by immunoblotting. The positions of MTAF600, hyper- (P-RB) and hypo- (RB) phosphorylated RB, and E2F-1 are indicated. MTAF600 (lane 1) and E2F-1 (lane 3) Control rabbit IgG (lane 2) and Control mouse IgG (lane 4).
FIG. 2b shows that MTAF600 does not interact directly with E2F- 1. Left, GST-RB- (lane 2) and MTAF600- (lane 3) immobilized matrices as well as control matrix (lane 4) were incubated with the E2F-1/DP complex. After washing, bound E2F-1 was detected by immunoblotting. 10% of the E2F-1/DP complex used for immunoprecipitation was also analyzed (lane 1). Right Panel, as a positive control for experiments shown in the left panel, MTAF600-immobilized (lane 1) and control matrixes were incubated with GST-RB. After washing, bound GST-RB was detected by immunoblotting.
FIG. 3 shows that RB sites are required for MTAF600 interaction.
FIG. 3a is a schematic of RB constructs used for measuring interaction with MTAF600 and E2F-1. Positions of RB domains A, B, and C are indicated. The constructs that interact with MTAF600 are shaded. Numbers indicate amino acid position of RB from the N-terminus. RB9 is the triple alanine-substituted mutant (I753A, N757A, M761A) lacking LXCXE-binding activity (Dick et al., “Mutagenesis of the pRB pocket reveals that cell cycle arrest functions are separable from binding to viral oncoproteins”, Mol. Cell. Biol., Vol. 20, pp. 3715–3727, 2000), whereas, R661W, C706F, and S567L are single substitution mutants derived from tumor (Kaye et al., “A single amino acid substitution results in a retinoblastoma protein defective in phosphorylation and oncoprotein binding”, Proc. Natl. Acad. Sci. U.S.A., Vol. 87, pp. 6922–6926, 1990; Kratzke et al., “Functional analysis at the Cys706 residue of the retinoblastoma protein”, J. Biol. Chem., Vol. 267, pp. 25998–26003, 1992; Yilmaz et al., “Twelve novel RB1 gene mutations in patients with hereditary retinoblastoma”, Mutations in brief no. 206, Online. Hum. Mutat., Vol. 12, pp. 434, 1998).
FIG. 3b shows the interaction of RB mutants with MTAF600. GST alone (lane 1) and RB-GST fusions (lanes 2–11) expressed in E. coli were incubated with a cell extract. After GST-pull down, bound MTAF600 and E2F-1 were detected by immunoblotting.
FIG. 4 shows that MTAF interacts with all RB family members.
FIG. 4a shows that MTAF600 interacts with all RB family members in vitro. The large pocket domains of RB (lane 2), p107 (lane 3), and p130 (lane 3) were expressed as GST-fusions. These fusions as well as GST control (lane 1) were incubated with a HeLa nuclear extract and purified by GST pull-down. Bound MTAF600 (top) and GST proteins (bottom) were analyzed by immunoblotting with anti-MTAF600 and anti-GST antibodies, respectively.
FIG. 4b shows that MTAF600 interacts with all RB family members in vivo. Immunoprecipitation experiments were carried out from 3T3 (lanes 1 ̃4) and HeLa (lanes 5 ̃8) extracts with control (lanes 1 and 5), RB (lanes 2 and 6), p107 (lanes 3 and 7), and p130 (lanes 4 and 8) antibodies. Immunoprecipitated materials were analyzed by immunoblotting with anti-MTAF600 antibody (top) and a mixture of anti-RB, anti-p107, and anti-p130 antibodies (bottom).
FIG. 5 shows MTAF600 sites required for RB binding.
FIG. 5a provides a schematic of MTAF600 constructs used for interaction experiments. Top, the constructs that interact with RB are shaded. Numbers indicate the amino acid position of MTAF600 from the N-terminus. The region (residues 4463–4512) that possesses sequence similarity to human papillomavirus is indicated (top sequence; SEQ ID NO:2). Bottom, sequence alignment of MTAF600 with human papillomavirus E7 (types 16, 18, and 22), SEQ ID NOs 3–5, respectively. Conserved residues are shaded. The LXCXE motif and putative zinc finger domain (asterisks) of E7 are shown. Note that MTAF600 has no LXCXE motif.
FIG. 5b shows the interaction of GST-RB with various MTAF600 fragments expressed in COS-7 cells. Various MTAF600 fragments indicated in the Figure were expressed as FLAG-tagged proteins by transfecting into COS-7 cells. Extracts prepared from transfected cells were incubated with recombinant GST-RB (379–928). MTAF600 fragments were analyzed by immunoblotting with anti-FLAG antibody before (bottom) and after (top) GST-pull down. Immuno-reactive bands that correspond to estimated molecular weights are indicated by asterisks.
FIG. 5c shows the fine mapping of the RB-binding sites with bacterially expressed MTAF600 fragments. The MTAF fragments were expressed as His-tagged proteins in E. coli and incubated with recombinant GST-RB (379–928). After GST pull-down, proteins were detected by Coomassie brilliant blue R250 staining (top) or immunoblotting with anti-His antibody (middle). His-tagged proteins before GST pull-down were also analyzed by immunoblotting. Immuno-reactive bands that correspond to estimated molecular weights are indicated by asterisks.
FIG. 5d shows that E7 inhibits binding of RB with MTAF600 as does E2F-1. GST-RB (379–928) was pre-incubated with (lane 2) and without (lane 1) recombinant HPV-16 E7. After GST pull-down, samples were resolved by SDS-PAGE and stained with Coomassie brilliant blue R250, confirming that the interaction between RB and E7 is stoichiometric (top). GST-RB (379–928) on matrix with and without pre-binding of E7 was incubated with a cell extract prepared from NIH3T3 cells. After washing, bound proteins were detected by immunoblotting with anti-MTAF600 (middle) and anti-E2F-1 antibodies (bottom).
FIG. 6 shows that MTAF600 is involved in repression of E2F-responsive genes by the RB family members.
FIG. 6(a) shows that MTAF600 (3910–4851) represses E2F-responsive promoter activity in U2OS osteosarcoma cells.
FIG. 6(b) MTAF600 (3910–4851) actively represses E2F-1 promoter activity.
FIG. 6(c) shows that MTAF600 (3910–4851) represses E2F-responsive promoter activity in both RB+/+ 3T3 fibroblasts.
FIG. 6(d) shows that MTAF600 (3910–4851) represses E2F-responsive promoter activity in both RB−/− 3T3 fibroblasts.
FIG. 7 shows that MTAF600 is involved in cell cycle arrest mediated by RB family members.
FIG. 8 shows how loss of MTAF600 induces transformation in NIH 3T3 fibroblasts using MTAF600 siRNA to knock out the expression of MTAF600 as determined by observation of the cell culture by microscopy.
FIG. 9 shows how down-regulation of MTAF600 induces formation of “micro-foci” in NIH 3T3 fibroblasts, as determined by foci/dish.
FIG. 10 shows how loss of MTAF600 results in formation of polynucleated cells resulting from inhibition of cytokinesis using phase microscopy and fluorescent microscopy.
FIG. 11 shows the effect of loss of MTAF600 on alignment of chromosomes along the spindle in replicating cells.
FIG. 12 shows a gel in which increased amounts of MTAF600 siRNA associated with decreased MTAF600 protein does not alter amounts of RB protein or alpha tubulin in cells. This correlates with levels of MTAF600 RNA and alpha tubulin RNA.
FIG. 13 shows how MTAF600 localizes at the Kinetochore of the nucleus.
FIG. 14 shows the co-localization of MTAF600 with alpha tubulin along the spindle during telophase.
FIG. 15 shows co-localization of MTAF600 and RB with microtubules in Metaphase.
FIG. 16 shows co-localization of MTAF600 with alpha tubulin along the spindle in metaphase
FIG. 17 shows MTAF600 associated with microtubules in interphase.
FIG. 18 shows the protein binding domains in MTAF600 amino acid sequence.
FIG. 19 shows calmodulin binding sites mapped on the MTAF600 amino acid Sequence (SEQ ID NO:6).
FIG. 20 shows how E7 inhibits binding of MTAF600 to RB.
FIG. 21 shows how the RB-binding domain (SEQ ID NO:2) of MTAF600 has sequence similarity to E7 (SEQ ID NO:4) comparing amino acid sequences.
FIG. 22 shows how the RB-binding domain of MTAF600 has sequence similarity to E7.
FIG. 23 shows how MTAF600 sites are required for RB binding.



DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS
Definitions. As used in this description and the accompanying claims, the following terms shall have the meanings indicated, unless the context otherwise requires:
The term “substantially similar” or “substantially homologous” refers to MTAF600, means a protein, peptide or small molecule having MTAF600 sequence.
A “high degree of homology” refers to at least approximately 70% amino acid homology.
A “retinoblastoma complex” refers to a complex that contains proteins and/or other factors necessary for tumor suppressor activity to occur. The complex may include for example any or all of MTAF600, RB, P107, P130, E2F1–5, DP1 or 2, calmodulin or calcium ions. The proteins and/or factors in the complex are characterized by their role in modulating tumor suppressor activity associated with RB. The retinoblastoma complex of proteins does not exclude other proteins or non-protein factors than those recited above provided that it can be shown using any of the assays provided herein (see FIGS. 1 through 7) or using other assays known in the art that the protein or non-protein factor play a role in tumor suppression associated with RB.
“Retinoblastoma” is a malignant tumor of the eye. Mutations that affect both alleles of the retinoblastoma susceptibility gene are a prerequisite for the development of this tumor. The retinoblastoma susceptibility gene (RB gene) consists of 27 exons scattered over 180 kb at chromosome 13q14 (Lohmann, Hum. Mutat., Vol. 14, pp. 283–288; 1999). Retinoblastoma protein appears to inhibit G1-S transition by inhibiting E2F activity and to affect genome stability.
The term “administration to a subject” encompasses any of the methods of drug delivery known in the art. For example, gene therapy is one form of delivery in which the gene encoding the agent is expressed in the subject either because it is introduced or because regulatory sequences are manipulated by an exogenous promotor, enhancer or operator to alter the endogenous expression. Various methods of transferring or delivering DNA to cells for expression of the gene product protein, otherwise referred to as gene therapy. (Yang, Crit. Rev. Biotechnol. 12(4): 335–356; 1992 which is hereby incorporated by reference). A more detailed description of a variety of approaches to gene therapy can be found in U.S. Pat. No. 5,854,221 herein incorporated by reference.
The term “vector” refers to a carrier that can contain or associate with specific nucleic acid sequences, which functions to transport the specific nucleic acid sequences into a cell. Examples of vectors include plasmids and infective microorganisms such as viruses, or non-viral vectors such as ligand-DNA conjugates, liposomes, lipid-DNA complexes. It may be desirable that a recombinant DNA molecule comprising a tumor suppressor activating factor DNA sequence is operatively linked to an expression control sequence to form an expression vector capable of expressing the inhibitor. The transfected cells may be cells derived from the patient's normal tissue, the patient's diseased tissue, or may be non-patient cells.
For example, mammalian expression vectors which bind to the RB family proteins and suppress cell growth can be prepared by inserting the MTAF600 nucleotides 11,728–14,553 (encode amino acids 3,910–4,851) or MTAF600 nucleotides 14,767–13,611 (encode amino acids 4,293–4,534) for MTAF600 into the vector pFLAG-CMV2 (Sigma-Aldrich). (Ogryzko et al., Cell, Vol. 94, pp. 35–44, 1998). Alternatively MTAF600 cDNA can be obtained using overlapping cDNA clones that encoded an assembled open reading frame with the potential to encode a 5,183 amino acid polypeptide were isolated from a human fetal liver cDNA library (CLONTECH Laboratories Inc.). The insert fragments encoding MTAF600 are excised from λ phage by digesting with endonuclease NotI, and then subcloned into pBluescript SK+ vector (Stratagene). MTAF600 DNA may then be recloned into appropriated vectors as required.
Mouse models of knockout mice for MTAF600 are provided herein as research tools for studying the mechanism of tumor suppression during development and in the adult. These mouse models further serve as an in vivo screen for therapeutic agents or vectors that are directed to correcting defects in tumor suppression.
Active agents described herein can be administered to the subject by any of the methods known in the art. Routes of administration of agents include oral, rectal, ophthalmic (including intravitreal or intracameral), nasal, topical (including buccal and sublingual), intrauterine, vaginal or parenteral (including subcutaneous, intraperitoneal, intramuscular, intravenous, intradermal, intracranial, intratracheal, and epidural), dermal, transdermal, or mucosal routes of administration. Other delivery approaches also contemplated include sustained release formulations contained within a biodegradable matrix. Treatments may include a combination of procedures and compositions.
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. Alternatively, the agent can be formulated by means of sustained-release biodegradable polymers similar to those described in U.S. Pat. No. 5,854,221 herein incorporated by reference.
Formulations may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. Dosage units include those that contain a daily dose or unit, daily sub-dose, or an appropriate fraction thereof, of the administered ingredient.
Reagents for detecting target molecules, assaying their concentration and determining their binding kinetics may include antiserum, polyclonal antibodies, monoclonal antibodies or fragments of monoclonal antibodies that have binding specificity for MTAF600, peptide fragments of MTAF600 or analogs of the same or compete with MTAF600 for binding with other molecules in the RB complex (including RB, P107, P130, E2F1–5 and DP 1 or 2).
Methods for making antisera, polyclonal antibodies and monoclonal antibodies or Fab fragments are well established in the art (see for example, the description of techniques in U.S. Pat. No. 5,854,221).
For example, antigens for polyclonal and monoclonal antibodies against MTAF600 nucleotides 9,028–10,110 (encoding amino acids 3,010–3,370) were prepared using bacterial expression vector pET28a(+), information for which can be found on the internet at “www.novagen.com”. Recombinant MTAF600 (amino acid residues 3,010–3,370) were expressed in E. coli BL21(DE3) as an N-terminal His-tagged protein and purified by Ni-NTA agarose (QIAGEN) according to the manufacturers's protocol. The resulting protein will be used to raise polyclonal and monoclonal antibodies. Kits for measurement of the active agent that provide rapid, reliable, sensitive, and specific measurement and localization of molecules include competitive and non-competitive assays, radioimmunoassay, bioluminescence and chemiluminescence assays, fluorometric assays, sandwich assays, immunoradiometric assays, dot blots, enzyme linked assays including ELISA, microtiter plates, antibody coated strips or dipsticks for rapid monitoring of urine or blood, and immunocytochemistry. For each kit the range, sensitivity, precision, reliability, specificity and reproducibility of the assay are established. The kit is useful for the measurement of MTAF600 in animals and humans with and without tumors.
Another kit useful for both research and clinical analysis uses immunohistochemistry or laser scanning cytometry techniques, which are well known to those skilled in the art. These techniques permit localization of MTAF600 in tissues and cells using both light and electron microscopy. For example, tumors are biopsied or collected and tissue sections cut with a microtome to examine sites of inhibitor production. Such information is useful for diagnostic and possibly therapeutic purposes in the detection and treatment of cancer.
Assays may utilize extracts of various tissues, including but not limited to primary and secondary tumors, including retinoblastoma, osteosarcoma and cancers of the lung, breast and bladder.
Here, we report the purification of RB in its native form and we demonstrate that RB is present in the complex with P107 and P130. The 600 kDa subunit, referred to as MT-associated factor (MTAF) 600, interacts directly with RB and microtubules and plays a role in active repression of E2F-responsive genes, cell cycle arrest, and genomic stability.
Loss of RB binding to the E2(1–5) complex for an RB mutant can give rise to the tumor phenotype. Correction of that defect either by repair of the RB lesion or by substituting an E2(105) binding molecule that can reconstitute a normal phenotype for the abnormal one has therapeutic value. The interaction between MTAF600 and the RB complex as well as the interaction between the RB complex and the E2(1–5) complex provides the basis for an assay that may be used to identify small molecules that can be used to manipulate the cell cycle. For example, small molecules may be identified that disrupt the association of MTAF600 with the RB complex (RB, P107 and P130) and hence disrupt E2F-dependent transcription.
The RB Complex
It is here demonstrated for the first time that RB forms a stoichiometric (or nearly stoichiometric) complex with MTAF600. Immunoprecipitation experiments indicate that MTAF600 binds to RB regardless of the phosphorylation status of RB. Moreover, MTAF600 binds to RB without disrupting the interaction between RB and E2F. While not wishing to be limited by theory, it is proposed here that it is likely that the hypophosphorylated form of RB gains access to E2F as a complex with MTAF600, rather than as free RB. In support of this view, E2F and DP proteins, in addition to MTAF600, are copurified with RB when the FLAG-epitope tag is attached at the protein's C-terminal end. Further, MTAF600 appears to be required for repression of E2F-mediated transcription by RB. In all, we conclude that the RB complex, containing RB and MTAF600, is a natural and functional form of RB.
While there might be other RB-containing complexes, the MTAF600-containing complex represented here could be the dominant form, given that we purified RB by immunoprecipitation from nuclear extracts without fractionation and then identified MTAF600 as a major component in immunoprecipitated materials. Our purified complex does not include RB-binding proteins that have been previously reported, except for E2F and DP, which copurified with C-terminally tagged RB. However, previously identified RB-binding proteins are not excluded. (Reviewed in Mulligan et al., Trends Genet., Vol. 14, pp. 223–229, 1998; Lipinski et al., Oncogene, Vol. 18, pp. 7873–7882, , 1999; and Dick et al., Mol. Cell. Biol., Vol. 20, pp. 3715–3727, 2000). We believe that the RB complex, rather than RB alone, is a minimum functional unit and that previously identified RB-binding proteins interact with the RB complex to regulate functions.
Drosophila Homolog of MTAF600
The protein push, whose gene encodes the Drosophila homolog of MTAF600, has been isolated as a gene that is involved in neuronal excitability. (Richards et al., Genetics, Vol. 142, pp. 1215–1223, 1996). Mutations in push cause increased release of transmitter at the neuromuscular junction, which might be caused by altered microtubule function. Moreover, push has been independently identified as a gene that affects meiosis and male sterility. (Sekelsky et al., Genetics, Vol. 152, pp. 529–542, 1999). Mutants of push are defective in meiotic chromosome segregation and spindle formation. These results suggest that MTAF600 plays a role not only in retardation of S-phase entry but also regulation of chromosomal segregation in meiosis and mitosis.
Further, push has been independently identified as the calmodulin-binding protein calo. (Xu et al., J. Biol. Chem., Vol. 273, pp. 31297–31307, 1998). Consistent with this report, we have found that calmodulin as a third subunit of the RB-complex, although calmodulin cannot be detected in the gel shown in FIG. 1A due to its small molecular weight. Calmodulin directly interacts with MTAF600.
Plant Homolog of MTAF600
A protein of exceptional size that is associated with auxin transport has been named BIG. BIG has been associated with positioning of auxin efflux carrier at the plasma membrane via control of vehicle transport or fusion. It appears to have significant identity with Drosophila protein Calossin/Pushover. (Gil et al., Genes and Development, Vol. 15, pp. 1985–1997). We determine here that BIG is related to MTAF600 and that where BIG provides vehicle transport relating to auxins, MTAF600 functions in microtubule mediated transport. Interestingly, Drosophila Pushover is also associated with transport in particular synaptic trnasmission at the neuromuscular junction
The MTAF600-binding Sites on RB
The RB region (residues 379 ̃772; see FIG. 3A), which contains the evolutionally conserved domains A and B, is referred to as the “small pocket” domain. Crystal structure of the small pocket demonstrates that the A and B boxes each contain a helical structural motif, which is referred to as the “cyclin-fold”. (Lee et al., Nature, Vol. 391, pp. 859–865, 1998) Although RB, cyclin A, and transcription initiation factor TFIIB have quite distinct biological functions, each has 2 cyclin motifs and interacts with target proteins in a similar fashion. (Bagby et al., Cell, Vol. 82, pp. 857–867, 1995; Jeffrey et al., Nature, Vol. 376, pp. 313–320, 1995; Nikolov et al., Nature, Vol. 377, pp. 119–128, 1995). While the first cyclin fold of cyclin A and TFIIB respectively interact with CDK2 and TBP, the second cyclin fold of RB (B domain) interacts with the LXCXE motif of E7 The LXCXE-binding domain of RB is highly conserved among species as well as among RB family members, (Lee et al., Nature, Vol. 391, pp. 859–865, 1998) and thus is considered to be a target for various cellular factors. The following cellular RB-binding proteins possess LXCXE or a related sequence that may be involved in RB-binding: histone deacetylase 1 (HDAC1); HDAC2; BRG1; hBrm; retinoblastoma binding protein 1 (RBP1); RBP2; AhR; Bog; CtIP; cyclin D1; cyclin D2; cyclin D3; Elf-1; HBP1; HEC1; hsp75; Rim; RIZ; and UBF. (Reviewed in Dick et al., Mol. Cell. Biol., Vol. 20, pp. 3715–3727, 2000). Some of these proteins contribute to active repression of E2F-mediated transcription by RB (discussed below).
While the LXCXE-binding domain of RB is a potential target for various cellular proteins, binding of E2F is independent of the LXCXE-binding domain of RB. Moreover, E2F does not bind to the small pocket but to the large pocket (residues 379 ̃928), (Huang et al., DNA Cell Biol., Vol. 11, pp. 539–548, 1992; Shan et al., Proc. Natl. Acad. Sci. U.S.A., Vol. 93, pp. 679–684, 1996) suggesting that E2F may interact with both the small pocket and the C domain. Alternatively, E2F may bind to only C-domain, but the small pocket might be required for proper folding of the C domain or vice versa. Interestingly, binding properties of MTAF600 to RB appear to be similar to those of E2F, namely, independence of the LXCXE-binding domain and dependence on the large pocket. However, given that MTAF600 and E2F do not compete for binding to RB, the RB surface for MTAF600-binding could be distinct from that for E2F-binding. Active repression of E2F-responsive transcription by RB
In the early days of research on inhibitory mechanisms by RB, it was thought that RB simply neutralizes the activator function of E2F. However, deletion of the E2F-binding sites in some promoters leads to up-regulation of transcription activity in G0/1 rather than down-regulation in S-phase. (Neuman et al., Mol. Cell. Biol., Vol. 14, pp. 6607–6615, 1994). We propose here that E2F functions as a repressor of transcription in G0/G1 that is mediated through the RB family members. Support for the link between RB and chromatin modifications include the following:
First, histone acetylases (HDAC1, 2 and 3) have been shown to interact directly with RB. (Brehm et al., Nature, Vol. 391, pp. 597–601, 1998; Ferreira et al., Proc. Natl. Acad. Sci. U.S.A., Vol. 95, pp. 10493–10498, 1998; Luo et al., Cell, Vol. 92, pp. 463–473, 1998; Magnaghi-Jaulin et al., Nature, Vol. 391, pp. 601–605, 1998). Acetylation of core histone tails plays an important role in transcriptional activation in chromatin contexts. Recruitment of histone deacetylases to promoters via E2F and RB could allow them to alter acetylation status and maintain chromatin in a hypoacetylated state. Moreover, RB and DNA methyltransferase appear to be functionally related. (Fuks et al., Nat. Genet., Vol. 24, pp. 88–91, 2000; Robertson et al., Nat. Genet., Vol. 25, pp. 338–3342, 2000). Although the molecular mechanisms are unclear, methylation of the CpG island is associated with transcriptional silencing and the formation of high-ordered chromatin structures enriched in hypoacetylated histones. The finding that the DNA methyltransferase DNMT1 copurifies with HDAC1, RB, and E2F (Robertson et al., Nat. Genet., Vol. 25, pp. 338–3342, 2000) suggests that targeted methylation as well as deacetylation in E2F-responsive promoters may contribute to active repression.
Another model of active repression involves chromatin remodeling. ATP-dependent chromatin remodeling factors regulate transcription in both positive and negative ways by changing the positioning of nucleosomes to transcription-competent and -incompetent states, respectively. (Kingston et al., Genes Dev., Vol. 13, pp. 2339–2352, 1999) The BRG1 subunit (a human homolog of SWI2/SNF2) of the chromatin-remodeling complex has been shown to interact with RB and contribute to active repression. (Zhang et al., Cell, Vol. 101, pp. 79, 2000). In addition to these chromatin modifiers, RBP1, HBP1, RIZ, and RBP2 have shown to contribute to active repression by interacting with RB. (Reviewed in Dick et al., Mol. Cell. Biol., Vol. 20, pp. 3715–3727, 2000).
FIGS. 10–17 illustrate the colocalization of MTAF600 with microtubules and alpha-tubulin, its association with spindles and centrosomes and the role of MTAF600 in cytokinesis. MTAF600 plays various roles in events mediated by microtubules for example, chromatin segregation, nuclear division, cytokinesis and microtubule mediated transport.
We have shown for the first time that MTAF600 is responsible for active repression in an RB-interaction-dependent manner. While we believe that this repressive activity in the RB-binding domain of MTAF600 reflects a role in the full-length protein, it is highly possible that this activity is regulated, for instance, by an allosteric effect in the full-length MTAF600. Further, interaction between MTAF600 and RB is a potential regulatory point. As we demonstrated, viral transforming factor E7, by interacting with RB, inhibits not only access of E2F, but also that of MTAF600 to RB. Likewise, cellular factors may regulate the MTAF600-RB interaction by a mechanism distinct from regulation of the RB-E2F interaction.
RB mutants lacking LXCXE-binding activity were constructed and were found not to bind to LXCXE-containing viral transforming factors. However, importantly, they still retain the ability to arrest the cell cycle at G1 (Chen and Wang, Mol. Cell. Biol., Vol. 20, pp. 5571–5580, 2000; Dahiya et al., Mol. Cell. Biol., Vol. 20, pp. 6799–6805, 2000; Dick et al., Mol. Cell. Biol., Vol. 20, pp. 3715–3727, 2000) and to actively repress E2F-mediated transcription. (Dahiya et al., Mol. Cell. Biol., Vol. 20, pp. 6799–6805, 2000). Thus, LXCXE-binding sites of RB are not essential for active repression and cell cycle arrest. However, this does not necessarily mean that LXCXE-binding factors are not functional or significant. While LXCXE-binding factors appear to be nonessential for active repression, it is highly possible that LXCXE-binding factors cooperatively function with other factors for active repression. In addition, LXCXE-binding factors may play crucial roles in other biological functions, such as establishment of irreversible growth arrest in myogenic differentiation. (Chen et al., Mol. Cell. Biol., Vol. 20, pp. 5571–5580, 2000).
While HDAC1, HDAC2, RGB1, RBP1, HBP1, RIZ, and RBP2 have LXCXE motifs, (reviewed in Dick et al., Mol. Cell. Biol., Vol. 20, pp. 3715–3727, 2000) this does not necessarily mean that of these factors bind to RB in a LXCXE-binding site-dependent manner because LXCXE motifs of these factors may not be interaction sites or may not be sole binding sites. Indeed, RGB1 interacts with RB mutants lacking LXCXE-binding activity. (Dahiya et al., Mol. Cell. Biol., Vol. 20, pp. 6799–6805, 2000)
Our findings provide new insights into the mechanisms by which the RB family members function as tumor suppressors and the interaction between RB and MTAF600 to give rise to tumor suppression. The chromosomal location of MTAF600 on chromosome 1p36, which is a hot spot for genes associated with tumors (P73 and P53), further implicates this protein in its mutated form with cancer susceptibility. This is illustrated further in FIGS. 8 and 9 in which cells which have been subjected to loss of MTAF600 expression in the presence of RNAsi transform NIH 3T3 fibroblasts to form foci which are a marker of tumor formation.
Like the E2F-RB interaction, the RB-MTAF600 interaction could be regulated by cellular and viral factors. Moreover, evidence that MTAF600 is a calmodulin-binding protein strongly suggests that MTAF600 is involved in signaling.
The examples provided below are to illustrate embodiments of the invention but are not intended to be limiting. All references cited herein are incorporated by reference.
EXAMPLES
Example 1
Purification and Characterization of the RB Complex
HeLa cells were transduced with a recombinant retrovirus expressing a bicistronic mRNA that encoded FLAG-epitope tagged RB at the N-terminus linked to the ILR2α-subunit surface marker, a construct that was driven by the internal ribosome entry site, and the transduced subpopulation was purified by repeated cycles of affinity cell sorting (Ogryzko et al Cell, Vol. 94, pp. 35–44, 1998). The RB complex was purified from nuclear extracts prepared from the resulting cells by immunoprecipitated with M2 anti-FLAG agarose as described (Ogryzko et al., Cell, Vol. 94, pp. 35–44, 1998). Specific protein bands were excised from Coomassie brilliant blue R250-stained SDS-PAGE gels, digested with trypsin in the presence of 50% [18O]-water to label the C-termini of the tryptic peptides, extracted and sequenced by tandem mass spectrometry (MS/MS) as described previously (Ogryzko et al., Cell, Vol. 94, pp. 35–44, 1998 and therein).
Overlapping cDNA clones that encoded an assembled open reading frame with the potential to encode a 5,183 amino acid polypeptide were isolated from a human fetal liver cDNA library (CLONTECH Laboratories Inc.). MTCTM Panels (CLONTECH Laboratories Inc.) were used to amplify mRNA for MTAF600 with the following primers correspond to the nucleotide positions 15153–15179 and 15403–15403, respectively 5′ GAGAGCCACACGTGTGGAAATCTTGCG-3′ (SEQ ID No:7); and 5′-TCAGGGCTTTGTCGGCAGCTTCGTA-3′ (SEQ ID No:8).
Protein Expression in E. coli 
Various regions of RB as well as the large pocket domain of p107 and p130 were amplified by PCR and subcloned into pGEX6P-1 (Amersham Pharmacia Biotech Inc.) to express as GST fusions in E. coli. Tumor-derived point mutations were introduced into the large pocket domain of RB in pGEX6P-1 by PCR-based mutagenesis, whereas the large pocket domain of the RB9 construct was amplified by PCR to subclone into pGEX6P-1. The MTAF600 fragments and HPV-16 E7 were amplified by PCR and subcloned into pET28c (Novagen Inc.) to express as His-tagged proteins. Internal deletions of MTAF600 were introduced by PCR-based mutagenesis. GST- and His-fusion proteins were expressed and purified with glutathione-Sepharose (Amersham Pharmacia Biotech Inc.) and Ni-NTA agarose (QUIAGEN), respectively, according to the manufacturers' protocols.
Protein Expression in Mammalian Cells
Various fragments of MTAF600 and HPV-16 E7 were subcloned into pFLAG-CMV2 (Sigma-Aldrich). For stable expression, MTAF600 fragments were subcloned into pCMV-Tag2 (Stratagene). The full-length RB was subcloned into pTB701, which contains the SV40 promoter linked to an HA epitope tag (Ono et al., 1988).
Plasmids were transfected into mammalian cells with TransIT®-HeLaMONSTERTM (PanVera Corp.) or LipofectAMINE® (Life Technologies Inc.), except that COS-7 cells were transfected by electroporation using the Gene-Pulsar (Bio-Rad Laboratories).
Binding Assays
Interaction experiments were carried out in Buffer B (20 mM Tris-HCl buffer [pH 8.0]; 5 mM MgCl2; 10% glycerol; 100 mM KCl; 1 mM PMSF; 10 mM 2-mercaptoethanol; 0.1% Tween 20) containing 1% Triton X-100 and 0.1 M KCl in a total volume of 20 μl. For GST-pull down experiments,  ̃5 pmol of GST-RB derivatives were incubated with  ̃1 mg of cell extracts or  ̃50 pmol of His-tagged MTAF600 derivatives for 30 min at 4° C. Samples were further incubated with 10 μl of glutathione-Sepharose (50% slurry) with rotation, washed 4 times, and eluted with 10 μl of SDS-PAGE sample buffer.
For immunoprecipitation experiments, cell extracts were incubated with 1 μg of antibody for 1 hr. Samples were further incubated with 10 μl of protein G-Sepharose (50% slurry) (Amersham Pharmacia Biotech Inc.) with rotation, washed 4 times, and eluted with 10 μl of SDS-sample buffer.
To test binding of MTAF600 to E2F-1 (FIG. 2B), MTAF600 was immunoprecipitated from a HeLa nuclear extract so that  ̃1 pmol of MTAF600 was immobilized per 5 μl of protein G-Sepharose (packed volume). The matrix was extensively washed with Buffer B containing 1% Triton X-100 and 0.3 M KCl extensively until RB was completely removed from the matrix. GST-RB (379–928) was also immobilized through anti-RB antibody at  ̃1 pmol per 5 μl of protein G-Sepharose (packed volume). 5 μl of the resulting beads (packed volume) were incubated with  ̃1 pmol of E2F-1/DP for 30 min at 4° C., washed 4 times with Buffer B containing 1% Triton X-100 and 0.1 M KCl, and eluted with 10 μl of SDS-PAGE sample buffer.
Antibodies
Rabbit polyclonal antibody against MTAF600 was raised against a fragment containing residues 3,010–3,370. For anti-human RB antibodies, clone XZ-77 (Upstate Biotechnology Inc.) was used for immunoprecipitation, whereas clone G3-245 (BD Sciences-PharMingen) and RB C-15 (Santa Cruz Biotechnology Inc.) were used for immunoblotting. Anti-FLAG M2 antibody-conjugated agarose (Sigma-Aldrich) was used for immunoaffinity purification, whereas anti-FLAG M5 antibody was used for immunoblotting. Suppliers for the other antibodies are as follows: HA 12CA5 (Roche Molecular Biochemicals); 6xHis (CLONTECH Laboratories Inc.); GST (Amersham Pharmacia Biotech Inc.); p107 C-18 (Santa Cruz Biotechnology Inc.); p130 C-20 (Santa Cruz Biotechnology Inc.); and E2F-1 KH20/KH95 (Upstate Biotechnology Inc.).
Reporter Experiments
Cells were transfected with various effector and reporter plasmids as indicated in the Legend to FIG. 5. The cells were harvested after 48 hr of transfection, and luciferase activity was measured by using Enhanced Luciferase Assay Kit (BD Sciences-PharMingen) according to the manufacturers' protocols. Luciferase activity was normalized against that of β-galactosidase and expressed as a relative activity.
Cell Cycle Analysis
Stably transfected U2OS cells were grown in DMEM containing 10% fetal bovine serum and kept for 36 hr at confluency to enrich G0/G1 cells. Cells were replated at 5×104/cm2 and were further incubated. Cells were harvested at the indicated periods, analyzed by fluorescence-activated cell sorter (FACScan, BD Biosciences), and the data were analyzed using the ModFitLT program.
RNA Interference (RNAsi)
Double stranded RNA is used to target specific mRNA (MTAF600 mRNA) for degradation thereby silencing its expression. This technique has been described by Zamore, Nature, Vol. 8, pp. 746–750, 2001.
Example 2
Purification of the RB Complex
To purify RB we first established cells that stably express FLAG-epitope-tagged RB (f:RB). Although HPV-18 E7 expressed in HeLa cells is believed to inactivate RB family proteins, we risked employing HeLa cells for the following reasons: first, exogenous expression of RB in HeLa cells suppresses cell growth, indicating that RB is functional, at least in part, when exogenously expressed. Moreover, among different kinds of cells we have tested, we were only able to grow HeLa cells on a large scale when RB was exogenously expressed.
To establish HeLa cells expressing f:RB, we transduced recombinant retrovirus expressing a bicistronic mRNA that encoded f:RB linked to the ILR2 α-subunit surface marker (Ogryzko et al., Cell, Vol. 94, pp. 35–44, 1998) driven by the internal ribosome entry site. The transduced subpopulation was purified by repeated cycles of affinity cell sorting (Ogryzko et al., Cell, Vol. 94, pp. 35–44, 1998) with anti-ILR2 antibody-conjugated magnetic beads. (Ogryzko et al., Cell, Vol. 94, pp. 35–44, 1998) Using anti-FLAG antibody-conjugated agarose, f:RB was purified from nuclear extracts of the resulting cells (FIG. 1A, lane 2). As a control, mock purification was performed from non-transduced HeLa cells (lane 1). Immunoprecipitated materials were eluted with FLAG peptide and analyzed by SDS-PAGE. In addition to f:RB, the 600 kDa protein MT-associated factor (MTAF) 600 was specifically purified (lane 2).
In the course of this study, we found that most precipitated RB is hyperphosphorylated when the FLAG-tag is attached at the N-terminus, whereas most precipitated RB is hypophosphorylated when the tag is attached at the C-terminus (data not shown). Since the N-terminally and C-terminally tagged RB are both present as hyperphosphorylated and hypophosphorylated forms in HeLa cells, accessible surfaces of the protein could be drastically different between hyperphosphorylated and hypophosphorylated forms of RB, perhaps due to conformational alterations. In support of this notion, we observed that, when RB was tagged at its N-terminus, E2F and DP family members were not detected, whereas when RB was tagged at its C-terminus, DP-1 and E2F-1 were copurified with MTAF600.
Example 3
Identification of the Novel RB-associated Factor MTAF600
Using an analysis of tryptic fragments of MTAF600 by tandem mass spectrometry (MS/MS), several EST clones that encode uncharacterized polypeptides were identified. A human liver cDNA library was screened with one of the EST clones (GenBank accession number T66125) as a probe. By “cDNA walking,” we isolated overlapping clones that enabled us to compose an open reading frame with the potential to encode a protein (5183 residues) with an estimated molecular mass of 573,536 Da (FIG. 1B). RT PCR analysis of RNA isolated from various human tissues indicated that MTAF600 is ubiquitously expressed (FIG. 1C).
A database search with PSI BLAST program (Altschul et al., Nucleic. Acids. Res., Vol. 25, pp. 3389–3402, 1997) identified proteins homologous to ones found in Drosophila melanogaster, Arabidopsis thaliana, and Caenorhabditis elegans. While the C. elegans sequence is incomplete, the predicted peptides of the Drosophila and Arabidopsis counterparts appear to be complete, consisting of 5322 and 5079 residues, respectively. While all of them were reported by genome projects, the Drosophila clone was independently isolated by genetic screening known as pushover. (Richards et al., Genetics, Vol. 142, pp. 1215–1223, 1996; Sekelsky et al., Genetics, Vol. 152, pp. 529–542, 1999). Moreover, it has also been isolated as a calmodulin binding protein, calo (Xu et al., J. Biol. Chem., Vol. 273, pp. 31297–31307, 1998) (see Discussion) where calmodulin is an abundant cell protein that is activated in the presence of calcium.
Example 4
MTAF600 Interacts with Hypophosphorylated and Hyperphosphorylated RB
The hypophosphorylated form of RB, which is abundant in quiescent or differentiating cells, inhibits E2F-dependent transcription through direct interaction with E2F proteins. In contrast, the hyperphosphorylated form of RB, which is abundant in proliferating cells, does not possess the ability to interact with E2F proteins, thus allowing the activation of E2F-dependent genes that are required for S-phase entry (Weinberg, Cell, Vol. 81, pp. 323–330, 1995; Dyson, Genes Dev., Vol. 12, pp. 2245–2262, 1998).
To elucidate the mechanism whereby MTAF600 participates in the RB-E2F pathway, we determined which form of RB, whether hyperphosphorylated or hyperphosphorylated, binds to MTAF600. Further, we examined whether MTAF600 affects the RB-E2F interaction.
When MTAF600 was immunoprecipitated from a HeLa extract, both hypophosphorylated and hyperphosphorylated RB were copurified (FIG. 2A, lane 1). Moreover, E2F-1 was also immunoprecipitated along with MTAF600. We next performed reciprocal immunoprecipitation with anti-E2F-1 antibody (lane 3). As expected, only hypophosphorylated RB coprecipitated with E2F-1. Consistent with the reciprocal experiments, MTAF600 coprecipitated with E2F-1.
Given that MTAF600 interacts with E2F-1, we tested whether the interaction of these factors is direct or indirect. GST-RB and MTAF600 immobilized on matrix were incubated with the purified E2F-1/DP complex to determine interaction (FIG. 2B, left). No detectable E2F-1 was retained on MTAF600 agarose (lane 3), while a considerable amount of E2F-1 was retained on GST-RB agarose. The control experiments showing that GST-RB binds to MTAF600 agarose demonstrate that the immobilized MTAF600 employed for these experiments is functional (FIG. 2B, right). Thus, these results exclude the direct interaction of MTAF600 with E2F-1. In all, we conclude that MTAF600 interacts with both hypophosphorylated and hyperphosphorylated RB, and further that MTAF600 interacts with RB without interfering RB binding to E2F-1.
Example 5
The Large Pocket Domain of RB is Responsible for Interaction with MTAF600
To map RB sites required for MTAF600 binding, various RB constructs shown in FIG. 3A were expressed as GST fusions in E. coli. 
The GST-RB fusions were incubated with HeLa nuclear extracts, and then bound MTAF600 and E2E-1 were analyzed by Western blotting after GST precipitation (FIG. 3B). The RB residues 379 ̃928, referred to as RB (379–928), which encompass the large pocket domain, interacted with both MTAF600 and E2F-1. However, further deletions (379 ̃772, 646 ̃928, and 379 ̃646, lanes 9 ̃11) led to loss of interactions with MTAF600 and E2F-1, indicating that the large pocket domain of RB is required for interaction with both MTAF600 and E2F-1.
Viral transforming factors such as HPV E7, adenovirus E1A, and simian virus 40 large T antigen bind to RB through the conserved LXCXE motifs that are critical for transforming properties. (Reviewed in Zalvide et al., Mol. Cell. Biol., Vol. 15, pp. 5800–5810, 1995; Flint et al., Annu. Rev. Genet., Vol. 31, pp. 177–212, 1997). The LXCXE-binding sites are located within the B region (see FIG. 3A) and are well conserved among the RB family proteins. However, RB mutants lacking LXCXE-binding activity are still able to bind to E2F and repress transcriptional activity, indicating that binding of E2F to RB is independent of the LXCXE-binding sites. (Chen et al, Mol. Cell. Biol., Vol. 20, pp. 5571–5580, 2000; Dahiya et al., Mol. Cell. Biol., Vol. 20, pp. 6799–6805, 2000; Dick et al., Mol. Cell. Biol., Vol. 20, pp. 3715–3727, 2000). To test the requirement of the LXCXE-binding sites for MTAF600 interaction, we employed the RB mutant, RB9, (Dick et al., Mol. Cell. Biol., Vol. 20, pp. 3715–3727, 2000) which possesses 3 amino acid substitutions in the LXCXE contact surface and thus lacks the ability to bind to E7 or E1A. The large pocket domain of RB9 was expressed as a GST-fusion protein and tested for its ability to bind to MTAF600. Consistent to the previous report, (Dick et al., “Mutagenesis of the pRB pocket reveals that cell cycle arrest functions are separable from binding to viral oncoproteins”, Mol. Cell. Biol., Vol. 20, pp. 3715–3727, 2000) RB9 interacted with E2F-1 as does wild-type RB. Likewise, RB9 also interacted with MTAF600 (FIG. 3B, lane 4). Almost equimolar amounts of GST proteins were recovered after GST pull-down.
In all, we conclude that both MTAF600 and E2F bind to the large pocket of RB independently of the LXCXE-binding sites. However, given that MTAF600 and E2F can simultaneously bind to RB (FIG. 2), these factors could target distinct surfaces of the large pocket of RB.
We next tested whether tumor-derived RB mutants (R661W, C706F, and S567L) (Kaye et al., Proc. Natl. Acad. Sci. U.S.A., Vol. 87, pp. 6922–6926, 1990; Kratzke et al., J. Biol. Chem., Vol. 267, pp. 25998–26003, 1992; Yilmaz et al, Mutations in brief no. 206, Online. Hum. Mutat., Vol. 12, pp. 434, 1998) bind to MTAF600 (FIG. 3B, lanes 5 ̃7). Consistent with the previous reports, these mutants did not interact with E2F-1. (Kratzke et al., Oncogene , Vol. 9, pp. 1321–1326, 1994; Sellers et al., Genes Dev., Vol. 12, pp. 95–106, 1998) Importantly, none of these mutants showed interaction activity with MTAF600, suggesting that these mutations cause conformational alterations of RB, which lead to loss of interaction with MTAF600 and E2F-1.
We further tested whether the other RB family proteins p107 and p130 interact with MTAF600. The large pocket domain of RB, p107, and p130 were expressed as GST fusions and tested for interactions. As expected from sequence and functional conservations of the large pocket domain among RB family proteins, RB, p107, and p130, all interacted with MTAF600 in vitro (FIG. 3C).
Example 6
RB Binds to the MTAF600 Fragment with E7-like Sequence
To determine MTAF600 sites that are responsible for RB interaction, various MTAF600 fragments (1 ̃833, 1241 ̃1649, 1681 ̃2401, 2660 ̃3359, 3214 ̃3899, 3910 ̃4851, and 4480 ̃5183; FIG. 5A) were expressed as FLAG-tagged proteins by transfecting into COS-7 cells. Extracts containing recombinant MTAF600 fragments were prepared from resulting cells and tested for GST interaction. As shown in FIG. 5B, only MTAF600 (3910 ̃4851) interacted with RB in vitro. This interaction was confirmed in COS-7 cells by immunoprecipitation of exogenously expressed MTAF600 (3910 ̃4851) and RB.
For more precise mapping of the MTAF600 sites required for RB-binding, further deletions were introduced in the MTAF600 residues 3910 ̃4851 and were expressed as His-tagged proteins in E. coli (FIG. 5C). The MTAF600 (4293 ̃4534) interacted with RB (lane 1), while MTAF600 (4529 ̃4851) did not (lane 2). Importantly, binding of the MTAF600 (4293 ̃4534) with RB is almost stoichiometric judging from the Coomassie brilliant blue-stained SDS-PAGE gel. On the other hand, the internal deletions (Δ1 and Δ2; FIG. 5A) within MTAF600 (4293 ̃4534) resulted in loss of interaction (FIG. 5C, lanes 3 and 4).
A BLAST database search with the RB-binding domain of MTAF600 as query revealed that the MTAF600 residues, located within the essential region for RB-binding, possess sequence similarity to HPV E7 (FIG. 5A). While HPV-22 (type 22) E7 shows the best conservation, E7 of HPV-16 and -18, high-risk types which link to subset of malignant tumors, (For reviews, see Flint et al., “Viral transactivating proteins”, Annu. Rev. Genet., Vol. 31, pp. 177–212, 1997) also display significant conservations. The conservation is observed in E7 residues 27–77 (based on the HPV-22 E7 sequence) adjacent to the LXCXE motif. While the LXCXE motif of E7 is sufficient for specific binding to RB, (Lee et al., Nature, Vol. 391, pp. 859–865, 1998 and therein) the contiguous C-terminal region is responsible for stable binding to RB and transforming activity. (For review, see Flint et al., Annu. Rev. Genet., Vol. 31, pp. 177–212, 1997) (see Discussion). The sequence similarity between E7 and the RB binding domain of MTAF600 suggests that E7 and MTAF600 bind competitively to RB. GST-RB was pre-incubated with E7 (FIG. 5D) and then tested for interaction with MTAF600. As predicted, pre-incubation of RB with E7 significantly reduced binding of both MTAF600 and E2F-1, suggesting that not only E2F-1 but also MTAF600 are targeted by E7.
Example 7
MTAF600 Functions as a Corepressor of RB
An important role of RB is negative regulation of E2F-dependent transcription, allowing repression of various genes required for S-phase entry. (For reviews, see Weinberg, “The retinoblastoma protein and cell cycle control”, Cell, Vol. 81, pp. 323–330, 1995, Dyson, “The regulation of E2F by pRB-family proteins”, Genes Dev., Vol. 12, pp. 2245–2262, 1998) Recent studies have shown that RB does not simply neutralize the function of E2F activation domain; instead, RB functions as an active repressor when RB in recruited on E2F-dependent promoters via interaction with E2F, and thus, deletion of E2F-binding sites results in gene activation in some promoters. (Neuman et al., “Transcription of the E2F-1 gene is rendered cell cycle dependent by E2F DNA-binding sites within its promoter”, Mol. Cell. Biol., Vol. 14, pp. 6607–6615, 1994). Because formation of the stoichiometric complex between RB and MTAF600 suggest that functions of RB, at least in part, could be contributed by MTAF600, we tested whether MTAF600 contributes to transcriptional repression by RB.
We first tested a luciferase reporter gene driven by three E2F-binding sites linked to the TK core promoter. (Magnaghi-Jaulin et al., Nature, Vol. 391, pp. 601–605, 1998). In FIG. 6A, U2OS cells were transiently transfected with the 3×E2F-TK-luciferase reporter (0.5 mg) (Magnaghi-Jaulin et al., Nature, Vol. 391, pp. 601–605, 1998) and expression vectors for RB, MTAF600 (3910–4851), MTAF600 (3910–4851)D1, and HPV-16 E7 (0.1 mg) as indicated. All transfection mixtures include 0.1 mg of CMV-β-galactosidase reporter, which is not under regulation of E2F, for normalization of transfection efficiency. After 48 hr of transfection, cells were harvested and processed for reporter assays. Luciferase activity is plotted as relative activity±standard division after normalizing against b-galactosidase activity. The control reporter construct lacking E2F-binding sites displayed <5% of the activity displayed by the 3×E2F-TK-luciferase reporter.
Strong transactivation from this reporter was observed by endogenous E2F in U2OS cells in an E2F-binding site-dependent manner (FIG. 6A). This activation was inhibited by co-transfecting RB in a dose-dependent manner. Likewise, co-transfection of MTAF600 (3910 ̃4851) significantly inhibited E2F-dependent transcription. In addition to MTAF600 (3910 ̃4851), MTAF600 (4300 ̃4534) similarly inhibited E2F-dependent transcription. Inhibition of E2F-dependent transcription by MTAF600 (3910 ̃4851) appears to be dependent on endogenous RB family proteins, since the MTAF600 mutant lacking RB-binding activity, MTAF600 (3910 ̃4851)Δ1, did not inhibit E2F-dependent transcription. Moreover, HPV E7 counteracted inhibitory activity by MTAF600 (3910 ̃4851). This shows that a likely pathway for the role of MTAF600 in E2F-dependent transcription is through interaction with RB.
To explore the role of MTAF600 in active repression, we employed the E2F-1 promoter linked to a luciferase reporter gene (FIG. 6B). Experiments were performed as described above except that a luciferase reporter under regulation of the E2F-1 promoter was employed.
The E2F-1 promoter has putative binding sites for E2F, Sp-1, ATF, E4F, and NF-κB (Neuman et al., Mol. Cell. Biol., Vol. 14, pp. 6607–6615, 1994). Given that mutation in the E2F-binding sites leads to derepression of the promoter activity during G1In the E2F-1 promoter, the E2F-binding sites must play a role in active repression of transcription. The E2F-1-luciferase gene was strongly expressed by endogenous transcription factors in U2OS cells. Importantly, this activation was efficiently repressed by transfecting either RB or MTAF600 (3910 ̃4851). However, the activity was not inhibited by the MTAF600 mutant lacking RB binding activity, MTAF600 (3910 ̃4851)Δ1. These results indicate that MTAF600 (3910 ̃4851) contributes to active repression of the E2F-1 promoter in conjunction with endogenous RB family proteins.
To explore whether MTAF600 inhibits E2F-mediated transcription in RB-deficient cells, we employed wild type and RB-deficient NIH3T3 fibroblasts. (Classon et al., Proc. Natl. Acad. Sci. U.S.A., Vol. 97, pp. 10820–10825, 2000). FIGS. 6c and 6d show results obtained when experiments were performed as described for FIG. 6a except that RB+/+ (FIG. 6C) and RB−/− (FIG. 6D) 3T3 fibroblasts were employed (Classon et al, Proc. Natl. Acad. Sci. U.S.A., Vol. 97, pp. 10820–10825, 2000).
Given that only weak transactivation from this reporter was observed by endogenous E2F in NIH3T3 fibroblasts, we cotransfected E2F-1 expression vector to study the repressive effect (FIGS. 6C and D). In both wild type and RB-deficient NIH3T3 fibroblasts, MTAF600 (3910 ̃4851) repressed E2F-mediated transcription, although repression is less effective in the RB-deficient fibroblasts. Whereas, MTAF600 mutant lacking RB binding activity did not, suggesting that MTAF600 silences E2F-mediated transcription in collaboration with p107 and/or p130 in the RB-deficient fibroblasts. These results support the data showing binding of MTAF600 to all RB family members (FIG. 4).
Example 8
MTAF600 Inhibits Cell Proliferation
We examined whether expression of MTAF600 (3910 ̃4851) leads to inhibition of S-entry of the cell cycle. Stably transfected U2OS cells were synchronized at G0/G1 by contact inhibition. In the control cells, 64% of the cells were in S-phase at 24 hr after induction of cell growth by replating. In contrast, in the cells expressing MTAF600 (3910 ̃4851), G0/G1 cells were predominant and only 37% of the cells were found in S-phase after 24 hr. On the other hand, no significant delay in entering S-phase was observed in cells expressing the mutant form of MTAF600 (3910 ̃4851), although expression level of the mutant protein was comparable to that of the wild-type MTAF600 (3910 ̃4851) (data not shown). From these results, we conclude that exogenous expression of MTAF600 (3910 ̃4851) inhibits progression of cells into S-phase in collaboration with endogenous RB family proteins.
In other experiments, U2OS cells were stably transfected with the expression vectors for MTAF600 (3910–4851) and MTAF600 (3910–4851) D1, as well as the control vector, and were synchronized at G0/G1 by contact inhibition. Cells were replated and harvested at 0, 24, and 36 hrs. Cell cycle stage was analyzed by FACS, as indicated in FIG. 7, which shows that MTAF600 is involved in cell cycle arrest mediated by RB family members.
Using MTAF600 siRNA to knock out the expression of MTAF600, FIG. 8 shows how loss of MTAF600 induces transformation in NIH 3T3 fibroblasts, as determined by observation of the cell culture by microscopy.
Using foci/dish analysis, down-regulation of MTAF600 was found to induce formation of “micro-foci” in NIH 3T3 fibroblasts, as shown in FIG. 9.
Phase microscopy and fluorescent microscopy was used to examine the effect of loss of MTAF600 on cytokinesis. As seen in FIG. 10, the loss of MTAF600 results in inhibition of cytokinesis and formation of polynucleated cells, when compared to normal cells.
Determination of the effect of loss of MTAF600 on alignment of chromosomes along the spindle in replicating cells is shown in FIG. 11. Green represents MTAF600 protein which is present in the control cells and absent in MTAF600 SiRNA cells. In the control cells, RB is organized in the nucleus whereas in the MTAF600 SiRNA treated cells, RB is dispersed. Red is RB protein, Blue is DNA.
As seen in FIG. 12, increased amounts of MTAF600 siRNA associated with decreased MTAF600 protein does not alter amounts of RB protein or alpha tubulin in cells, as determined by PAGE analysis. This correlates with levels of MTAF600 RNA and alpha tubulin RNA.
Localization of MTAF600 at the Kinetochore of the nucleus is shown in FIG. 13. Dynein, shown in red, binds to the microtubule and uses the energy in ATP molecules to move from the positive (+) end of the microtubule (where new tubulin dimers are adding to the microtubule) toward the minus (−) end of the microtubule. Each small step requires the hydrolysis of one ATP molecule. Dynein pulls subcellular materials toward the center of the cell, or in the case of mitosis, toward the poles of the spindle and thus toward the centers of the two new daughter cells. Fluorescent staining reveals the colocalization of MTAF600 with Dynein.
Telephase colocalization of MTAF600 with alpha tubulin along the spindle is seen in FIG. 14. Localization of MTAF600 is enhanced in the green/blue image, compared to α-tubulin enhancement in the red/blue image, followed with the merged image (yellow/red/blue/green).
Co-localization of MTAF600 and RB with microtubules during Metaphase is shown in FIG. 15. MTAF600 enhancement is shown in the green/blue image; RB enhancement is shown in the red/blue image; and the merge image is seen with yellow.
Co-localization of MTAF600 with alpha tubulin along the spindle in metaphase is shown in FIG. 16. MTAF600 enhancement is shown in green, α-tubulin enhancement is shown in red, and the merged image is shown in yellow.
Association of MTAF600 with microtubules, specifically α-tubulin, during interphase, is shown in Figure. MTAF600 enhancement is shown in green; α-tubulin enhancement is shown in red; and the merged image is shown in yellow.
FIG. 18 shows Protein binding domains in the MTAF600 amino acid sequence are shown in FIG. 18. A zinc finger domain is found in the 1650–1730 region of the sequence, in yellow; a calmodulin (CaM) binding domain is found at region 4076–4122, in green; and a retinoblastoma (RB) binding domain is found at the 4293–4534 region of the sequence, in red.
Calmodulin (CaM) binding sites are mapped on the MTAF600 amino acid sequence in FIG. 19. The 4089 to 4112 region is expanded, to show the actual amino acid sequence in that region.
E7 inhibition of MTAF600 binding to RB is shown in FIG. 20. Coomasie brillian blue staining indicates the presence of a GST-RB fusion protein band in the presence and absence of E7, as well as a band for E7 in the E7 lane, and Western blot analysis shows the complete absence of an MTAF600 band or an E2F-1 band in the presence of E7 after incubation with nuclear extracts.
Similarity of the RB-binding domain of MTAF600 with E7 is shown in FIG. 21, where the amino acid sequences of the two proteins are compared. MTAF600 is shown in red, and E7 is shown in blue. The E7-like and MTAF600-like similar sequences are indicated generally in yellow, and the specific sequences for the MTAF600 E7-like region and the relevant E7 segment are shown below with specific amino acid matches highlighted in red.
The RB-binding domain of MTAF600, and its sequence similarity to E7, is shown generally in FIG. 22, with the E7-like region highlighted in yellow.
MTAF600 sites required for RB binding are shown in FIG. 23.

## Claims

1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide comprising SEQ ID NO:1.

2. The isolated nucleic acid molecule of claim 1 wherein the polyp eptide consists of SEQ ID NO:1.

3. A vector comprising any of the isolated nucleic acid molecules of claims 1 and 2.

4. An expression vector comprising any of the isolated nucleic acid molecules of claims 1 and 2.

5. A cultured cell containing any of the isolated nucleic acid molecules of claims 1 and 2.

6. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide wherein the amino acid sequence of the polypeptide is at least 90% identical to SEQ ID NO: 1, wherein the polypeptide binds to R13.

7. The isolated nucleic acid molecule of claim 6, wherein the amino acid sequence comprises 5,183 residues.