WO2003020758A2

WO2003020758A2 - Cdk regulator proteins called ringo

Info

Publication number: WO2003020758A2
Application number: PCT/IB2002/003799
Authority: WO
Inventors: M. Angel Rodriguez Nebreda; Ingvar Mats Ferby; Gustavo J. Gutierrez; Laurent H. Perez
Original assignee: European Molecular Biology Laboratory
Priority date: 2001-09-05
Filing date: 2002-09-04
Publication date: 2003-03-13
Also published as: WO2003020758A3; AU2002330705A1; GB0121477D0

Abstract

The invention relates to proteins termed Ringo or Speedy that regulate the activity of cyclin-dependent kinases (cdks). The invention also relates to methods for the regulation of cdks, which have great promise in the development of strategies to prevent and treat diseases and conditions in which the expression or activity of a cdk is defective.

Description

REGULATOR MOLECULES

The present invention relates to proteins that regulate the activity of cyclin-dependent kinases (cdks). The invention also relates to methods for the regulation of cdks, which have great promise in the development of strategies to prevent and treat diseases and conditions in which the expression or activity of a cdk is defective.

All publications, patents and patent applications cited herein are incorporated in full by reference.

Cdks form a family of kinases and have essential roles in the regulation of cell proliferation, in neuronal function and in transcription. The first cdk to be identified, cdc2, was initially discovered as a gene essential for both Gl/S and G2/M transitions in the yeast cell cycle (Nurse et al, 1981, Nature 292: 558). A human cdc2 homologue, cdkl, was subsequently cloned (Lee et al, 1987, Nature 237: 31) and cdc2 homologues were later found to be present in all eukaryotes. It was at this time realised that cdkl was just one of a large family of kinase regulators present in eukaryotes. To date, 9 cdks have been identified in humans. It is now known that some of those cdks play an essential role in cell division. For example, in mitosis, different cdks control the steps between different phases of the cell cycle through phosphorylation of cell proteins such as histones, cytoskeletal proteins, transcription factors and tumour suppressor genes. The kinase activity of cdks generally requires their association with regulatory subunits known as cyclins. The association with cyclins appears to induce a conformational change in the cdk, enabling it to be phosphorylated by cdk-activating kinase (CAK), a trimeric complex of the proteins cdk7, cyclinH and MAT1. In the early to mid Gl phase of the cell cycle, extracellular signals modulate the activation of cyclinDl/cdk4 and cyclinD3/cdk6. These complexes initiate a phosphorylation cascade which results in the expression of genes whose products are required for Gl/S transition and S phase progression, such as cdk2, cyclin E and cyclinA. The S/G2 transition is regulated by cdkl , in association with cyclinA and the G2/M transition is promoted by cdkl/cyclinB (see Morgan, 1997, Annu Rev Cell Dev Biol 13:261 for review).

The cdks which are involved in the regulation of meiosis are also often in the form of cyclin/cdk complexes. For example, fully grown Xenopus oocytes are arrested at the G2/M boundary of the first meiotic division until maturation is initiated by the hormone progesterone which activates a cdkl/cyclinB complex, so resulting in G2/M progression (Kobayasgi et al, 1991 , Cold Spring Harbor Symp. Quant. Biol., 56: 437). In addition, some cdks associated with cyclins are known to directly influence transcription. For example, the cdk7/cyclinH/MATl complex is a component of the basal transcription factor TFIIH. Furthermore, the cdk8/cyclin C complex has been found in a multiprotein complex with RNA polymerase II and CDK9/cyclinT is a component of the positive transcription elongation factor P-TEFb.

However, contrary to initial belief, activation of cdks does not always appear to require the presence of cyclins. In 1999, a protein named Ringo, also known as Speedy, was identified in Xenopus oocytes (Ferby et al, 1999, Genes & Development, 13(16): 2177; Lenormand et al, 1999, EMBO J., 18(7): 1869). Ringo exhibited no sequence homology to cyclins but could bind and activate both cdkl and cdk2 in the absence of progesterone. Further work demonstrated that Ringo binds and activates cdkl and cdk2 in Xenopus oocytes, bypassing the phosphorylation generally required for activation (Karaiskou et al, 2001 , J. Biol. Chem., published July 18, 2001 as 10.1074/jbc.M 104722200). In addition, cdkl/RINGO and cdk2/RINGO were only weakly inhibited by inhibitors of cdk-cyclin complexes. The cdk/RINGO complexes might therefore be active under condition where cyclin-bound cdks were inhibited and hence play different regulatory roles.

In addition, cdk5 has been shown to be activated by proteins other than cyclins. cdk5 is not involved in cell cycle regulation but appears to have an important role in neuronal function. It has been demonstrated that a p35-cdk5 complex is important for normal development and function of the brain. In the brains of Alzheimer's patients, the cdk5 protein is associated with p25, a protein derived from p35 by proteolytic cleavage. Association with the p25 protein leads to constitutive activation of cdk5 and it is believed that cdk5-p25 may be involved in the cytoskeletal abnormalities and neuronal death associated with Alzheimer's (Tsai et al, 1999, 402: 615). In addition, it has been suggested that cdk5 is involved in neurodegeneration in Amyotrophic Lateral Sclerosis (Nguyen et al, 2001 , Neuron, 30(1): 135). It has also been demonstrated that cocaine induces increased level and activity of cdk5 in dopamine-sensitive brain cells, leading to the suggestion that cdk5 may mediate structural changes in neurons which result in cocaine addiction (Bibb et al, 2001 , Nature 4210: 376).

In view of the essential roles played by cdks in the body, there is a need to identify regulators of both cdk levels and activity. The identification of new cdk regulators will facilitate the understanding of the roles of different cdks and the effect of loss of regulation by cdks more fully. In turn, this will enable targets to be identified for the development of drugs that will restore normal control of cell processes where such control has been lost. Summary of the invention

Accordingly, the invention provides a cdk regulator molecule comprising a core region from a RINGO protein of mammalian origin, or a functional equivalent thereof.

The inventors have discovered that a region found in RINGO proteins, herein described as a "core region", has activity in modulating the function of cdk proteins. As indicated previously, cdks play an essential role in the regulation of a variety of different processes within cells. The ability to increase the activity of cdks will therefore enable researchers to understand these processes more fully and, furthermore, will have a wide variety of practical applications. For example, abnormally high cell cycle progression is often associated with proliferative disorders such as cancer. By promoting cell cycle progression, the cdk regulator molecules according to the invention may be implicated in cancer. Accordingly, activators of cdk proteins such as cdkl and cdk2, which are involved in cell cycle regulation, are likely to be valuable targets for the development of drugs to modulate cell proliferation and differentiation. Furthermore, modulators, in particular inhibitors, of the cdk regulator molecules according to the invention may be useful agents from which anti-cancer drugs that target these cdk regulator molecules may be developed.

Activators of cdks involved in meiosis may enable treatments for male and female infertility to be developed. Furthermore, activators of cdk5 may be useful in the development of treatments for neurodegenerative diseases in which abnormal cdk5 activity is implicated. By a "core region" of a RINGO protein of mammalian origin is meant a protein sequence of between 90 and 1 10 amino acids having at least 40% identity, preferably at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or greater identity, with any one of the regions of the RINGO proteins that are specifically exemplified herein in Figure 6. The proteins that are specifically exemplified are the human proteins Ringol and Ringo2, and the mouse proteins Ringo 3 and Ringo 4.

In the case of Ringol, the core region stretches from amino acid residues 151-252 inclusive. In the case of Ringo2, the core region stretches from amino acid residues 36-138 inclusive. In the case of Ringo3, the core region stretches from amino acid residues 66-168 inclusive. In the case of Ringo4, the core region stretches from amino acid residues 133-234 inclusive. As the skilled reader will be aware, as and when new Ringo proteins are identified and characterised, the core regions from these proteins may also be utilised as cdk regulator molecules according to the invention. By "cdk regulator molecule" is meant a protein that modulates the activity of one or more cyclin-dependent kinases (cdks). Cdks form a family of kinases that are activated by cyclin binding and include proteins such as cdc28, cdc2, p34^cdc2, cdkl , cdk2 and cdk5.

A cdk regulator molecule according to the invention may increase (activate) or decrease (inhibit) the activity of a cdk protein. Preferably, cdk regulator molecules according to the invention have an activating effect.

The regulator molecule may modulate the activity of the cdk protein directly, for example, through interacting physically with the cdk protein, or may act through an intermediary, such as another regulator protein. Alternatively, the regulator molecule may modulate the activity of the cdk protein indirectly, for example, through altering the level of expression of the cdk protein in a cell.

Preferably, the regulator molecules of the invention regulate, more preferably activate, one or more cdks selected from the group consisting of cdkl , cdk2 and cdk5.

The cdk regulator molecules according to the invention may preferably be derived from a mouse or from a human. If from a human, the core region from the Ringo protein preferably comprises the core region of Ringo 1 or the core region of Ringo 2 presented in Figure 6 or may be a functional equivalent thereof. If from a mouse, the core region from the Ringo protein preferably comprises the core region of Ringo 3 or the core region of Ringo 4 presented in Figure 6 or may be a variant thereof. According to a further aspect of the invention, there is provided a cdk regulator molecule consisting of a core region from a RINGO protein, as this is defined above, or a functional equivalent thereof. The core region may be isolated from a RINGO protein of any origin, including eukaryotes such as vertebrates (particularly mammals and fish), invertebrates (such as amphibians and insects) and yeast, and prokaryotes. Specific examples of cdk regulator molecules according to this aspect of the invention include proteins consisting of the core region of Ringo 1 (stretching between amino acid residues 151 and 252 inclusive), the core region of Ringo2 (stretching between amino acid residues 36 and 138 inclusive), the core region of Ringo3 (stretching between amino acid residues 66 and 168 inclusive), the core region of Ringo4 (stretching between amino acid residues 133 and 234 inclusive), and the core region of laevis RINGO (stretching between amino acid residues 60 and 163). The precise boundaries given here are, of course, approximate, in that proteins that have been truncated beyond these boundaries at either or both of the C and N termini of the core region may still be effective as cdk regulators. Furthermore, as and when new Ringo proteins are identified and characterised, the core regions from these proteins may also be utilised as cdk regulator molecules according to the invention.

The cdk regulator molecules of the invention include the complete mammalian Ringo proteins from which the core region is derived. If from a human, the Ringo protein may comprise the Ringol sequence presented in Figure 1 or the Ringo 2 sequence presented in Figure 2. If from a mouse, the Ringo protein may comprise the Ringo 3 sequence presented in Figure 3 or the Ringo 4 sequence presented in Figure 4. Murine homologues of the Ringo 1 sequence presented in Figure 1 and the Ringo 2 sequence presented in Figure 2, and human homologues of the Ringo 3 sequence presented in Figure 3 and the Ringo 4 sequence presented in Figure 4 are also included in the invention.

The cdk regulator molecules of the invention also include active fragments of the complete mammalian Ringo proteins, provided that such active fragments retain a biological activity that is associated with the full length protein. For example, it is considered that certain regions of the Ringo proteins other the core region itself may be involved in cdk modulation. Indeed, the inventors have demonstrated herein that expression of Ringol blocks progesterone-induced oocyte maturation. Overexpressed Ringol was found to bind and activate endogenous cdkl , but the complex appears unable to trigger G2/M progression. Although they do not wish to be bound by any specific theory, the inventors consider that this inhibitory effect may be exerted by any one of the LI and L2 amino acid sequences upstream of the core region and/or the Rl , R2 and R3 amino acid sequences downstream of the core region, as indicated on Figure 6.

According to a further aspect of the invention, there is therefore provided a cdk regulator molecule comprising an active fragment of a Ringo protein or a functional equivalent thereof. The fragments may increase (activate) or decrease (inhibit) the activity of a cdk protein. Preferably, the cdk regulator molecules according to this aspect of the invention have an inhibitory effect. More preferably, the cdk regulator molecules according to this aspect inhibit the activity of cdkl , cdk2 or cdk5. In a preferred embodiment of this aspect, the cdk regulator molecule comprises all or part of any one of the LI and L2 amino acid sequences of Ringol and/or the Rl , R2 and R3 amino acid sequences of Ringol presented in Figure 6.

The term "functional equivalent" is used herein to describe variants, derivatives or fragments of the cdk regulator molecules of the invention that retain the ability to regulate the activity of cdk proteins.

Functional equivalents of the cdk regulator molecules of the invention thus include natural biological variants (e.g. allelic variants or geographical variants within the species from which the cdk regulator molecules are derived). Functionally-equivalent variants of the cdk regulator molecules of the invention also include, for example, mutants containing amino acid substitutions, insertions or deletions from the wild type protein sequences presented herein. Such mutants may include polypeptides in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code. Typical such substitutions are among Ala, Val, Leu and He; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; among the basic residues Lys and Arg; or among the aromatic residues Phe and Tyr. Particularly preferred are variants in which several, i.e. between 5 and 10, 1 and 5, 1 and 3, 1 and 2 or just 1 amino acids are substituted, deleted or added in any combination. Especially preferred are silent substitutions, additions and deletions, which do not alter the properties and activities of the protein. Also especially preferred in this regard are conservative substitutions.

Functionally-equivalent variants with improved function from that of the wild type sequence may also be designed through the systematic or directed mutation of specific residues in the protein sequence. Improvements in function that may be desired will include improvements such as a greater ability to regulate the activity of a cdk, either by increasing or by decreasing the activity or the level of expression of a cdk protein.

The term "functional equivalent" also refers to molecules that are structurally similar to the cdk regulator molecules of the invention or that contain similar or identical tertiary structure. In particular, the term "functional equivalent" refers to molecules that are structurally similar to proteins comprising or consisting of the core region sequences presented in Figure 6 which are responsible for cdk regulation. Such functional equivalents may be derived from the natural cdk regulator molecules or they may be prepared synthetically or using techniques of genetic engineering. In particular, synthetic molecules that are designed to mimic the tertiary structure or active site of the naturally-occurring cdk regulator molecules of the invention are considered to be functional equivalents.

The term "functional equivalent" also refers to homologues of the cdk regulator molecules of the invention. By "homologue" is meant a protein exhibiting a high degree of similarity or identity to the amino acid sequence of the cdk regulator molecules of the invention. By "similarity" is meant that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. By "identity" is meant that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences. Preferably, homologues possess greater than 40% identity with the naturally-occurring cdk regulator molecule sequences. More preferably, homologues according to the invention show greater than 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98% or 99% sequence identity with the sequence of the natural protein. Percentage identity, as referred to herein, is as determined using BLAST version 2.1.3 using the default parameters specified by the NCBI (the National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/) [Blosum 62 matrix; gap open penalty=l l and gap extension penalty=l]. Tools such as PROSITE (http://expasy.hcuge.ch/sprot/prosite.html), PRINTS http://iupab.leeds.ac.uk/bmb5dp/prints.html), Profiles (http://ulrec3.unil.ch/software/ PFSCAN_form.html), Pfam (http://www.sanger.ac.uk/software/pfam), Identify (http://dna.stanford.edu/identify/) and Blocks (http://www.blocks.fhcrc.org) databases may also be used to identify homologues, as well as hidden Markov models (HMMs; preferably profile HMMs). Such homologues may include proteins in which one or more of the amino acid residues are substituted with another amino acid residue. Any such substituted amino acid residue may or may not be a naturally occurring amino acid.

According to a further aspect of the invention, there is provided a fusion protein comprising a cdk regulator molecule of the invention or functional equivalent thereof, as described in any one of the embodiments recited above. The cdk regulator molecule or functional equivalent may be genetically or chemically fused to one or more peptides or polypeptides. Preferably, the cdk regulator molecule or functional equivalent is fused to a marker domain. Preferably, the marker domain is a fluorescent tag, an epitope tag that allows purification by affinity binding, an enzyme tag that allows histochemical or fluorescent labelling, or a radiochemical tag. In a preferred embodiment, the fluorescent tag is a green fluorescent protein (GFP) or a fluorescent derivative thereof such as YFP or CFP (see Prasher et al, (1995), Trends in Genetics, 1 1 (8), 320).

Such fusion proteins will be useful in a variety of methods for establishing the role of the cdk regulator molecules of the invention. For example, they can be used to establish the subcellular location of the cdk regulator molecules of the invention.

Methods for the generation of fusion proteins are standard in the art and will be known to the skilled reader. For example, most general molecular biology, microbiology recombinant DNA technology and immunological techniques can be found in Sambrook et al, (Molecular

Cloning, A Laboratory Manual, Cold Harbor-Laboratory Press, Cold Spring Harbor, N.Y., 2000) or Ausubel et al, (Current Protocols in Molecular Biology, Wiley Interscience, New York, 1991).

Generally, fusion proteins may be most conveniently generated recombinantly from nucleic acid molecules in which two nucleic acid sequences are fused together in frame. These fusion proteins will be encoded by nucleic acid molecules that contain the relevant coding sequence of the fusion protein in question.

The cdk regulator molecules, functional equivalents and fusion proteins of the invention may be prepared in recombinant form by expression in a host cell. Suitable expression methods are well known to those of skill in the art and many are described in detail by Sambrook J. et al Molecular cloning: a laboratory manual New York: Cold Spring Harbour Laboratory Press, 2000) and Fernandez J.M. & Hoeffler J.P. (Gene expression systems. Using nature for the art of expression ed. Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto, 1998.) The proteins and functional equivalents of the present invention can also be prepared using conventional techniques of protein chemistry, for example by chemical synthesis.

According to a further embodiment, the invention provides antibodies that bind to a cdk regulator molecule or functional equivalent, as described above. Antisera and monoclonal antibodies can be made by standard protocols using the cdk regulator molecule or functional equivalent as an immunogen (see, for example, Antibodies: A Laboratory Manual ed. By Harlow and Lane, Cold Spring Harbor Press, 1988). As used herein, the term "antibody" includes fragments of antibodies that also bind specifically to a cdk regulator molecule or functional equivalent thereof. The term "antibody" further includes chimeric and humanised antibody molecules having specificity for the cdk regulator molecules of the invention and for functional equivalents thereof. Antibodies that bind to cdk regulator molecules are useful in a variety of methods for elucidating the function of cdks. For example, they can be used to demonstrate the presence of a cdk regulator molecule bound to cdk. They can also be used to measure the quantity of a cdk regulator molecule in a cell extract. In some cases, it will be desirable to attach a label group to the antibody in order to facilitate detection. Preferably, the label is an enzyme, a radiolabel or a fluorescent tag. The antibodies can also be used to inhibit the binding of a cdk regulator molecule to a cdk, thereby preventing the activator from increasing the activity of that cdk. These antibodies may be useful in methods for elucidating the function of the cdk and may also have medical applications. The invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding a cdk regulator molecule, functional equivalent thereof, or fusion proteins as described above. Specific examples of nucleic acid molecules encoding cdk regulator molecules according to the invention include those nucleic acid sequences presented in Figures 1-5 herein. However, included in this aspect of the invention are nucleic acid molecules that are at least 70% identical, more preferably, at least 80%, 90%, 95%, 98% or 99% identical over their entire length to a nucleic acid molecule encoding a cdk regulator molecule according to any one of the embodiments of the invention described above. Such nucleic acid molecules include single- or double-stranded DNA, cDNA and RNA, as well as synthetic nucleic acid species. Preferably, the nucleic acid molecules are DNA or cDNA molecules. According to one embodiment, the invention provides a nucleic acid molecule including the nucleic acid sequence presented in Figure 5, or a nucleic acid molecule that forms part of a gene identified from this nucleic acid sequence, and which encodes a cdk regulator molecule.

The invention also includes cloning and expression vectors incorporating the nucleic acid molecules. Such expression vectors may additionally incorporate regulatory sequences such as enhancers, promoters, ribosome binding sites and termination signals in the 5' and 3' untranslated regions of genes, that are required to ensure that the coding sequence is properly transcribed and translated, or to regulate the expression of the protein relative to the growth of the cell in which it is expressed. Also, control sequences may be included that encode signal peptides or leader sequences. These leader or control sequences may be removed by the host during post-translational processing. In some cases, the vectors may incorporate signal sequences which direct the expressed proteins to specific subcellular locations. For example, it may be desirable to assess the effect of a cdk regulator molecule in the nucleus, in which case a nuclear localisation signal may be included in the vector. Vectors according to the invention include plasmids and viruses (including both bacteriophage and eukaryotic viruses), as well as other linear or circular DNA carriers, such as those employing transposable elements or homologous recombination technology. Many such vectors and expression systems are known and documented in the art (see, for example, Fernandez J.M. & Hoeffler J.P. in Gene expression systems. Using nature for the art of expression ed. Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto, 1998). Suitable viral vectors include baculovirus-, adenovirus- and vaccinia virus- based vectors.

Suitable hosts for recombinant expression include commonly used prokaryotic species, such as E. coli, or eukaryotic yeasts that can be made to express high levels of recombinant proteins and that can easily be grown in large quantities. Mammalian cell lines grown in vitro are also suitable, particularly when using virus-derived expression systems. Another suitable expression system is the baculovirus expression system that involves the use of insect cells as hosts. An expression system may also constitute host cells that have the appropriate encoding nucleic acid molecules incorporated into their genome. Proteins may also be expressed in vivo, for example in insect larvae or in mammalian tissues.

A variety of techniques may be used to introduce the vectors according to the present invention into prokaryotic or eukaryotic host cells. Suitable transformation or transfection techniques are well described in the literature (see, for example, Sambrook et al, Molecular cloning: a laboratory manual New York: Cold Spring Harbour Laboratory Press, 2000; Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience, New York, 1991 ; Spector, Goldman & Leinwald, Spector et a,l Cells, a laboratory manual; Cold Spring Harbour Laboratory Press, 1998). In eukaryotic cells, expression systems may either be transient (e.g. episomal) or permanent (such as by chromosomal integration) according to the needs of the system.

The invention also provides antisense nucleic acid molecules which hybridise under high stringency hybridisation conditions to the nucleic acid molecules encoding the cdk regulator molecules or functional equivalents thereof. High stringency hybridisation conditions are defined herein as overnight incubation at 42°C in a solution comprising 50% formamide, 5XSSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5xDenhardts solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 X SSC at approximately 65 °C.

In a preferred embodiment of this aspect of the invention, a label capable of being detected is attached to these antisense nucleic acid molecules. Preferably, the label is selected from the group consisting of radioisotopes, fluorescent compounds and enzymes.

These antisense nucleic acid molecules may be used as probes to detect defects in the expression of a cdk regulator molecule. For example, these probes can be used to measure the level of a RNA encoding a cdk regulator molecule in a cell to establish whether there is a defect in the transcription of the gene encoding the cdk regulator molecule. The antisense nucleic acid molecules of the invention may therefore be useful in diagnostic assays. The antisense nucleic acid molecules can also be used in antisense therapy, inhibiting the transcription or translation of a gene encoding a cdk regulator molecule or a functional equivalent thereof by hybridising to mRNA or genomic DNA encoding the cdk regulator molecule. Antisense nucleic acid molecules for use in antisense therapy may be delivered to the cell directly using suitable transfection or transformation techniques known in the literature. Alternatively, an expression vector, such as a plasmid may be delivered to the cell, such that when the nucleic acid in the plasmid is transcribed in the cell, it produces an RNA molecule which is complementary to at least a unique portion of the cellular mRNA which encodes the cdk regulator molecule or a functional equivalent thereof. The ability to inhibit the expression of a cdk regulator molecule will enable researchers to establish the role of the cdk regulator molecule and the cdk to which it binds. Antisense nucleic acid molecules may also have medical applications. The invention also includes transformed or transfected prokaryotic or eukaryotic host cells containing a nucleic acid molecule encoding a cdk regulator molecule or functional equivalent thereof as described above or an antisense nucleic acid molecule which hybridises to such a nucleic acid molecule.

A further aspect of the invention provides a method for preparing a cdk regulator molecule or a functional equivalent thereof or a fusion protein as defined above, which comprises culturing a host cell containing a nucleic acid molecule encoding a cdk regulator molecule or functional equivalent thereof or a fusion protein according to the invention under conditions whereby said protein is expressed and recovering said protein thus produced.

As the skilled reader will appreciate, the identification of the cdk regulator molecules of the present invention paves the way for the development of compounds that modulate the level or activity of these cdk regulator molecules. Although the inventors do not wish to be bound by any specific theory, it is considered that the precise equilibrium of the cdk regulator molecules of the invention in the cell may prove to be of considerable importance in the normal functioning of the cell. Accordingly, molecules that modulate the level or activity of cdk regulator molecules of the invention in a cell, whether they act to upset or to restore the normal balance of cdk regulator molecules in the cell, are therefore likely to be of great diagnostic and therapeutic value. Accordingly, a further aspect of the invention provides a method of identifying a compound that modulates the level or the activity of a cdk regulator molecule of the invention. Such compounds may activate or inhibit either the level of expression of the gene or the activity of the cdk regulator molecule of the invention and form a further aspect of the present invention. As the skilled reader will be aware, a variety of screening techniques may be used to identify such compounds; candidate compounds may be isolated from, for example, cells, cell-free preparations, chemical libraries or natural product mixtures.

The candidate compound may be selected as a result of a screening method for proteins that interact with the cdk regulator molecules or functional equivalents thereof. Such interactions can be detected, for example, using a yeast 2-hybrid screen.

The effect of candidate compounds on the level or the activity of a cdk regulator molecule or functional equivalent thereof can be assessed in a variety of ways.

Alterations in the level of a cdk regulator molecule may be assessed either by looking at the level of the proteins themselves or by looking at the level of the mRNA encoding the cdk regulator protein.

For example, the level of cdk regulator molecule in a cell can be measured by separating a cell extract on a matrix, such as a gel, and then adding an antibody which binds to the cdk regulator molecule. Following removal of unbound antibody, the level of the cdk regulator molecule can be determined by assessing the level of the antibody bound to it. This may be done by using a second labelled antibody which binds to the first antibody. Alternatively, the first antibody may be labelled directly, for example with a fluorescent label, to enable direct detection. This method may be used to compare the level of cdk regulator molecule in a cell before and after incubation with a candidate compound. The level of mRNA encoding the cdk regulator molecule can be assessed, for example, by traditional blotting techniques described in Sambrook et al [supra]. Messenger RNA can be purified and separated using gel electrophoresis. The nucleic acids on the gel are then blotted onto a solid support, such as nitrocellulose. The solid support is exposed to a labelled probe and then washed to remove any unhybridized probe. Next, the duplexes containing the labelled probe are detected. Typically, the probe is labelled with a radioactive moiety. Alternatively, the polymerase chain reaction (PCR) may be used to amplify the amount of mRNA encoding the cdk regulator molecule or functional equivalent thereof. The assay is described in: Mullis et al. [Meth. Enzymol. (1987) 155: 335-350]; US patents 4,683,195 & 4,683,202. After a threshold amount of mRNA is generated by PCR, the level can be detected by Southern blots, as described above. The level of the cdk regulator molecule or functional equivalent thereof in cells contacted with the candidate compound can be compared to the level in cells which have not been contacted with a candidate compound. An alteration in the activity of cdk regulator molecule can also be assessed in a number of ways. Preferably, the activity of the cdk regulator molecule is assessed by measuring its ability to activate the kinase activity of the cdk to which it binds.

Suitable compounds for testing in the methods of the invention may be any one of a number of different compounds. Where the screening method is being conducted in an organism, particularly suitable compounds are small molecules that are suitable for oral delivery, including natural or modified substrates, hormones, small organic molecules, such as small natural or synthetic organic molecules of up to 2000Da, preferably 800Da or less, and inorganic molecules. Other suitable compounds might include polypeptides, such as enzymes, receptors, antibodies, and structural or functional mimetics of these polypeptides, including peptides and peptidomimetics, although these compounds would not be suitable for oral delivery.

As will be apparent to the skilled reader, candidate polypeptide and peptide compounds for testing in the method of the invention may either be natural compounds, isolated from natural sources, or may be synthetic or recombinant.

The invention further provides a compound that is a modulator of a cdk regulator molecule, identified or identifiable by any one of the screening methods mentioned above. It will be understood that modulators of the invention are not limited to those identified by the above methods but include any molecules which increase or decrease the level or activity of a cdk regulator molecule of the invention or a functional equivalent thereof.

Such modulator compounds may alter the level of a cdk regulator molecule or functional equivalent thereof by modulating expression at the level of transcription or translation. For example, a modulator may be a transcription factor which promotes the transcription of a gene encoding a cdk modulator. Alternatively, a modulator compound may be a ribozyme specific for the mRNA sequence of a cdk regulator molecule, which inhibits the expression of the cdk regulator molecules of the invention . Ribozymes are catalytically active RNAs that can be natural or synthetic (see for example Usman, N, et al., Curr. Opin. Struct. Biol (1996) 6(4), 527-33.) Modulator compounds may act to modulate the level of the proteins directly. For example, modulator compounds may include enzymes that digest the cdk regulator molecule, thereby reducing the level of cdk regulator molecule. Modulator compounds may actually target the cdk regulator molecules for degradation by binding to them.

Modulator compounds may alter the activity of a cdk regulator molecule of the invention or a functional equivalent thereof in a number of ways. Preferably, modulator compounds which alter the activity of a cdk regulator molecule may do so by binding to the molecule. Modulator compounds therefore include antibodies, as described previously, which bind to the cdk regulator molecules of the invention and inhibit their activity.

The identification of the core region of RINGO proteins as possessing activity in regulating cdk activity allows a number of physiological processes, particularly cell cycle progression, to be regulated. Such regulation may be effect in vivo, for example, as some form of therapy, or may be effected in vitro, for example, to modulate the activity of cells in tissue culture. According to one aspect of this embodiment of the invention, there is thus provided a method of modulating cell cycle progression comprising providing a cell with a cdk regulator molecule, a functional equivalent thereof or a modulator compound as described above. The cdk regulator molecules, functional equivalents and modulator compounds of the invention may either activate or inhibit cell cycle progression at any stage in the cell cycle. Preferably, the cdk regulator molecules, functional equivalents and modulator compounds of the invention modulate cell cycle progression by modulating G2/M progression. Preferably, cdk regulator molecules, functional equivalents and modulator compounds of the invention which modulate cell cycle progression do so by modulating the activity of cdkl or cdk2.

The ability to modulate cell cycle progression will have a variety of applications, particularly in the medical field. Abnormally high cell cycle progression is often associated with proliferative disorders such as cancer. The cdk regulator molecules of the invention which are involved in promoting cell cycle progression may be implicated in cancer and this method will enable researchers to establish whether cdk regulator molecules result in neoplastic cells. If so, modulators of cdk regulator molecules, in particular inhibitors, may be useful starting points for the development of cancer drugs which target these cdk regulator molecules.

According to a further aspect of the invention, there is provided a method of modulating the rate of meiosis in a germ cell by providing the cell with a cdk regulator molecule, functional equivalent or modulator, as described above. Preferably, the cdk regulator molecules and functional equivalents act by modulating G2/M progression in meiosis. The cdk regulator molecules may either activate or inhibit G2/M progression. Preferably, the cdk regulator molecules and functional equivalents modulate G2/M progression through activating cdkl and cdk2. The cdk regulator molecules and functional equivalents of the invention may modulate the rate of meiosis in male germ cells or in female germ cells or in both.

It has also been discovered that a protein including the core region of Ringo3 may act to increase G2/M progression in female germ cells in the absence of progesterone. According to a specific embodiment of this aspect of the invention, there is thus provided a method of increasing G2/M progression in a female germ cell in the absence of progesterone comprising supplying the cell with a cdk regulator molecule comprising the core region of Ringo3 as this is described herein, or a functional equivalent thereof. It has also been found that a protein including the core region of Ringo2 may act to accelerate progesterone-mediated G2/M progression in a female germ cell. Accordingly, a further specific embodiment of the invention provides a method of accelerating progesterone-mediated G2/M progression in a female germ cell by providing the cell with a cdk regulator molecule comprising or consisting of the core region of Ringo2 or a functional equivalent thereof. It has also been found that a protein including the core region of Ringol may act to inhibit progesterone-mediated G2/M progression in a germ cell, particularly a female germ cell. Accordingly, a further specific embodiment of the invention provides a method of inhibiting G2/M progression in a germ cell comprising providing the cell with a cdk regulator molecule comprising or consisting of the core region of Ringo 1 or a functional equivalent thereof. Similar methods may involve providing a cell with one or more modulator compounds that modulate the activity or level of a cdk regulator molecule according to the invention. Of course, the effect of the modulator on the rate of meiosis will depend on whether the cdk regulator molecule increases or decreases the rate of meiosis and whether the modulator acts to activate or inhibit the cdk regulator molecule. The ability to modulate the rate of meiosis will enable researchers to study the meiotic process in more detail. It may also enable them to establish whether any of the cdk regulator molecules or modulator compounds of the invention will be useful in treatments for male and female infertility.

It has also been found that a protein including the core region of a RINGO protein may have potential in the modulation of neuronal function. According to this aspect of the invention, there is provided a method of modulating neuronal function comprising providing a neuronal cell with a cdk regulator molecule, a functional equivalent thereof or a modulator of the invention. Preferably, the cdk regulator molecule or functional equivalent thereof activates cdk5. This method will enable researchers to establish the role of cdk regulator molecules in neurodegeneration. If cdk regulator molecules are shown to be involved in promoting neurodegeneration via the activation of cdk5, it may be possible to develop drugs based on modulators that inhibit the cdk5 activators for use in the treatment of disorders such as Alzheimer's Disease, ALS and cocaine addiction.

As referred to above, the cdk regulator molecules, functional equivalents and modulators of the invention have a wide variety of potential medical applications. Accordingly, the invention further provides a cdk regulator molecule or a functional equivalent thereof, a nucleic acid molecule encoding said cdk regulator molecule or functional equivalent, an antisense nucleic acid molecule which hybridises to the nucleic acid molecule encoding said cdk regulator molecule or functional equivalent, or a modulator compound, as described above, for use as a pharmaceutical. A further aspect of the invention includes a pharmaceutical composition comprising a cdk regulator molecule or a functional equivalent thereof, a nucleic acid molecule encoding said cdk regulator molecule or functional equivalent, an antisense nucleic acid molecule which hybridises to the nucleic acid molecule encoding said cdk regulator molecule or functional equivalent, or a modulator compound, according to any one of the embodiments of the invention recited above, in conjunction with a pharmaceutically-acceptable carrier molecule.

Carrier molecules may be genes, polypeptides, antibodies, liposomes, polysaccharides, polylactic acids, polyglycolic acids and inactive virus particles or indeed any other agent provided that the carrier does not itself induce toxicity effects or cause the production of antibodies that are harmful to the individual receiving the pharmaceutical composition. Carriers may also include pharmaceutically acceptable salts such as mineral acid salts (for example, hydrochlorides, hydrobromides, phosphates, sulphates) or the salts of organic acids (for example, acetates, propionates, malonates, benzoates). Pharmaceutically acceptable carriers may additionally contain liquids such as water, saline, glycerol, ethanol or auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like. Carriers may enable the pharmaceutical compositions to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions to aid intake by the patient. A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

The amount of the active compound in the composition should also be in a therapeutically- effective amount. The phrase "therapeutically effective amount" used herein refers to the amount of agent needed to treat or ameliorate a targeted disease or condition. An effective initial method to determine a "therapeutically effective amount" may be by carrying out assays in the transgenic organism model, although more accurate tests must be carried out on the target organism if initial tests are successful. The transgenic organism model may also yield relevant information such as the preferred routes of administration that will lead to maximum effectiveness. The exact therapeutically-effective dosage will generally be dependent on the patient's status at the time of administration. Factors that may be taken into consideration when determining dosage include the severity of the disease state in the patient, the general health of the patient, the age, weight, gender, diet, time and frequency of administration, drug combinations, reaction sensitivities and the patient's tolerance or response to the therapy. The precise amount can be determined by routine experimentation but may ultimately lie with the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg (mass of drug compared to mass of patient) to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones.

Uptake of a pharmaceutical composition by a patient may be initiated by a variety of methods including, but not limited to enteral, intra-arterial, intrathecal, intramedullary, intramuscular, intranasal, intraperitoneal, intravaginal, intravenous, intraventricular, oral, rectal (for example, in the form of suppositories), subcutaneous, sublingual, transcutaneous applications (for example, see WO98/20734) or transdermal means. Gene guns or hyposprays may also be used to administer pharmaceutical compositions. Typically, however, the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Direct delivery of the compositions can generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Dosage treatment may be a single dose schedule or a multiple dose schedule. The invention also includes the use of a cdk regulator molecule or a functional equivalent thereof, a nucleic acid molecule encoding said cdk regulator molecule or functional equivalent thereof, an antisense nucleic acid molecule which hybridises to the nucleic acid molecule encoding said cdk regulator molecule or functional equivalent thereof, a modulator or a composition, as described above, in the manufacture of a medicament for treating a disease or a condition associated with aberrant cdk activity.

The cdk regulator molecules and functional equivalents of the invention , nucleic acid molecules encoding these cdk regulator molecules and functional equivalents and modulator compounds that activate the cdk regulator molecules and functional equivalents will be useful in the manufacture of medicaments for treating diseases associated with low cdk activity. In particular, activators of cdkl and cdk2, nucleic molecules encoding such activators and modulators which increase the activity of such activators, may be useful in the development of drugs to treat conditions such as male or female infertility by inducing meiosis in germ cells, as described previously. Antisense nucleic acid molecules and other modulators which inhibit the level or activity of the cdk regulator molecules and functional equivalents of the invention will be useful in the manufacture of medicaments for treating diseases associated with high cdk activity. In particular, cdk regulator molecules which promote cell cycle progression, for example by activating cdkl and cdk2, may be involved in cell proliferation which leads to cancer. Accordingly, antisense molecules and modulator compounds which inhibit the level or activity of the cdk regulator molecules of the invention may be useful in the development of drugs to treat proliferative disorders such as cancer. In addition, the antisense nucleic acid molecules and other modulators which inhibit the ability of the cdk regulator molecules to activate cdk5 may be useful in the manufacture of medicaments to treat conditions such as Alzheimer's Disease, ALS and cocaine addiction in which abnormally high cdk5 levels are implicated.

According to a still further aspect of the invention, there is provided a method of treating a disorder or disease associated with abnormal cdk activity in a patient, comprising administering to the patient a cdk regulator molecule or functional equivalent thereof, a nucleic acid molecule encoding said cdk regulator molecule or functional equivalent, an antisense nucleic acid molecule which hybridises to the nucleic acid molecule encoding said cdk regulator molecule or functional equivalent thereof, a modulator compound, or a composition as described above in a therapeutically-effective amount. Preferred patients are mammals, more preferably humans.

The invention further comprises a method of diagnosing a condition associated with an abnormal level or activity of a cdk regulator molecule of the invention comprising measuring the level of a cdk regulator molecule of the invention in a cell sample obtained from a patient. Preferably, the level of cdk regulator molecule in the sample is measured using antibodies against the cdk regulator molecule, as described previously. The level of the cdk regulator molecule can also be measured indirectly by assaying the activity or levels of a binding partner of the cdk regulator molecule. Antibodies to the cdk regulator proteins of the invention have been found to co-immunoprecipitate binding partners, such as cdks, in mammalian tissues. The level of the cdk regulator in a cell sample may therefore be measured by measuring the level of activity of these cross-reacting binding partner proteins. For example, the level of the cdk regulator molecule may be measured by measuring the HI kinase activity of cdks in the immunoprecipitate.

The level of the cdk regulator molecule in the sample can then be compared to a standard. For example, a low level of a cdk regulator molecule in germ cells may be a diagnostic marker for infertility. Conversely, a high level of cdk 1 or cdk 2 activator in other cells may be a marker for cell proliferation and cancer. A high level of cdk5 activator in brain cells may be a marker of a neurodegenerative disease such as Alzheimer's Disease.

In another embodiment of the invention, a nucleic acid molecule as described above may be used to create a transgenic animal, most commonly a rodent. The modification of the animal's genome may either be done locally, by modification of somatic cells or by germ line therapy to incorporate inheritable modifications. Such transgenic animals may be particularly useful in the generation of animal models for screening for drug molecules that are effective as modulators of the cdk regulator molecules of the invention.

Various aspects and embodiments of the present invention will now be described in more detail by way of example. It will be appreciated that modification of detail may be made without departing from the scope of the invention.

Brief description of the Figures:

Figure 1 : human Ringo 1

Figure 2: human Ringo 2 Figure 3: mouse Ringo 3

Figure 4: mouse Ringo 4

Figure 5: human cDNA clone (BG22374) identified as having 89% identity with cDNA encoding mouse Ringo 3

Figure 6: The amino acid sequences of the core region of Xenopus Ringo, Ringo 1 , Ringo2, Ringo and Ringo 4. The percentage identity of the core region sequences of Ringo 1 , Ringo 2, Ringo 3 and Ringo 4 with the core region sequence of Xenopus Ringo is indicated.

Figure 7: Xenopus Ringo, Ringo 1 , Ringo 2 and Ringo 3 bind cdkl in vitro.

Figure 8: Xenopus Ringo, Ringo 1 , Ringo 2 and Ringo 3 activate cdk2 in vitro.

Figure 9: Effect of mammalian Ringo proteins on Xenopus oocyte maturation A: Ringo 3 induces oocyte maturation and Ringo 2 accelerates progesterone-induced oocyte maturation

B: Ringo 1 inhibits progesterone-induced oocyte maturation

Figure 10: Ringo 1 binds and activates cdkl in oocytes Figure 1 1 : Overexpression of cdk 1 rescues the inhibition of oocyte maturation by Ringo 1

Figure 12: Expression of Ringo 1 in human tissues

Figure 13: Detection of histone HI kinase activiy and cdk protein in anti-Ringo immunoprecipitates from mouse tissues

Figure 14: Xenopus Ringo core region binds and activates cdkl in oocytes Figure 15: Amino acid sequence alignement of Xenopus Ringo, Ringo 1 , Ringo 2, Ringo 3 and Ringo 4.

Figure 16: Activation of cdk5 in vitro by Xenopus Ringo, Ringo 2 and Ringo 3.

Examples

We have now identified four RINGO-related mammalian cDNAs, which can also bind and activate Cdkl and Cdk2 in vitro and in Xenopus oocytes. We have named these genes Ringol (Figure 1), Ringo2 (Figure 2), Ringo3 (Figure 3) and Ringo4 (Figure 4). Ringol and Ringo2 are of human origin, while Ringo3 and Ringo4 are from the mouse. We have also identified in the databases human sequences (ESTs) that are about 90% identical to mouse Ringo3 (Figure 5). The similarity between the mammalian and Xenopus RINGO proteins is especially strong in one region of about 120 amino acids, which we have named the RINGO "core". In this region the amino acid sequences are between 44 and 63% identical (see Figure 6).

We have characterised 3 of these mammalian RINGO cDNAs. Human Ringol and human Ringo2 were provided by EST suppliers; the EST corresponding to Ringol was truncated at the N-terminus and we assembled the full length sequence using genomic sequences from the databases. We cloned mouse Ringo3 by RT-PCR using specific PCR primers and mouse testis mRNA.

First, we confirmed that the three mammalian RINGO proteins bind and activate Cdc2 and

Cdk2 in vitro, as the Xenopus RINGO protein does. For these experiments, the Ringol cDNA was cloned in the bacterial expression vector pMalc2, downstream of the malE gene. The

Ringo2 and Ringo3 were cloned in the bacterial expression vector pGEX-KG downstream of the GST gene. The fusion proteins malE-Ringol , GST-Ringo2 and GST-Ringo3 were expressed in E.Coli and purified by affinity chromatography.

To address whether these proteins can directly associate with Cdkl , we performed pull-down experiments. For this purpose, extracts prepared from insect cells infected with Cdkl- expressing baculovirus were incubated with either malE-Ringol , GST-Ringo2 or GST-Ringo3 and as control GST or malE alone all bound to the corresponding beads. After extensive washing, the proteins that remained bound to the beads were analysed by immunoblotting with anti-Cdkl antibodies. We found that the three mammalian RINGO proteins bound to Cdkl in this assay (Figure 7). In a parallel series of experiments, the three bacterially-produced mammalian RINGO proteins were incubated with purified recombinant GST-Cdk2 and its kinase activity assayed using histone HI as in vitro substrate. We found that the three purified mammalian RINGO proteins were able to increase the histone HI kinase activity of Cdk2, in the absence of any additional factors (Figure 8). We also investigated the ability of the three mammalian RINGO proteins to regulate the meiotic G2/M progression of the cell cycle when expressed in Xenopus oocytes. For these experiments, the three cDNAs were cloned into the FTX5 expression vector and the resulting constructs were transcribed in vitro to obtain mRNAs, which were injected into stage VI oocytes. Expression of Ringo3 was able to induce oocyte maturation in the absence of progesterone stimulation (Figure 9A).

Expression of Ringo2 was not able to induce oocyte maturation on its own, but when these Ringo2-expressing oocytes were incubated with progresterone, they enter meiotic M-phase much faster than normal oocytes (injected with water instead of Ringo2 mRNA). Thus, Ringo2 also has also a positive (albeit weaker) effect and strongly accelerates progesterone-oocyte maturation (Figure 9A).

Surprisingly, expression of Ringol not only was unable to induce oocyte maturation but it had the opposite effect and completely blocked oocyte maturation induced by progesterone (Figure 9B). We confirmed that the overexpressed Ringol was able both to bind the endogenous Cdkl and to activate its kinase activity on histone HI (Figure 10), but the oocytes remained arrested in G2 phase of the cell cycle. Interestingly, overexpression in the oocytes of Cdkl was able to rescue the inhibition of progesterone-induced oocyte maturation by Ringol (Figure 1 1). This demonstrates that Ringol can interact with Cdkl in the oocytes, but the complex is somehow unable to trigger G2/M progression.

By Northern blot hybridisation, we detected high level expression of the Ringol mRNA in human testis, but we did not detect expression in eight other human tissues including ovary, spleen, thymus and prostate (Figure 12). This suggests that Ringol may function in male meiosis. Analysis of the expression pattern of mammalian Ringo2 and Ringo3 is in progress.

We also used an antibody raised against the "core" of the Xenopus RINGO protein to detect cross-reacting proteins in mammalian tissues. We detected proteins that were recognised by anti-PSTAIR antibodies (raised against a peptide sequence that is highly conserved in Cdk proteins) in anti-RINGO immunoprecipitates prepared from several mouse tissues. Some of these RDNGO-associated proteins probably correspond to Cdkl and/or Cdk2, depending on the tissues (Figure 13). Accordingly, we could measure histone HI kinase activity associated with the RINGO immunoprecipitates.

We also found that the "core" of the Xenopus RINGO protein fused to GST can bind and activate Cdkl in the oocytes (Figure 14), indicating that this region, that is highly conserved between all RINGO proteins, is likely to be important for the regulation of Cdk activity by RINGO.

We also found that the mammalian proteins can directly activate cdk5 in vitro (see Figure 16).

Claims

1. A cdk regulator molecule comprising a core region from a RINGO protein of mammalian origin, or a functional equivalent thereof.

2. A cdk regulator molecule according to claim 1 wherein the Ringo protein is derived from a human.

3. A cdk regulator molecule according to claim 2 comprising the core region amino acid sequence of human Ringo 1 presented in Figure 6 or a functional equivalent thereof.

4. A cdk regulator molecule according to claim 3 comprising the amino acid sequence of human Ringol presented in Figure 1 or a functional equivalent thereof.

5. A cdk regulator molecule according to claim 2 comprising the core region amino acid sequence of human Ringo 2 presented in Figure 6 or a functional equivalent thereof.

6. A cdk regulator molecule according to claim 5 comprising the amino acid sequence of of human Ringo 2 presented in Figure 2 or a functional equivalent thereof.

7. A cdk regulator molecule according to claim 1, wherein the Ringo protein is derived from a mouse.

8. A cdk regulator molecule according to claim 7 comprising the core region amino acid sequence of Ringo 3 presented in Figure 6 or a functional equivalent thereof.

9. A cdk regulator molecule according to claim 8 comprising the amino acid sequence presented in Figure 3 or a functional equivalent thereof.

10. A cdk regulator molecule according to claim 7 comprising the core region amino acid sequence of Ringo 4 presented in Figure 6 or a functional equivalent thereof.

11. A cdk regulator molecule according to claim 10 comprising the amino acid sequence of Ringo4 presented of Figure 4 or a variant thereof.

12. A cdk regulator molecule that consists of the core region amino acid sequence of a RINGO protein.

13. A cdk regulator molecule according to claim 12, which consists of the core region of Ringo 1 (stretching between amino acid residues 151 and 252 inclusive), the core region of Ringo2 (stretching between amino acid residues 36 and 138 inclusive), the core region of Ringo3 (stretching between amino acid residues 66 and 168 inclusive), the core region of Ringo4 (stretching between amino acid residues 133 and 234 inclusive), or the core region of X laevis RINGO (stretching between amino acid residues 60 and 163)

14. A cdk regulator molecule or functional equivalent according to any one of the preceding claims which is a recombinant protein.

15. An antibody which binds to a cdk regulator molecule or functional equivalent according to any one of claims 1 to 14.

16. A fusion protein comprising a cdk regulator molecule or functional equivalent thereof according to any one of claims 1 to 14 that is genetically or chemically fused to one or more peptides or polypeptides.

17. A fusion protein according to claim 16 wherein the cdk regulator molecule or functional equivalent thereof is fused to a marker domain.

18. A fusion protein according to claim 17 wherein said marker domain is a fluorescent tag, an epitope tag that allows purification by affinity binding, an enzyme tag that allows histochemical or fluorescent labelling, or a radiochemical tag.

19. A fusion protein according to claim 18 wherein said fluorescent tag is a green fluorescent protein (GFP) or a fluorescent derivative thereof.

20. A nucleic acid molecule encoding a cdk regulator molecule or functional equivalent thereof, or a fusion protein according to any one of claims 1 to 14 or claims 16 to 19.

21. An antisense nucleic acid molecule that hybridises under conditions of high stringency to a nucleic acid molecule according to claim 20.

22. A vector comprising a nucleic acid molecule according to claim 20 or claim 21.

23. A host cell transformed or transfected with a nucleic acid molecule of claim 20 or claim 21 or a vector of claim 22.

24. A method of preparing a cdk regulator molecule or functional equivalent thereof according to any one of claims 1 -14 or a fusion protein according to any one of claims 16-19, comprising introducing a vector comprising a nucleic acid molecule according to claim 23 into a host cell and culturing said host cell under conditions wherein said cdk regulator molecule or functional equivalent or fusion protein is expressed and recovering said cdk regulator molecule or functional equivalent or fusion protein.

25. A method of identifying compounds that modulate the level or activity of a cdk regulator molecule or functional equivalent thereof according to any one of claims 1-14 comprising supplying a cell with a candidate compound and assessing the effect of said compound on the level or the activity of a cdk regulator molecule or a functional equivalent thereof.

26. A compound that is a modulator of a cdk regulator molecule or a functional equivalent thereof identifiable by a method according to claim 25.

27. A modulator compound according to claim 26 which is an antibody as recited in claim 15.

28. A method of modulating cell cycle progression comprising providing a cell with a cdk regulator molecule or a functional equivalent thereof according to any one of claims 1 -14 or a modulator according to claim 26 or claim 27.

29. A method of modulating the rate of meiosis of a germ cell by providing the cell with a cdk regulator molecule or functional equivalent according to any one of claims 1-14 or a modulator according to claim 26 or claim 27.

30. A method of modulating neuronal function comprising providing a neuronal cell with a cdk regulator molecule or functional equivalent according to any one of claims 1-14 or a modulator according to claim 26 or claim 27.

31. A cdk regulator molecule or a functional equivalent thereof according to any one of claims 1 -14, a nucleic acid molecule according to claim 20, an antisense nucleic acid molecule according to claim 21 or a modulator according to claim 26 or claim 27, for use as a pharmaceutical.

32. A pharmaceutical composition comprising a cdk regulator molecule or a functional equivalent thereof according to any one of claims 1 -14, a nucleic acid molecule according to claim 20, an antisense nucleic acid molecule according to claim 21 or a modulator compound according to claim 26 or claim 27.

33. Use of a cdk regulator molecule or a functional equivalent thereof according to any one of claims 1 -14, a nucleic acid molecule according to claim 20, an antisense nucleic acid molecule according to claim 21 , a modulator compound according to claim 26 or claim 27 or a composition according to claim 32 in the manufacture of a medicament for the treatment a disease or a condition associated with abnormal cdk activity.

34. A method of treating a disorder or disease associated with abnormal cdk activity in a patient, comprising administering to the patient a cdk regulator molecule or a functional equivalent thereof according to any one of claims 1 -14, a nucleic acid molecule according to claim 20, an antisense nucleic acid molecule according to claim 21 , a modulator compound according to claim 26 or claim 27 or a composition according to claim 32.

35. Use according to claim 33 or a method according to claim 34 wherein said disease or condition is a proliferative condition.

36. Use or a method according to claim 35 wherein said proliferative condition is cancer.

37. Use according to claim 33 or a method according to claim 34 wherein said disease or 5 condition is male or female infertility.

38. Use according to claim 33 or a method according to claim 34 wherein said disease or condition is a neurodegenerative condition.

39. Use or method according to claim 38 wherein said neurodegenerative condition is selected from Alzheimer's Disease, ALS and cocaine addiction.

10 40. A method of diagnosing a physiological condition associated with an abnormal level of cdk regulator molecule or a functional equivalent thereof according to any one of claims 1-14 comprising measuring the level of a cdk regulator molecule in a cell sample.

41. A transgenic animal comprising a cdk regulator molecule according to any one of claims 1- 14 or a fusion protein according to any one of claims 16-19.