WO2001011042A1 - Dapp1, adaptateur double pour phosphotyrosine et 3-phosphoinositides - Google Patents

Dapp1, adaptateur double pour phosphotyrosine et 3-phosphoinositides Download PDF

Info

Publication number
WO2001011042A1
WO2001011042A1 PCT/GB2000/002599 GB0002599W WO0111042A1 WO 2001011042 A1 WO2001011042 A1 WO 2001011042A1 GB 0002599 W GB0002599 W GB 0002599W WO 0111042 A1 WO0111042 A1 WO 0111042A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
fragment
variant
fusion
derivative
Prior art date
Application number
PCT/GB2000/002599
Other languages
English (en)
Inventor
Dario Renato Alessi
Simon Dowler
Richard Currie
Original Assignee
Medical Research Council
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Medical Research Council filed Critical Medical Research Council
Priority to AU28070/01A priority Critical patent/AU2807001A/en
Publication of WO2001011042A1 publication Critical patent/WO2001011042A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to related polypeptides, polynucleotides and uses thereof, in particular to members of the PH and SH2 domain protein families.
  • PI 3-kinases members of the phosphoinositide 3-kinase family which phosphorylate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) at the D-3 position of the inositol ring to generate the lipid second messenger, PtdIns(3,4,5)P 3 [3].
  • PtdIns(3,4,5)P 3 [4] and its immediate breakdown product, PtdIns(3,4)P 2 which is also thought to be a signalling lipid [3], leading to the recruitment of these molecules to the plasma membrane and the activation of signal transduction pathways that regulate cell growth, proliferation, survival, differentiation and cytoskeletal changes ( ameh, L. E. and Cantley, L. C. (1999) The role of phosphoinositide 3- kinase lipid products in cell function. J. Biol. Chem. 274, 8347-50).
  • Proteins possessing such PH domains include the serine/threonine protein kinases, PKB [5] and PDK1 [6], the Bruton's tyrosine kinase BTK [7], the Rho/Rac GTP exchange factor VAV [8], the adaptor protein Gabl (Rodrigues, G. A., Falasca, M., Zhang, Z., Ong, S. H. and Schlessinger, J. (2000) A novel positive feedback loop mediated by the docking protein Gabl and phosphatidylinositol 3-kinase in epidermal growth factor receptor signaling. Mol. Cell Biol. 20, 1448-59) and the ARF GTP exchange factor GRP1 [9].
  • PtdIns(3,4,5)P3 Interaction of PtdIns(3,4,5)P3 with PKB, BTK, Gabl and VAV not only causes their translocation to the membrane of cells, but also induces a conformational change which results in them becoming phosphorylated and hence activated by specific upstream protein kinases.
  • PKB is phosphorylated and activated by PDK1 [10]
  • BTK [6] and VAV [7] are phosphorylated and activated by members of the Src-family of tyrosine kinases
  • Gabl is phosphorylated by receptor tyrosine kinases (Rodrigues et al (2000)).
  • DAPP1 Dual Adaptor for Phosphotyrosine and 3-Phosphoinositides
  • DAPP1 may interact with both tyrosine phosphorylated proteins and 3-phosphoinositides and may therefore play a role in regulating the location and/or activity of such proteins(s) in response to agonists that elevate PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 .
  • DAPP1 becomes tyrosine phosphorylated in response to insulin and growth factors and this phosphorylation is prevented by inhibitors of PI 3-kinase.
  • DAPPl may play a role in regulating signal transduction pathways downstream from PI 3- kinase and receptor tyrosine kinases. Modulators of DAPPl phosphorylation and/or interactions may be useful in the treatment of conditions including cancer, diabetes, stroke, immune disorders and inflammation.
  • a first aspect of the invention provides a substantially pure polypeptide comprising the amino acid sequence
  • the polypeptides whose amino acid sequences are shown above are considered to comprise a SH2 domain and a PH domain.
  • the polypeptides may interact with tyrosine-phosphorylated proteins and/or with 3-phosphoinositides.
  • the polypeptides with the amino acid sequence as shown above are herein referred to as human Dual Adaptor for Phosphotyrosine and 3- Phosphoinositide (hDAPPl; the first sequence shown) or mouse Dual Adaptor for Phosphotyrosine and 3-Phosphoinositide (mDAPPl; the second sequence shown).
  • the amino acid sequence of human DAPPl is also shown in Figure 1 and Figure 2.
  • the mouse DAPPl is a variant of the human DAPPl and is also shown in Figure 1.
  • Standard IUPAC one and three letter codes are used for amino acid sequences used in the specification, and the amino acid sequences are listed N-terminal to C-terminal as is conventional.
  • substantially pure we mean that the said polypeptide is substantially free of other proteins.
  • any composition that includes at least 30% of the protein content by weight as the said polypeptide, preferably at least 50%, more preferably at least 70%, still more preferably at least 90% and most preferably at least 95% of the protein content is the said polypeptide.
  • the invention also includes compositions comprising the said polypeptide and a contaminant wherein the contaminant comprises less than 70% of the composition by weight, preferably less than 50% of the composition, more preferably less than 30% of the composition, still more preferably less than 10% of the composition and most preferably less than 5% of the composition by weight.
  • the invention also includes the substantially pure said polypeptide when combined with other components ex vivo, said other components not being all of the components found in the cell in which said polypeptide is found.
  • the polypeptides of the invention can be produced using recombinant DNA technology.
  • Variants may be made using the methods of protein engineering and site-directed mutagenesis well known in the art using the recombinant polynucleotides described below.
  • fragment of said polypeptide we include any fragment which retains activity or which is useful in some other way, for example, for use in raising antibodies or in a binding or other assay, or which fragment may have other functions as described in more detail below. Preferred fragments of DAPPl are discussed further below.
  • fusion of said polypeptide we include said polypeptide fused to any other polypeptide.
  • the said polypeptide may be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A in order to facilitate purification of said polypeptide. Examples of such fusions are well known to those skilled in the art. Further examples are also described in Example 1.
  • the said polypeptide may be fused to an oligo-histidine tag such as His6 or to an epitope recognised by an antibody such as the well known Myc tag epitope or the FLAG (DYKDDDDK) epitope, as described in Example 1. Fusions to any variant, fragment or derivative of said polypeptide are also included in the scope of the invention.
  • fusions or variants, fragments, derivatives or fusions thereof which retain desirable properties, such as binding properties (for example, the ability to bind to a tyrosine-phosphorylated protein or to a 3- phosphoinositide) or the ability to be phosphorylated on a tyrosine residue in response to insulin or growth factor signalling (in an intact cell) or other biological functions, of DAPPl are particularly preferred. It is also particularly preferred if the fusions are one which are suitable for use in the screening assays described later.
  • variants of the polypeptide we include insertions, deletions and substitutions, either conservative or non-conservative. In particular we include variants of the polypeptide where such changes do not substantially alter the activity, for example the binding activity (for example to a tyrosine- phosphorylated protein or to a 3-phosphoinositide) of the said polypeptide.
  • variants of DAPPl do not include polypeptides which have the amino acid sequence of known polypeptides comprising a SH2 and/or PH domain. It will be appreciated that the protein termed APS [20-22] is not a variant of DAPPl.
  • Plecktrin Homology (PH) domain is well known to those skilled in the art. These domains of " 100 residues are found in over 70 other proteins and are predicted to fold into a similar 3-dimensional structures and may mediate protein-lipid, protein-protein interactions, or both (Gibson, T . et al (1994) Trends Biochem. Sci. 19, 349-353; Shaw, G. (1996) Bioessays 18, 35- 46). Polypeptides with PH domains of determined tertiary sructure include plecktrin, spectrin, dynamin, and phospholipase C- ⁇ . Although the percentage identity is poor between PH domains in general there are certain positions that show high levels of residue type conservation.
  • SH2 domain is similarly well known to those skilled in the art.
  • Polypeptides comprising a SH2 domain include the following (with NCBI accession numbers): Src (P12931); PLC- ⁇ l (P19174); PI 3-kinase p85 ⁇ subunit (P27986); PTP-2C (Q06124); APS (BAA 22514); Ras GAP (P20936).
  • Src Src
  • PLC- ⁇ l P19174
  • PI 3-kinase p85 ⁇ subunit P27986)
  • PTP-2C Q06124
  • APS BAA 22514
  • Ras GAP Ras GAP
  • substantially all is meant at least 80%, preferably 90%, still more preferably 95%, 98% or 100% (ie all) of the said sequence.
  • substantially full-length is meant comprising at least 80%, preferably 90%, still more preferably 95%, 98% or 100% (ie all) of the sequence of the full length polypeptide.
  • polypeptide variant has an amino acid sequence which has at least 65% identity with either amino acid sequence given above, more preferably at least 75%, still more preferably at least 90%, yet more preferably at least 95%, and most preferably at least 98% or 99% identity with either amino acid sequence given above, most preferably with the amino acid sequence given above for human DAPPl .
  • the percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequences have been aligned optimally.
  • the alignment may alternatively be carried out using the Clustal W program (Thompson, J.D., Higgins, D.G. and Gibson, T . (1994), Clustal-W - improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nuc. Acid Res. 22, 4673-4680).
  • the parameters used may be as follows:
  • Fast pairwise alignment parameters K-tuple(word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent.
  • a particular embodiment of the invention provides a substantially pure human DAPPl polypeptide which consists of the amino acid sequence MGRAELLEGKMSTQDPSDL SRSDGEAELLQDLG YHGNLTRHAAEALLLSNG CDGSYLLRDSNETTGLYSLSVRAKDSVKHFHVEYTGYSFKFGFNEFSSLKDFV KHFANQPLIGSETGTLMVLKHPYPRKVEEPSIYESVRVHTAMQTGRTEDDLVP TAPSLGTKEGYLTKQGGLVKTWKTRWFTLHRNELKYFKDQMSPEPIRILDLTE CSAVQFDYSQERVNCFCLVFPFRTFYLCAKTGVEADE IKILRWKLSQIRKQL NQGEGTIRSRSFIFK or naturally occurring allelic variants thereof.
  • a further particular embodiment of the invention provides a substantially pure mouse DAPPl polypeptide which consists of the amino acid sequence MGRAELLGGNMSTQDPSELWGRADGGTDLLQDLG YHGNLTRHAAEALLLSNG RDGSYLLRDSNEQTGLYSLSVRAKDSVKHFHVEYTGYSFKFGFNEYSSLKDFV KHFANQPLIGSETGTLMVLKHPYPREVEEPCIYESVRVHTAMQTGRTENDLVP TAPSLGTKEGYLTKQGGLVKT KTRWFTLQRNELKYFKDQMSPEPIRILDLTE CSAVQFDYSQERVNCFCLVFPFRTFYLCAKTGVEADEWIKILRWKLSKIRKQL DQGEDTVRSRSFIFK or naturally occurring allelic variants thereof.
  • a preferred fragment of the polypeptide of the invention comprises the amino acid sequence of amino acids 1 to 166 of either of the given amino acid sequences, preferably of the given amino acid sequence for human DAPPl. This fragment comprises the SH2 domain of DAPPl . It is further preferred that the fragment does not comprise the amino acid sequence of amino acids 167 to 280 of the given amino acid sequence. This fragment comprises the SH2 domain of DAPPl and does not comprise the PH domain of DAPPl.
  • a further preferred fragment of the polypeptide of the invention comprises the amino acid sequence of amino acids 125 to 280 of either of the given amino acid sequences, preferably of the given amino acid sequence for human DAPPl. This fragment comprises the PH domain of DAPPl. It is further preferred that the fragment does not comprise the amino acid sequence of amino acids 1 to 124 of the given amino acid sequence. This fragment comprises the PH domain of DAPPl and does not comprise the SH2 domain of DAPPl.
  • Preferred fusions of these fragments include fusions as described in Example 1 , for example fusions in which the said fragment has an N-terminal GST tag followed by a FLAG (DYKDDDDK) epitope tag fused to the N-terminus of the said fragment.
  • a preferred variant of the polypeptide or fragment of the invention is a variant or fragment wherein the residue equivalent to Lys 173 of the given amino acid sequence (preferably the given amino acid sequence of human DAPPl) is mutated, for example to a leucine residue, and/or wherein the residue equivalent to Trp250 of the given amino acid sequence is mutated, for example to a leucine residue and/or wherein the residue equivalent to Arg61 is mutated, for example to a glutamate residue and/or wherein the residue equivalent to to Tyrl39 is mutated, for example to a phenylalanine or acidic (for example glutamate or aspartate) residue and/or wherein the residue equivalent to Gly2 is mutated, for example to an alanine residue.
  • the residue equivalent to Lys 173 of the given amino acid sequence preferably the given amino acid sequence of human DAPPl
  • the residue equivalent to Trp250 of the given amino acid sequence is mutated, for example to a leucine residue and/or wherein
  • mutation of the said residue equivalent to Lysl73 and/or the residue equivalent to Trp250 may prevent or reduce the interaction between the DAPPl polypeptide and a 3-phosphoinositide, as described in Example 1. It will further be appreciated that mutation of a different residue that is conserved or invariant in PH domains may similarly prevent or reduce the interaction between the DAPPl polypeptide and a 3-phosphoinositide.
  • mutation of the said residue equivalent to Arg61 may prevent or reduce the interaction between the DAPPl polypeptide and a tyrosine phosphorylated protein. It will further be appreciated that mutation of a different residue that is conserved or invariant in SH2 domains may similarly prevent or reduce the interaction between the DAPPl polypeptide and a tyrosine phosphorylated protein.
  • Mutation of the said residue equivalent to Tyrl39 to phenylalanine may prevent phosphorylation of the DAPPl polypeptide on the residue equivalent to Tyrl39, as discussed in Examples 2 and 4.
  • Mutation of the said residue to an acidic residue, for example glutamate or aspartate may have a similar effect (ie may mimic) the phosphorylation of a DAPPl polypeptide on a tyrosine residue equivalent to Tyrl39 of the given amino acid sequence.
  • Mutation of the said residue equivalent to Gly2 may prevent or reduce any myristoylation of the DAPPl polypeptide.
  • fragments and variants may be useful in screening assays, medicine and/or in investigating the involvement of DAPPl in normal and diseased cells.
  • a fragment of DAPPl comprising the PH domain but not the SH2 domain or a fragment of DAPPl comprising the SH2 domain but not the PH domain may be capable of acting as an inhibitor, for example a dominant-negative inhibitor, of signalling via a signalling pathway in which DAPPl may be involved, as discussed further below, for example signalling via a growth factor receptor.
  • a fragment may be useful, for example, as an anti-cancer agent or in the promotion of apoptosis. Promotion of apoptosis may be beneficial in the resolution of inflammation.
  • a variant of DAPPl in which the residue equivalent to Tyrl39 is mutated to an acidic residue, for example an aspartate or glutamate residue may be useful in increasing signalling via a signalling pathway in which DAPPl may be involved, as discussed further below, for example signalling via the insulin receptor.
  • a variant may be useful, for example, in the treatment of diabetes.
  • over-expression of a substantially full- length native DAPPl polypeptide may also be useful in increasing signalling in which DAPPl is involved and therefore may also be useful in the treatment of diabetes. It may also be useful in reducing apoptosis; thus, it may be useful in treating a patient in need of protection against apoptosis. Reducing apoptosis may be useful following ischaemic injury, for example stroke or myocardial infarction, and in tissue repair. It may also be useful in the treatment of patient before, after or during heart surgery.
  • a fusion of a polypeptide, variant or fragment of the invention wherein the fusion comprises a GST and/or FLAG epitope portion may be particularly useful.
  • a GST tag may be useful in purifying or detecting the fusion protein, as described in Example 1, for example in detecting the interaction between the fusion protein and a phospholipid.
  • the variant or fragment or derivative or fusion of the said polypeptide, or the fusion of the variant or fragment or derivative has at least 30% of the tyrosine-phosphorylated protein binding affinity and/or at least 30% of the 3-phosphoinositide (for example PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 ) binding activity of DAPPl.
  • variant or fragment or derivative or fusion of the said polypeptide, or the fusion of the variant or fragment or derivative has at least 50%, preferably at least 70% and more preferably at least 90% of the tyrosine-phosphorylated protein binding affinity and/or at least 50%, preferably at least 70% and more preferably at least 90% of the 3- phosphoinositide binding activity of DAPPl .
  • variants or fusions or derivatives or fragments which are devoid of one or more binding activities as set out above may nevertheless be useful, for example as described above or by interacting with another polypeptide, or as antigens in raising antibodies.
  • Methods of measuring the binding affinity with 3-phosphoinositides are described, for example, in Example 1 below. Methods of measuring protein-protein interactions are well known to those skilled in the art and are discussed further below.
  • residue equivalent to a particular residue, for example the residue Lysl73 of full-length human DAPPl
  • amino acid residue occupies a position in the native two or three dimensional structure of a polypeptide equivalent to the position occupied by the said particular residue, for example Lysl73, in the native two or three dimensional structure of full-length human DAPPl .
  • the residue equivalent to a particular residue may be identified by alignment of the sequence of the polypeptide with that of full-length human DAPPl in such a way as to maximise the match between the sequences.
  • the alignment may be carried out by visual inspection and/or by the use of suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group, which will also allow the percent identity of the polypeptides to be calculated, or using the Align program (Pearson (1994) in: Methods in Molecular Biology, Computer Analysis of Sequence Data, Part II (Griffin, AM and Griffin, HG eds) pp 365-389, Humana Press, Clifton).
  • residues identified in this manner are also "equivalent residues”.
  • a further aspect of the invention provides a polynucleotide, preferably a recombinant polynucleotide, encoding the polypeptide of the invention or encoding a variant, fragment, derivative or fusion thereof or fusion of a variant, fragment or derivative thereof.
  • Preferences and exclusions for the said polynucleotide variant are the same as in the first aspect of the invention, except that a polynucleotide encoding a fragment consisting of an amino acid sequence present in the C-terminal 156 residues of the given amino acid sequence for human DAPPl or in the C-terminal 160 residues of the given amino acid sequence for mouse DAPPl is excluded.
  • ESTs Expressed Sequence Tags
  • a further aspect of the invention provides a recombinant polynucleotide suitable for expressing a polypeptide as defined in the first aspect of the invention or suitable for expressing a variant or fragment or derivative of fusion of said polypeptide or a fusion of a said variant or fragment or derivative.
  • Preferences and exclusions for the said polynucleotide variant are preferences for, and exclusions of, polynucleotides which encode the same polypeptides as in the first aspect of the invention.
  • the polynucleotide is a polynucleotide that may be translated to form the polypeptide, for example RNA, or that the polynucleotide (which is preferably DNA) encoding the polypeptide of the invention is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression.
  • an expression vector such as a plasmid
  • the polynucleotide may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by any desired host; such controls may be incorporated in the expression vector.
  • a further aspect of the invention is a replicable vector suitable for expressing a polypeptide as defined in the first aspect of the invention or suitable for expressing a variant or fragment or derivative of fusion of said polypeptide or a fusion of a said variant or fragment or derivative.
  • Preferences and exclusions for the said polynucleotide variant are preferences for, and exclusions of, polynucleotides which encode the same polypeptides as in the first aspect of the invention.
  • the replicable vector may be suitable for expressing a fusion of the polypeptide as defined in the first aspect of the invention, in particular a GST fusion.
  • a further aspect of the invention is a polynucleotide encoding a fusion of the polypeptide as defined in the first aspect of the invention, or a fusion of a variant or fragment or derivative, in particular a GST fusion.
  • a still further aspect is a vector suitable for replication in a eukaryotic, preferably mammalian, cell, comprising a polynucleotide encoding the polypeptide, or a variant or fragment or derivative or a fusion of the polypeptide, as defined in the first aspect of the invention, or a fusion of a variant or fragment or derivative, in particular a GST fusion. It is not considered that any of the ESTs excluded from other aspects of the invention are vectors as defined above; however, it will be appreciated that any other of the ESTs clones that may be such a vector are also excluded.
  • vectors suitable for replication in mammalian/eukaryotic cells are well known to those skilled in the art. It will be appreciated that a vector may be suitable for replication in both prokaryotic and eukaryotic cells.
  • nucleotide sequence encoding human DAPPl is also shown in Figure 5.
  • mouse DAPPl The nucleotide sequence encoding mouse DAPPl is also shown in Figure 5.
  • sequences encoding other full length DAPPl polypeptides may be obtained by routine use of methods well known to those skilled in the art, making use of the sequences shown above.
  • PCR methods may be used, particularly methods developed to generate 5' cDNA sequences (for example, the "RACE” method, as well known to those skilled in the art).
  • RACE 5' cDNA sequences
  • sequence database analysis for example EST database analysis and sequencing, as well known to those skilled in the art.
  • an expressed sequence tag (EST) clone is not a recombinant polynucleotide as defined above as it lacks sequences necessary for the translation and therefore expression of the expressed sequence tag.
  • EST sequences may be cloned in the vector Uni-ZAP XR, pT7T3D-Pac, pBluescript SK-, Lafmid BA or pCMV-SPORT2 vector.
  • a polynucleotide comprising a fragment of the recombinant polynucleotide encoding a polypeptide of the invention or a variant, fragment, fusion or derivative may also be useful.
  • the polynucleotide comprises a fragment which is at least 10 nucleotides in length, more preferably at least 14 nucleotides in length and still more preferably at least 18 nucleotides in length.
  • Such polynucleotides are useful as PCR primers, as described in Example 1.
  • a polynucleotide complementary to the polynucleotide (or a fragment thereof) encoding a polypeptide of the invention or a variant, fragment, fusion or derivative may also be useful.
  • Such complementary polynucleotides are well known to those skilled in the art as antisense polynucleotides.
  • the polynucleotide or recombinant polynucleotide of the invention may be DNA or RNA, preferably DNA.
  • the polynucleotide may or may not contain introns in the coding sequence; preferably the polynucleotide is a cDNA.
  • a “variation" of the polynucleotide includes one which is (i) usable to produce a protein or a fragment thereof which is in turn usable to prepare antibodies which specifically bind to the protein encoded by the said polynucleotide or (ii) an antisense sequence equivalent to the gene or to a variation of type (i) as just defined.
  • different codons can be substituted which code for the same amino acid(s) as the original codons.
  • the substitute codons may code for a different amino acid that will not affect the activity or immunogenicity of the protein or which may improve or otherwise modulate its activity or immunogenicity.
  • site-directed mutagenesis or other techniques can be employed to create single or multiple mutations, such as replacements, insertions, deletions, and transpositions, as described in Botstein and Shortle, "Strategies and Applications of In Vitro Mutagenesis” Science, 229: 193-210 (1985), which is incorporated herein by reference. Since such modified polynucleotides can be obtained by the application of known techniques to the teachings contained herein, such modified polynucleotides are within the scope of the claimed invention.
  • polynucleotide sequence (or fragments thereof) encoding a polypeptide of the invention can be used to obtain other polynucleotide sequences that hybridise with it under conditions of high stringency.
  • Such polynucleotides includes any genomic DNA.
  • the polynucleotide of the invention includes polynucleotide that shows at least 60%, preferably 70%, and more preferably at least 80% and most preferably at least 90% homology with the polynucleotide identified in the method of the invention, provided that such homologous polynucleotide encodes a polypeptide which is usable in at least some of the methods described below or is otherwise useful.
  • Per cent homology can be determined by, for example, the GAP program of the University of Wisconsin Genetic Computer Group.
  • DNA-DNA, DNA-RNA and RNA-RNA hybridisation may be performed in aqueous solution containing between 0.1XSSC and 6XSSC and at temperatures of between 55°C and 70°C. It is well known in the art that the higher the temperature or the lower the SSC concentration the more stringent the hybridisation conditions. By “high stringency” we mean 2XSSC and 65°C. 1XSSC is 0.15M NaCl/0.015M sodium citrate. Polynucleotides which hybridise at high stringency are included within the scope of the claimed invention.
  • “Variations" of the polynucleotide also include polynucleotide in which relatively short stretches (for example 20 to 50 nucleotides) have a high degree of homology (at least 80% and preferably at least 90 or 95%) with equivalent stretches of the polynucleotide of the invention even though the overall homology between the two polynucleotides may be much less. This is because important active or binding sites may be shared even when the general architecture of the protein is different.
  • a desirable way to modify the DNA encoding a polypeptide of the invention is to use the polymerase chain reaction as disclosed by Saiki et ⁇ /(1988) Science 239, 487-491. This method may be used for introducing the DNA into a suitable vector, for example by engineering in suitable restriction sites, or it may be used to modify the DNA in other useful ways as is known in the art.
  • the DNA to be enzymatically amplified is flanked by two specific primers which themselves become incorporated into the amplified DNA.
  • the said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
  • the DNA (or in the case of retroviral vectors, RNA) is then expressed in a suitable host to produce a polypeptide comprising the compound of the invention.
  • the DNA encoding the polypeptide constituting the compound of the invention may be used in accordance with known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the polypeptide of the invention.
  • Such techniques include those disclosed in US Patent Nos. 4,440,859 issued 3 April 1984 to Rutter et al, 4,530,901 issued 23 July 1985 to Weissman, 4,582,800 issued 15 April 1986 to Crowl, 4,677,063 issued 30 June 1987 to Mark et al, 4,678,751 issued 7 July 1987 to Goeddel, 4,704,362 issued 3 November 1987 to Itakura et al, 4,710,463 issued 1 December 1987 to Murray, 4,757,006 issued 12 July 1988 to Toole, Jr. et al, 4,766,075 issued 23 August 1988 to Goeddel et aland 4,810,648 issued 7 March 1989 to Stalker, all of which are incorporated herein by reference.
  • DNA (or in the case of retroviral vectors, RNA) encoding the polypeptide constituting the compound of the invention may be joined to a wide variety of other DNA sequences for introduction into an appropriate host.
  • the companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.
  • the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression.
  • an expression vector such as a plasmid
  • the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector.
  • the vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells.
  • One selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance.
  • the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.
  • Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered.
  • bacteria for example E. coli and Bacillus subtilis
  • yeasts for example Saccharomyces cerevisiae
  • filamentous fungi for example Aspergillus
  • plant cells animal cells and insect cells.
  • the vectors include a prokaryotic replicon, such as the ColEl ori, for propagation in a prokaryote, even if the vector is to be used for expression in other, non-prokaryotic, cell types.
  • the vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E. coli, transformed therewith.
  • a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
  • Typical prokaryotic vector plasmids are pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA, USA) and pTrc99A and pKK223-3 available from Pharmacia, Piscataway, NJ, USA.
  • a typical mammalian cell vector plasmid is pSVL available from Pharmacia, Piscataway, NJ, USA. This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-1 cells.
  • an inducible mammalian expression vector is pMSG, also available from Pharmacia. This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.
  • the pEBG-2T expression vector may be used to express GST fusion proteins in eukaryotic cells, for example in 293 cells (human embryonic kidney cells).
  • Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA.
  • Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3.
  • Plasmids pRS413-416 are Yeast Centromere plasmids (YCps).
  • the present invention also relates to a host cell transformed with a polynucleotide vector construct of the present invention.
  • the host cell can be either prokaryotic or eukaryotic.
  • Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, MD, USA (No ATCC 31343).
  • Preferred eukaryotic host cells include yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic cell line.
  • a suitable cell line is the human 293 embryonic kidney cell line, as described in Example 1.
  • Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA.
  • Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, and monkey kidney-derived COS-1 cells available from the ATCC as CRL 1650.
  • Preferred insect cells are Sf9 cells which can be transfected with baculovirus expression vectors.
  • Transformation of appropriate cell hosts with a DNA construct of the present invention is accomplished by well known methods that typically depend on the type of vector used.
  • transformation of prokaryotic host cells see, for example, Cohen et al( ⁇ 912) Proc Natl. Acad. Sci. USA 69, 2110 and Sambrook et ⁇ /(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Transformation of yeast cells is described in Sherman et ⁇ /(1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, NY. The method of Beggs (1978) Nature 275, 104-109 is also useful.
  • reagents useful in transfecting such cells for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, MD 20877, USA.
  • Electroporation is also useful for transforming and/or transfecting cells and is well known in the art for transforming yeast cell, bacterial cells, insect cells and vertebrate cells.
  • bacterial species may be transformed by the methods described in Luchansky et ⁇ /(1988) Mol. Microbiol. 2, 637-646 incorporated herein by reference. The greatest number of transformants is consistently recovered following electroporation of the DNA-cell mixture suspended in 2.5X PEB using 6250V per cm at 25:FD.
  • Successfully transformed cells ie cells that contain a DNA construct of the present invention
  • cells resulting from the introduction of an expression construct of the present invention can be grown to produce the polypeptide of the invention.
  • Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975) J. Mol. Biol. 98, 503 or Berent et ⁇ /(1985) Biotech. 3, 208.
  • the presence of the protein in the supernatant can be detected using antibodies as described below.
  • successful transformation can be confirmed by well known immunological methods when the recombinant DNA is capable of directing the expression of the protein.
  • cells successfully transformed with an expression vector produce proteins displaying appropriate antigenicity. Samples of cells suspected of being transformed are harvested and assayed for the protein using suitable antibodies.
  • the present invention also contemplates a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium.
  • a further aspect of the invention provides a method of making the polypeptide of the invention or a variant, derivative, fragment or fusion thereof or a fusion of a variant, fragment or derivative the method comprising culturing a host cell comprising a recombinant polynucleotide or a replicable vector which encodes said polypeptide, and isolating said polypeptide or a variant, derivative, fragment or fusion thereof or a fusion of a variant, fragment or derivative from said host cell.
  • Methods of cultivating host cells and isolating recombinant proteins are well known in the art.
  • the said host cell is a eukaryotic cell.
  • native DAPPl ie DAPPl as present in cells in which it is naturally expressed
  • the said host cell is capable of myristoylating a said polypeptide of the invention or variant, fragment, derivative or fusion thereof or fusion of a said variant or fragment or derivative which is synthesised with the N-te ⁇ ninal amino acid sequence Met-Gly.
  • mammalian cells for example, are capable of performing myristolyation of suitable polypeptides.
  • the said host cell is a mammalian cell.
  • myristoy lation of the N-terminal glycine residue following cleavage of the initiator methionine residue may affect the biological properties of the polypeptide. It will be appreciated that prokaryotic cells may not be capable of performing myristoylation.
  • the myristoylation of a polypeptide may be investigated using methods known to those skilled in the art, for example using methods employing cells labelled with tritiated myristic acid or using mass spectrometry methods. Suitable methods may be described in Kaplan et al (1988) Mol Cell Biol 8, 2435-2441.
  • the invention also includes a polypeptide, or a variant, fragment, derivative or fusion thereof, or fusion of a said variant or fragment or derivative obtained or obtainable by the above method of the invention.
  • a still further aspect of the invention provides an antibody reactive towards a polypeptide of the invention. It is preferred that the antibody does not react substantially with another polypeptide comprising an SH2 and/or PH domain.
  • Antibodies reactive towards the said polypeptide of the invention may be made by methods well known in the art.
  • the antibodies may be poly clonal or monoclonal.
  • Suitable monoclonal antibodies which are reactive towards the said polypeptide may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniques and Applications", SGR Hurrell (CRC Press, 1982).
  • the antibody is raised using any suitable peptide sequence obtainable from the given amino acid sequence of human or mouse DAPPl as appropriate.
  • suitable peptides include, for example, RAELLEGKMSTQDPSDLWSR, HTAMQTGRTEDDLVPTAPSL and QIRKQLNQGEGTIRSRSFIFK.
  • an antibody may be raised using a peptide sequence comprising a residue of DAPPl that is capable of being phosphorylated or is phosphorylated.
  • an antibody may be raised using a peptide sequence comprising residue Tyrl39 of DAPPl.
  • a suitable sequence may be PRKVEEPSIYESVRVH. It will be appreciated that the antibody may be raised against the unphosphorylated peptide or against the phosphorylated peptide (ie the peptide phosphorylated on the tyrosine residue equivalent to Tyrl39 of full length DAPPl, underlined in the sequence shown above).
  • An antibody capable of reacting specifically with the phosphorylated peptide and/or with phosphorylated DAPPl may be particularly useful. Methods of preparing antibodies reactive with a phosphorylated peptide sequence are well known to those skilled in the art.
  • the antibody does not react substantially with another polypeptide comprising an SH2 and/or PH domain. Accordingly, it may be preferred if peptides based on the DAPPl sequence are used which vary significantly from any peptides found in any other PH or SH2 domains, for example in the polypeptides with which DAPPl is compared in Figure 2.
  • Peptides in which one or more of the amino acid residues are chemically modified, before or after the peptide is synthesised may be used providing that the function of the peptide, namely the production of specific antibodies in vivo, remains substantially unchanged.
  • modifications include forming salts with acids or bases, especially physiologically acceptable organic or inorganic acids and bases, forming an ester or amide of a terminal carboxyl group, and attaching amino acid protecting groups such as N-t- butoxycarbonyl. Such modifications may protect the peptide from in vivo metabolism.
  • the peptides may be present as single copies or as multiples, for example tandem repeats. Such tandem or multiple repeats may be sufficiently antigenic themselves to obviate the use of a carrier.
  • the peptide may be formed as a loop, with the N-terminal and C-terminal ends joined together, or to add one or more Cys residues to an end to increase antigenicity and/or to allow disulphide bonds to be formed.
  • a carrier preferably a polypeptide
  • the arrangement is preferably such that the peptide of the invention forms a loop.
  • a carrier function should be present in any immunogenic formulation in order to stimulate, or enhance stimulation of, the immune system. It is thought that the best carriers embody (or, together with the antigen, create) a T-cell epitope.
  • the peptides may be associated, for example by cross-linking, with a separate carrier, such as serum albumins, myoglobins, bacterial toxoids and keyhole limpet haemocyanin.
  • More recently developed carriers which induce T-cell help in the immune response include the hepatitis-B core antigen (also called the nucleocapsid protein), presumed T-cell epitopes such as Thr-Ala-Ser-Gly- Val-Ala-Glu-Thr-Thr-Asn-Cys, beta-galactosidase and the 163-171 peptide of interleukin-1.
  • the latter compound may variously be regarded as a carrier or as an adjuvant or as both.
  • several copies of the same or different peptides of the invention may be cross-linked to one another; in this situation there is no separate carrier as such, but a carrier function may be provided by such cross-linking.
  • Suitable cross-linking agents include those listed as such in the Sigma and Pierce catalogues, for example glutaraldehyde, carbodiimide and succinimidyl 4-(N- maleimidomethyl)cyclohexane-l-carboxylate, the latter agent exploiting the - SH group on the C-terminal cysteine residue (if present).
  • the peptide is prepared by expression of a suitable nucleotide sequence in a suitable host, then it may be advantageous to express the peptide as a fusion product with a peptide sequence which acts as a carrier. Kabigen's "Ecosec" system is an example of such an arrangement.
  • the peptide of the invention may be linked to other antigens to provide a dual effect.
  • Peptides may be synthesised by the Fmoc-polyamide mode of solid-phase peptide synthesis as disclosed by Lu et ⁇ /(1981) J. Org. Chem. 46, 3433 and references therein. Temporary N-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is effected using 20% piperidine in N,N- dimethylformamide.
  • Side-chain functionalities may be protected as their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), butyloxycarbonyl derivative (in the case of lysine and histidine), trityl derivative (in the case of cysteine) and 4- methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case of arginine).
  • glutamine or asparagine are C-terminal residues, use is made of the 4,4'-dimethoxybenzhydryl group for protection of the side chain amido functionalities.
  • the solid-phase support is based on a polydimethyl- acrylamide polymer constituted from the three monomers dimethylacrylamide (backbone-monomer), bisacryloylethylene diamine (cross linker) and acryloylsarcosine methyl ester (functionalising agent).
  • the peptide-to-resin cleavable linked agent used is the acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid derivatives are added as their preformed symmetrical anhydride derivatives with the exception of asparagine and glutamine, which are added using a reversed N,N-dicyclohexyl- carbodiimide/1-hydroxybenzotriazole mediated coupling procedure.
  • peptides are cleaved from the resin support with concomitant removal of side-chain protecting groups by treatment with 95 % trifluoroacetic acid containing a 50% scavenger mix.
  • Scavengers commonly used are ethanedithiol, phenol, anisole and water, the exact choice depending on the constituent amino acids of the peptide being synthesised.
  • Trifluoroacetic acid is removed by evaporation in vacuo, with subsequent trituration with diethyl ether affording the crude peptide.
  • Any scavengers present are removed by a simple extraction procedure which on lyophilisation of the aqueous phase affords the crude peptide free of scavengers.
  • Reagents for peptide synthesis are generally available from Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK. Purification may be effected by any one, or a combination of, techniques such as size exclusion chromatography, ion- exchange chromatography and (principally) reverse-phase high performance liquid chromatography. Analysis of peptides may be carried out using thin layer chromatography, reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis.
  • FAB fast atom bombardment
  • a further aspect of the invention provides a method of identifying a compound that modulates the phospholipid binding activity of a polypeptide of the invention, the method comprising contacting a compound with the said polypeptide or a suitable variant, fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative thereof and determining whether the phospholipid binding activity of the said polypeptide is changed compared to the phospholipid binding activity of the said polypeptide or said variant, fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative thereof in the absence of said compound.
  • a said suitable variant, fragment, derivative or fusion or a fusion of a variant, fragment or derivative comprises a PH domain, still more preferably a PH domain that is capable of binding to a 3- phosphoinositide.
  • a further aspect of the invention provides a method of identifying a compound that modulates the tyrosine-phosphorylated protein binding activity of a polypeptide of the invention, the method comprising contacting a compound with the said polypeptide or a suitable variant, fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative thereof and determining whether the tyrosine-phosphorylated protein binding activity of the said polypeptide is changed compared to the tyrosine- phosphorylated protein binding activity of the said polypeptide or said variant, fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative thereof in the absence of said compound.
  • a said suitable variant, fragment, derivative or fusion or a fusion of a variant, fragment or derivative comprises a SH2 domain, still more preferably a SH2 domain that is capable of binding to a tyrosine-phosphorylated protein.
  • Example 1 The binding of the polypeptide of the invention to phospholipids is described in Example 1. It is preferred that modulation of the binding to a 3- phosphoinositol, for example PtdIns(3,4,5)P 3 or PtdIns(3,4)P 2 is measured.
  • Methods of detecting binding of the polypeptide of the invention or fragment, variant, derivative or fusion thereof, or fusion of a variant, fragment or derivative to phospholipids are described in Example 1 and include a protein- lipid overlay assay in which the lipid is spotted onto a support, for example Hybond-C extra membrane, and protein bound to the support by virtue of interaction with the lipid is detected, for example using an antibody-based method, as well know to those skilled in the art.
  • a surface plasmon resonance assay for example as described in Example 1 or in Plant et al (1995) Analyt Biochem 226(2), 342-348, may alternatively be used. Methods may make use of a polypeptide of the invention or fragment, variant, derivative or fusion thereof, or fusion of a variant, fragment or derivative that is labelled, for example with a radioactive or fluorescent label.
  • a further aspect of the invention is a method of identifying a compound that is capable of binding to the polypeptide of the invention or fragment, variant, derivative or fusion thereof, or fusion of a variant, fragment or derivative wherein the said polypeptide of the invention or fragment, variant, derivative or fusion thereof, or fusion of a variant, fragment or derivative is exposed to the compound and any binding of the compound to the said polypeptide of the invention or fragment, variant, derivative or fusion thereof, or fusion of a variant, fragment or derivative is detected and/or measured.
  • the binding constant for the binding of the compound to the polypeptide may be determined.
  • Suitable methods for detecting and/or measuring (quantifying) the binding of a compound to a polypeptide are well known to those skilled in the art and may be performed, for example using a method capable of high throughput operation, for example a chip-based method in which the compounds to be tested are immobilised in a microa ⁇ ay on a solid support, as known to those skilled in the art.
  • the compound may be a drug-like compound or lead compound for the development of a drug-like compound.
  • drug-like compound is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament.
  • a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may have a molecular weight of less than 5000 daltons and which may be water-soluble.
  • a drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate target cellular membranes, but it will be appreciated that these features are not essential.
  • lead compound is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non-selective in its action, unstable, poorly soluble, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.
  • reagents including any fragment, derivative, variant or fusion of the polypeptide of the invention or fusion of a variant, fragment or derivative
  • conditions used in the method may be chosen such that the interactions between the said polypeptide and the phospholipid, for example 3- phosphoinositide or tyrosine-phosphorylated protein are substantially the same as between human DAPPl and the phospholipid in vivo.
  • the compound decreases the relevant binding activity of said polypeptide.
  • the compound may bind substantially reversibly or substantially irreversibly to the relevant binding site of said polypeptide.
  • the compound may bind to a portion of said polypeptide that is not the binding site so as to interfere with the binding of the said polypeptide to the phospholipid or tyrosine-phosphorylated protein.
  • the compound may bind to a portion of said polypeptide so as to decrease said polypeptide 's binding activity by an allosteric effect. This allosteric effect may be an allosteric effect that is involved in the natural regulation of the said polypeptide's activity.
  • the compound may act by interacting with the polypeptide of the invention and modulating, for example inhibiting, its phosphorylation, for example on a tyrosine residue.
  • the compound may, for example, change the configuration of DAPPl so that it is substantially unable to bind to a 3-phosphoinositide or to a tyrosine-phosphorylated polypeptide.
  • the compound may be capable of affecting the intracellular location of the DAPPl polypeptide; for example, it may inhibit or promote the translocation of DAPPl to a membrane.
  • the compound may modulate any interaction of a DAPPl molecule with further DAPPl molecule(s).
  • a compound that, for example, is capable of modulating the phosphorylation of DAPPl may thereby, for example, modulate the ability of DAPPl to bind to a phospholipid or to a tyrosine-phosphorylated protein.
  • a compound that is capable of modulating the ability of DAPPl to bind to a phospholipid may thereby modulate the intracellular location of the DAPPl molecule and/or modulate the phosphorylation of DAPPl .
  • the compound increases the binding activity of said polypeptide.
  • the compound may bind to a portion of said polypeptide that is not the relevant binding site so as to aid the binding of the said polypeptide to the phospholipid or tyrosine-phosphoryated protein, as appropriate.
  • the compound may bind to a portion of said polypeptide so as to increase said polypeptide's binding activity by an allosteric effect.
  • This allosteric effect may be an allosteric effect that is involved in the natural regulation of the said polypeptide's activity.
  • Examples of compounds that may inhibit binding of DAPPl to a tyrosine-phosphorylated protein may include phosphotyrosine and/or a peptide or co ⁇ esponding peptidomimetic compound consisting of or comprising an amino acid sequence derivable from a tyrosine-phosphorylated protein and including the or a phosphorylated tyrosine residue.
  • the peptide may have between about 2, 3, 4 or 5 and about 10, 15, 20 or 30 amino acids.
  • Example 1 the appropriate methods make use of the methods described in Example 1 for detecting and/or quantifying the interaction between a polypeptide and a phospholipid, for example a protein-lipid overlay or surface plasmon resonance method, as discussed above. It is preferred that a GST- tagged fusion of the polypeptide of the invention or a fragment therof is used. Methods in which radioactively or fluorescentiy labelled lipids are used may also be useful.
  • tyrosine-phosphorylated proteins examples include tyrosine-phosphorylated receptor tyrosine kinases, for example the insulin receptor or growth factor receptors, for example epidermal growth factor receptor, a member of the IRS family of protein tyrosine kinase substrates, isoforms of GRB (see Margolis (1994) Prog Biophys Mol Biol 62(3), 223-44 for a review), src or lck.
  • DAPPl may be capable of self-association; for example DAPPl may form dimers or trimers or higher multimers.
  • DAPPl self-association can be detected by co-expressing or mixing DAPPl fusions with different tags, for example FLAG-tagged and GST-tagged DAPPl; co- precipitation (for example, co-immunoprecipitation) of the different tags (using techniques well known to those skilled in the art) indicates that the molecules to which they are fused (ie DAPPl) are capable of association (ie self-association).
  • phosphorylation of DAPPl may promote or disrupt such self-association.
  • the ability of a DAPPl molecule to interact with other molecules for example other polypeptides or phospholipids, may be reduced if it is bound to another DAPPl molecule.
  • tyrosine-phosphorylated protein includes a tyrosine-phosphorylated peptide. Suitable methods include yeast two-hybrid interactions, co-purification, ELISA, co- immunoprecipitation methods and cellular response assays.
  • Cellular response assays may be carried out in a variety of cell types, for example in adipocytes or adipocyte cell lines, in a skeletal muscle cell line (such as the L6 myotubule cell line), liver cells or liver cell lines or cancer cells or cancer cell lines.
  • adipocytes or adipocyte cell lines in a skeletal muscle cell line (such as the L6 myotubule cell line), liver cells or liver cell lines or cancer cells or cancer cell lines.
  • the method may be performed in vitro, either in intact cells or tissues, with broken cell or tissue preparations or at least partially purified components. Alternatively, they may be performed in vivo.
  • the cells tissues or organisms in/on which the method is performed may be transgenic. In particular they may be transgenic for the polypeptide of the invention.
  • FRET Fluorescence Energy Resonance Transfer
  • an protein encoded by an RNA transcribed from a promoter regulated by DAPPl may be measured.
  • the protein may be one that is physiologically regulated by DAPPl or may be a "reporter" protein, as well known to those skilled in the art (ie a recombinant construct may be used).
  • a reporter protein may be one whose activity may easily be assayed, for example ( ⁇ -galactosidase, chloramphenicol acetylrransferase or luciferase (see, for example, Tan et ⁇ /(1996)).
  • Promoters whose activity may be regulated by a signalling pathway in which DAPPl may be involved include promoters which comprise an Insulin Response Element, as described below and/or are regulated by CREB and may comprise a CRE (cyclic AMP response element), which is regulated by the CREB protein.
  • CRE cyclic AMP response element
  • CREB control of COX2 expression is discussed in Montminy (1997) Ann Rev Biochem 66, 807-822.
  • CREB may also induce expression of the c-Fos (or Fos) gene. This gene is important in the control of proliferation and differentiation. Abe ⁇ ant control of c-Fos may be involved in cancer, and inhibition of Fos transcription may be useful as an anticancer treatment applicable to most cancers.
  • the promoter for IL-1 also contains a cyclic AMP-response element (Chandra et al (1995) J Immunol 155, 4535-4543). Thus, transcription of these genes may be assessed or the promoter for such a gene may be used in a reporter construct as described above. Phosphorylation of CREB, for example at Ser 133 may alternatively be measured as a means of measuring the activity of DAPPl .
  • Insulin exerts important effects on gene expression in multiple tissues (O'Brien, R. M. & Granner, D. K (1996) Physiol. Rev. 16, 1109-1161). In the liver, insulin suppresses the expression of a number of genes which contain a conserved insulin response sequence (IRS) 1 (CAAAAC/TAA), including insulin-like growth factor binding protein-1 (IGFBP-1), apolipoprotein CIII (apoCIII), phosphoenol-pyruvate carboxykinase (PEPCK) and glucose-6 phosphatase (G6Pase) (Goswami, R et al (1994) Endocrinol. 134, 2531-2539; Suwanickul, A et al (1993) J.
  • IGFBP-1 insulin-like growth factor binding protein-1
  • apoCIII apolipoprotein CIII
  • PEPCK phosphoenol-pyruvate carboxykinase
  • G6Pase glucose-6 phosphatas
  • the transcription of a gene indicated above may be measured by measurement of changes in the enzymatic or other activity of the said gene product, for example in a cell. Suitable methods will be well known to those skilled in the art.
  • a further aspect of the invention provides a method of identifying a compound which blocks the phosphorylation of a polypeptide of the invention by an interacting polypeptide the method comprising determining whether a compound enhances or disrupts the interaction between (a) a polypeptide according to claim 1 or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative and (b) the interacting polypeptide or a suitable variant, derivative, fragment or fusion thereof or a suitable fusion of a variant, derivative or fragment, or determining whether the compound substantially blocks phosphorylation of the said polypeptide according to claim 1 or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion by the interacting polypeptide or a suitable variant, derivative, fragment or fusion thereof.
  • the interacting polypeptide may be a receptor tyrosine kinase, for example the insulin receptor or the epidermal growth factor receptor.
  • the interacting polypeptide may also be Src or Lck or Lyn, as discussed further in Example 4.
  • Src, Lck and Lyn are Src- family protein tyrosine kinases.
  • the interacting polypeptide may be a Src-family protein tyrosine kinase.
  • the interacting polypeptide may be recombinant, for example as discussed in Example 4. It will be appreciated that such a method may be carried out in a cell.
  • DAPPl may be phosphorylated in response to stimulation of the cell by an agonist, for example a growth factor, such as emidermal growth factor or insulin-like growth factor- 1 or insulin, or exposure of the cell to a cellular stress such as exposure to hydrogen peroxide.
  • a growth factor such as emidermal growth factor or insulin-like growth factor- 1 or insulin
  • Other agents which may lead to cellular stress may include sodium arsenite or UV radiation.
  • DAPPl may also be phosphorylated in response to exposure of the cell to a tyrosine phosphatase inhibitor. It will be appreciated that the cell may be a recombinant cell.
  • the cell may be recombinant in relation to the polypeptide of the first aspect of the invention or it may be recombinant in relation to a polypeptide involved in the phosphorylation of DAPPl, for example a receptor tyrosine kinase, for example the insulin receptor, insulin-like growth factor- 1 receptor or epidermal growth factor receptor.
  • a polypeptide involved in the phosphorylation of DAPPl for example a receptor tyrosine kinase, for example the insulin receptor, insulin-like growth factor- 1 receptor or epidermal growth factor receptor.
  • the stimulated cells may be lysed and DAPPl immunoprecitated from the lysate using a DAPPl -specific antibody.
  • Phosphorylated DAPPl may then be detected in an ELISA-type assay using an antibody reacting with phosphotyrosine, as well known to those skilled in the art, or using an antibody specific for phosphorylated DAPPl.
  • a method using an antibody specific for phosphotyrosine may be more sensitive than one using an antibody reacting with phosphotyrosine but not specific for DAPPl.
  • the cells may be exposed to the compound in a 96 well plate or similar. Suitable cells are as indicated above, for example a cell line such as the 293 human embryonic kidney cell line.
  • a further aspect of the invention provides a method of identifying a polypeptide that interacts with a polypeptide according to the first aspect of the invention, the method comprising 1) contacting a) the polypeptide according to the first aspect of the invention or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative with b) a composition that may contain such an interacting polypeptide, 2) detecting the presence of a complex containing the polypeptide according to the first aspect of the invention or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative and an interacting polypeptide, and optionally 3) identifying any interacting polypeptide bound to the said polypeptide according to the first aspect of the invention or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative.
  • a still further aspect of the invention provides a method of identifying a polypeptide capable of phosphorylating the polypeptide according to the first aspect of the invention (DAPPl), preferably capable of phosphorylating DAPPl on a tyrosine residue, the method comprising 1) contacting a) the unphosphorylated polypeptide according to the first aspect of the invention or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative with b) a composition that may contain such a polypeptide capable of phosphorylating the polypeptide according to the first aspect of the invention, 2) detecting phosphorylation of the polypeptide according to the first aspect of the invention or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative, and optionally 3) identifying the polypeptide capable of phosphorylating the polypeptide according to the first aspect of the invention or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or
  • Methods of detecting phosphorylation of a polypeptide are well known to those skilled in the art.
  • antibodies capable of binding to phosphoserine, phosphothreonine or phosphotyrosine residues may be employed.
  • Radioactive phosphorous isotopes may be employed, for example 32 P or 33 P.
  • Methods of rendering the polypeptide of the invention unphosphorylated are known to those skilled in the art and may employ phosphatases, for example PP2A.
  • a further aspect of the invention provides a polypeptide identifiable by a method set out above, wherein the polypeptide is not src, lyn or lck.
  • the polypeptide is not a known member of the src family of protein tyrosine kinases. Examples of src-family protein tyrosine kinases are given, for example, in Liu et al (1999) Structural basis for selective inhibition of Src family kinases by PP Chem. Biol. 6, 671-8 and Hanke et al (1996) Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor.
  • an interacting polypeptide may be capable of phosphorylating DAPPl and similarly that a polypeptide capable of phosphorylating DAPPl may be a said interacting polypeptide.
  • An interacting polypeptide may in addition or alternatively comprise a phosphotyrosine residue ie be a tyrosine-phosphorylated polypeptide.
  • a further aspect of the invention provides a nucleic acid (or polynucleotide) encoding or capable of expressing an interacting polypeptide or polypeptide capable phosphorylating the polypeptide according to the first aspect of the invention, identifiable by a method set out above.
  • a still further aspect of the invention provides a nucleic acid complementary to a nucleic acid encoding or capable of expressing a an interacting polypeptide or polypeptide capable phosphorylating the polypeptide according to the first aspect of the invention, identifiable by a method set out above. Methods of preparing or isolating such a nucleic acid are well known to those skilled in the art.
  • the polypeptide may be cleaved, for example using trypsin, cyanogen bromide, V8 protease formic acid, or another specific cleavage reagent.
  • the digest may be chromatographed on a Vydac C18 column or subjected to SDS- PAGE to resolve the peptides.
  • the N-terminal sequence of the peptides may then be determined using standard methods.
  • sequences are used to isolate a nucleic acid encoding the peptide sequences using standard PCR-based strategies.
  • Degenerate oligonucleotide mixtures each comprising a mixture of all possible sequences encoding a part of the peptide sequences, are designed and used as PCR primers or probes for hybridisation analysis of PCR products after Southern blotting.
  • mRNA prepared from cells in which the polypeptide may be expressed is used as the template for reverse transcriptase, to prepare cDNA, which is then used as the template for the PCR reactions.
  • Positive PCR fragments are subcloned and used to screen cDNA libraries to isolate a full length clone for the polypeptide.
  • sequences of initial subcloned PCR fragments may be determined, and the sequence may then be extended by known PCR-based techniques to obtain a full length sequence.
  • the initial PCR sequence may be used to screen electronic databases of expressed sequence tags (ESTs) or other known sequences.
  • ESTs expressed sequence tags
  • related sequences may be identified which may be useful in isolating a full length sequence using the two approaches described above.
  • Sequences are determined using the Sanger dideoxy method.
  • the encoded amino acid sequences may be deduced by routine methods.
  • antibodies may be raised against the polypeptide.
  • the antibodies are used to screen a ⁇ gtll expression library made from cDNA copied from mRNA from cells in which the polypeptide may be expressed.
  • a transgenic animal in which a DAPPl gene is altered and/or a recombinant DAPPl gene is present for example a rodent, in particular a mouse, may be useful in, for example, identifying polypeptides that interact with DAPPl .
  • protein tyrosine kinases may be assayed, for example in vitro, for their ability to phosphorylate DAPPl.
  • the said polypeptide according to the first aspect of the invention or fragment, derivative, variant or fusion thereof used in the methods is one which is produced by recombinant DNA technology.
  • the interacting polypeptide or polypeptide capable of phosphorylating DAPPl used in the method of identifying compounds that modulate the interaction with or phosphorylation of DAPPl is one which is produced by recombinant DNA technology.
  • DAPPl phosphorylate DAPPl
  • DAPPl preferably recombinant DAPPl
  • an agonist such as insulin, insulin like growth factor- 1 or epidermal growth factor
  • a cellular stress for example hydrogen peroxide
  • the DAPPl may be expressed in a cell comprising a constitutively active form of PI 3-kinase, as discussed in Example 4.
  • DAPPl may then be isolated from the cell, for example using methods described in the examples.
  • DAPPl may be phosphorylated outside a cell using a tyrosine protein kinase, for example a src-family protein tyrosine kinase, preferably src, lye or lyn.
  • a tyrosine protein kinase for example a src-family protein tyrosine kinase, preferably src, lye or lyn.
  • the said components in the method are those that have interactions or activities which are substantially the same as those of DAPPl or an interacting polypeptide or polypeptide capable of phosphorylating DAPPl as the case may be but which may be more convenient to use in an assay.
  • fusions of DAPPl are particularly useful since said fusion may contain a moiety which may allow the fusion to be purified readily.
  • a further aspect of the invention provides a method of identifying a compound which blocks the phosphorylation or activation of a polypeptide according to the first aspect of the invention by a polypeptide as defined above (ie an interacting polypeptide or polypeptide capable of phosphorylating the polypeptide of the invention) the method comprising detemiining whether a compound enhances or disrupts the interaction between (a) a polypeptide according to the first aspect of the invention or a suitable variant, derivative, fragment or fusion thereof or a suitable fusion of a variant, derivative or fragment, and (b) said polypeptide as defined above (and in Claim 30) or a suitable variant, derivative, fragment or fusion thereof or a suitable fusion of a variant, derivative or fragment, or determining whether the compound substantially blocks activation of the said polypeptide according to the first aspect of the invention (as defined in Claim 1) or a suitable variant, derivative, fragment or fusion thereof or a suitable fusion of a variant, derivative or fragment, by said polypeptide as defined
  • reporter gene constructs may be prepared by methods known to those skilled in the art, using the teaching herein and as described above.
  • the expression of the reporter gene will be dependent on the activity of DAPPl and thus the effect of compounds can be measured.
  • the reporter gene may be fatal to the cells, or alternatively may allow cells to survive under otherwise fatal conditions. Cell survival can then be measured, for example using colorimetric assays for mitochondrial activity, such as reduction of WST-1 (Boehringer).
  • WST-1 is a formosan dye that undergoes a change in absorbance on receiving electrons via succinate dehydrogenase.
  • yeast two-hybrid system is used.
  • screening assays which are capable of high throughput operation will be particularly prefe ⁇ ed.
  • Examples may include the cell based assays described and protein-protein binding assays.
  • a further example is an SPA-based (Scintillation Proximity Assay) system as well known to those skilled in the art.
  • SPA-based systems may be particularly suitable for detecting 32 P or 33 P isotopes and may therefore be suitable for detecting or quantitating phosphorylation of a polypeptide, for example DAPPl.
  • beads comprising scintillant and an antibody capable of binding to a phosphotyrosine residue may be prepared.
  • the beads may be mixed with a sample comprising, for example, the polypeptide of the invention or fragment thereof, [ ⁇ 33 P]ATP or [ ⁇ PJATP, a polypeptide capable of phosphorylating the polypeptide of the invention on a tyrosine residue and with the test compound. Conveniently this is done in a 96-well format. The plate is then counted using a suitable scintillation counter, using known parameters for the particular radioactive label in an SPA assay. Only the radioactive label that is in proximity to the scintillant, ie only that bound to the polypeptide of the invention or fragment thereof that is bound to the anti- phosphotyrosine antibody anchored on the beads, is detected.
  • the beads may comprise an antibody that binds to the polypeptide of the invention.
  • a further aspect of the invention provides the use of a polypeptide according to the first aspect of the invention, variant, fragment, derivative or fusion thereof, or fusion of a variant, fragment or derivative in a screening assay for identifying a compound which modulates the activity of the polypeptide according to the first aspect of the invention or which modulates the activation of the said polypeptide according to the first aspect of the invention.
  • a further aspect of the invention provides a kit of parts comprising a polypeptide according to the first aspect of the invention, variant, fragment, derivative or fusion, or fusion of a variant, fragment or derivative and a means for carrying out a method of the invention as described above.
  • a kit may comprise, for example, a GST fusion of DAPPl or a fragment thereof and a tyrosine-phosphorylated protein, for example src or lck or lyn (or other src-family protein tyrosine kinase) or a tyrosine-phosphorylated fragment thereof.
  • a further aspect of the invention provides a compound identified or identifiable by any of the above screening methods. It will be appreciated that the compound is not a known inhibitor of PI 3-kinase or of a tyrosine kinase.
  • a further aspect of the invention provides a compound of the invention or a polypeptide of the invention or a variant, fragment, fusion or derivative or a fusion of a variant, fragment or derivative for use in medicine.
  • peptidomimetic compounds may also be useful.
  • polypeptide or “peptide” we include not only molecules in which amino acid residues are joined by peptide (-CO-NH-) linkages but also molecules in which the peptide bond is reversed.
  • Such retro-inverso peptidomimetics may be made using methods known in the art, for example such as those described in Meziere et al (1997) J. Immunol. 159, 3230-3237, incorporated herein by reference. This approach involves making pseudopeptides containing changes involving the backbone, and not the orientation of side chains. Meziere et al (1997) show that, at least for MHC class ⁇ and T helper cell responses, these pseudopeptides are useful.
  • Retro- inverse peptides which contain NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis.
  • the peptide bond may be dispensed with altogether provided that an appropriate linker moiety which retains the spacing between the C ⁇ atoms of the amino acid residues is used; it is particularly prefe ⁇ ed if the linker moiety has substantially the same charge distribution and substantially the same planarity as a peptide bond. It will be appreciated that the peptide may conveniently be blocked at its N- or C-terminus so as to help reduce susceptibility to exoproteolytic digestion.
  • polypeptide of the invention or a fragment thereof for example which comprises the SH2 domain but not the PH domain or vice versa, may be a peptidomimetic compound, as described above. Fragments or variants of DAPPl that may be useful in medicine are discussed above.
  • a derivative or fusion of a polypeptide of the invention or variant, fragment or fusion thereof which may be particularly useful, for example in medicine, may comprise the polypeptide of the invention or variant, fragment or fusion therof and a further portion. It is prefe ⁇ ed that the said further portion confers a desirable feature on the said molecule; for example, the portion may useful in detecting or isolating the molecule, or promoting cellular uptake of the molecule or the interacting polypeptide.
  • the portion may be, for example, a radioactive moiety, a fluorescent moiety, for example a small fluorophore or a green fluorescent protein (GFP) fluorophore, as well known to those skilled in the art.
  • the moiety may be an immunogenic tag, for example a Myc, FLAG or HA (haemagglutinin) tag, as known to those skilled in the art or may be a lipophilic molecule or polypeptide domain that is capable of promoting cellular uptake of the molecule or the interacting polypeptide, as known to those skilled in the art, for example as characterised for a Drosophila polypeptide (see, for example, Derossi et al (1998) Trends Cell Biol 8, 84-87).
  • Further useful tags include a tag that is capable of being phosphorylated, for example a tag capable of being phosphorylated by protein kinase A. Such a tag may be useful in introducing a radioactive label, for example 32 P or 33 P, onto the polypeptide.
  • a still further aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of the invention or a polypeptide of the invention or a variant, fragment, fusion or derivative or a fusion of a variant, fragment or derivative and a pharmaceutically acceptable carrier.
  • a suitable ca ⁇ ier will be known to those skilled in the art.
  • DAPPl Compounds, identifiable in the screening method, which mimic the effect of a 3-phosphoinositide, preferably phosphoinositol-3,4,5-rrisphosphate or phosphoinositol-3,4-bisphosphate, on DAPPl are believed to be useful in treating diabetes.
  • Compounds identifiable in the screening methods of the invention that inhibit phosphorylation of DAPPl are believed to be useful in treating cancer.
  • PKB is the cellular homologue of v-akt which is involved in leukaemias. DAPPl becomes tyrosine phosphorylated in response to insulin and growth factors and this phosphorylation is prevented by inhibitors of PI 3-kinase.
  • Compounds which induce or inhibit the tyrosine phosphorylation of DAPPl mau be useful in modulating the downstream signal transduction pathways that DAPPl regulates.
  • Compounds may be used, for example, for treatment of diabetes by switching on insulin-stimulated signal transduction pathways or for the treatment of cancer by inhibiting cell proliferation or promoting apoptosis.
  • Compounds may also be useful in the modulation or resolution of inflammation.
  • a further aspect of the invention provides a method of treating a patient in need of modulation of the activity of DAPPl or with an inflammatory or an ischaemic disease, cancer, diabetes or a defect in glycogen metabolism, the method comprising administering to the patient an effective amount of a compound of the invention or a polypeptide of the invention or a variant, fragment, fusion or derivative or a fusion of a variant, fragment or derivative.
  • inflammatory disease is included immune system disorders, for example autoimmune diseases, as will be apparent to those skilled in the art.
  • a further aspect of the invention provides the use of a compound of the invention or a polypeptide of the invention or a variant, fragment, fusion or derivative or a fusion of a variant, fragment or derivative in the manufacture of a medicament for treatment of a patient in need of modulation of the activity of DAPPl or with an inflammatory or an ischaemic disease, cancer, diabetes or a defect in glycogen metabolism.
  • a further aspect of the invention provides a compound capable of altering the expression of DAPPl .
  • the said compound may be an antisense molecule or ribozyme directed (for example, capable of binding to a polynucleotide encoding DAPPl under physiological conditions) against a polynucleotide encoding DAPPl.
  • a further aspect of the invention provides a compound capable of altering the expression of DAPPl for use in medicine.
  • a still further aspect of the invention provides the use of a compound capable of altering the expression of DAPPl in the manufacture of a medicament for the treatment of a patient in need of modulation of the activity of DAPPl or with an inflammatory or an ischaemic disease, cancer, diabetes or a defect in glycogen metabolism.
  • the nucleic acid of the invention may be an antisense oligonucleotide, for example an antisense oligonucleotide directed against a nucleic acid encoding a polypeptide of the invention such as the human DAPPl gene.
  • Antisense oligonucleotides are single-stranded nucleic acid, which can specifically bind to a complementary nucleic acid sequence.
  • RNA-RNA By binding to the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA-DNA duplex is formed.
  • RNA-RNA By binding to the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA-DNA duplex is formed.
  • antisense because they are complementary to the sense or coding strand of the gene. Recently, formation of a triple helix has proven possible where the oligonucleotide is bound to a DNA duplex. It was found that oligonucleotides could recognise sequences in the major groove of the DNA double helix. A triple helix was formed thereby. This suggests that it is possible to synthesise a sequence-specific molecules which specifically bind double-stranded DNA via recognition of major groove hydrogen binding sites.
  • the above oligonucleotides can inhibit the function of the target nucleic acid. This could, for example, be a result of blocking the transcription, processing, poly(A)addition, replication, translation, or promoting inhibitory mechanisms of the cells, such as promoting RNA degradations.
  • Antisense oligonucleotides are prepared in the laboratory and then introduced into cells, for example by microinjection or uptake from the cell culture medium into the cells, or they are expressed in cells after transfection with plasmids or retroviruses or other vectors carrying an antisense gene.
  • Antisense oligonucleotides were first discovered to inhibit viral replication or expression in cell culture for Rous sarcoma virus, vesicular stomatitis virus, herpes simplex virus type 1, simian virus and influenza virus. Since then, inhibition of mRNA translation by antisense oligonucleotides has been studied extensively in cell-free systems including rabbit reticulocyte lysates and wheat germ extracts.
  • Oligonucleotides are subject to being degraded or inactivated by cellular endogenous nucleases.
  • modified oligonucleotides eg having altered internucleotide linkages, in which the naturally occu ⁇ ing phosphodiester linkages have been replaced with another linkage.
  • Agrawal et al (1988) Proc. Natl. Acad. Sci. USA 85, 7079-7083 showed increased inhibition in tissue culture of HTV-1 using oligonucleotide phosphoramidates and phosphorothioates.
  • Oligonucleotides having artificial linkages have been shown to be resistant to degradation in vivo.
  • Shaw et al (1991) in Nucleic Acids Res. 19, 747-750 report that otherwise unmodified oligonucleotides become more resistant to nucleases in vivo when they are blocked at the 3' end by certain capping structures and that uncapped oligonucleotide phosphorothioates are not degraded in vivo.
  • the oligonucleotide is a deoxyribonucleic acid (DNA), although ribonucleic acid (RNA) sequences may also be synthesised and applied.
  • the oligonucleotides useful in the invention preferably are designed to resist degradation by endogenous nucleolytic enzymes. In vivo degradation of oligonucleotides produces oligonucleotide breakdown products of reduced length. Such breakdown products are more likely to engage in non-specific hybridization and are less likely to be effective, relative to their full-length counterparts. Thus, it is desirable to use oligonucleotides that are resistant to degradation in the body and which are able to reach the targeted cells.
  • the present oligonucleotides can be rendered more resistant to degradation in vivo by substituting one or more internal artificial internucleotide linkages for the native phosphodiester linkages, for example, by replacing phosphate with sulphur in the linkage.
  • linkages that may be used include phosphorothioates, methylphosphonates, sulphone, sulphate, ketyl, phosphorodithioates, various phosphoramidates, phosphate esters, bridged phosphorothioates and bridged phosphoramidates.
  • Such examples are illustrative, rather than limiting, since other internucleotide linkages are known in the art. See, for example, Cohen, (1990) Trends in Biotechnology.
  • oligonucleotides having one or more of these linkages substituted for the phosphodiester internucleotide linkages is well known in the art, including synthetic pathways for producing oligonucleotides having mixed internucleotide linkages.
  • Oligonucleotides can be made resistant to extension by endogenous enzymes by "capping" or incorporating similar groups on the 5' or 3' terminal nucleotides.
  • a reagent for capping is commercially available as Amino-Link JJTM from Applied BioSystems Inc, Foster City, CA. Methods for capping are described, for example, by Shaw et al (1991) Nucleic Acids Res. 19, 747-750 and Agrawal et al (1991) Proc. Natl. Acad. Sci. USA 88(17), 7595-7599, the teachings of which are hereby incorporated herein by reference.
  • oligonucleotides resistant to nuclease attack are for them to be "self-stabilised” as described by Tang et al (1993) Nucl. Acids Res. 21, 2729-2735 incorporated herein by reference.
  • Self-stabilised oligonucleotides have hairpin loop structures at their 3' ends, and show increased resistance to degradation by snake venom phosphodiesterase, DNA polymerase I and fetal bovine serum.
  • the self-stabilised region of the oligonucleotide does not interfere in hybridization with complementary nucleic acids, and pharmacokinetic and stability studies in mice have shown increased in vivo persistence of self-stabilised oligonucleotides with respect to their linear counterparts.
  • antisense agents also include larger molecules which bind to said interacting polypeptide mRNA or genes and substantially prevent expression of said interacting polypeptide mRNA or genes and substantially prevent expression of said interacting polypeptide.
  • expression of an antisense molecule which is substantially complementary to said interacting polypeptide is envisaged as part of the invention.
  • the said larger molecules may be expressed from any suitable genetic construct as is described below and delivered to the patient.
  • the genetic construct which expresses the antisense molecule comprises at least a portion of the said interacting polypeptide coding sequence operatively linked to a promoter which can express the antisense molecule in the cell.
  • Suitable promoters will be known to those skilled in the art, and may include promoters for ubiquitously expressed, for example housekeeping genes or for tissue- specific genes, depending upon where it is desired to express the antisense molecule.
  • the genetic construct can be DNA or RNA it is preferred if it is DNA.
  • the genetic construct is adapted for delivery to a human cell.
  • constructs of the invention may be introduced into the cells by any convenient method, for example methods involving retroviruses, so that the construct is inserted into the genome of the (dividing) cell.
  • Other methods involve simple delivery of the construct into the cell for expression therein either for a limited time or, following integration into the genome, for a longer time.
  • An example of the latter approach includes liposomes (Nassander et al (1992) Cancer Res. 52, 646-653).
  • Other methods of delivery include adenoviruses carrying external DNA via an antibody- polylysine bridge (see Curiel Prog. Med. Virol. 40, 1-18) and transferrin- polycation conjugates as ca ⁇ iers (Wagner et al (1990) Proc. Natl. Acad. Sci. USA 87, 3410-3414).
  • the DNA may also be delivered by adenovirus wherein it is present within the adenovirus particle.
  • naked DNA and DNA complexed with cationic and neutral lipids may also be useful in introducing the DNA of the invention into cells of the patient to be treated.
  • Non- viral approaches to gene therapy are described in Ledley (1995) Human Gene Therapy 6, 1129-1144.
  • Alternative targeted delivery systems are also known such as the modified adenovirus system described in WO 94/10323 wherein, typically, the DNA is ca ⁇ ied within the adenovirus, or adenovirus-like, particle.
  • Michael et al (1995) Gene Therapy 2, 660-668 describes modification of adenovirus to add a cell-selective moiety into a fibre protein.
  • a further aspect of the invention provides a virus or virus-like particle comprising a genetic construct of the invention.
  • suitable viruses or virus-like particles include HSV, AAV, vaccinia and parvovirus.
  • a ribozyme capable of cleaving the interacting polypeptide RNA or DNA may be administered in substantially the same and using substantially the same vehicles as for the antisense molecules.
  • Ribozymes which may be encoded in the genomes of the viruses or virus-like particles herein disclosed are described in Cech and Herschlag "Site-specific cleavage of single stranded DNA” US 5,180,818; Altman et al "Cleavage of targeted RNA by RNAse P" US 5,168,053, Cantin et al "Ribozyme cleavage of HTV-1 RNA” US 5,149,796; Cech et al "RNA ribozyme restriction endoribonucleases and methods", US 5,116,742; Been et al "RNA ribozyme polymerases, dephosphorylases, restriction endonucleases and methods", US 5,093,246; and Been et al "RNA ribozyme polymerases, dephospho
  • the genetic constructs of the invention can be prepared using methods well known in the art.
  • the compound of the invention or a polypeptide of the invention or a variant, fragment, fusion or derivative or a fusion of a variant, fragment or derivative, polynucleotide, composition or medicament of the invention may be administered in any suitable way, usually parenterally, for example intravenously, intraperitoneally or intravesically, in standard sterile, non- pyrogenic formulations of diluents and ca ⁇ iers.
  • the compound of the invention or a polypeptide of the invention or a variant, fragment, fusion or derivative or a fusion of a variant, fragment or derivative, polynucleotide, composition or medicament of the invention may also be administered in a localised manner, for example by injection.
  • the compound is administered orally, although this is not preferred for peptides.
  • the compound may be administered intravenously, parenterally or subcutaneously, although these are not prefe ⁇ ed.
  • a further aspect of the invention provides any novel polypeptide comprising a SH2 domain and a PH domain as herein disclosed.
  • FIG. 1 Amino acid sequence of human and mouse DAPPl. The amino acid residues equivalent to the SH2 domain are indicated by a solid line and those of the PH domain by a dashed line.
  • FIG. 1 Alignment of the SH2 and PH domains of DAPPl with other proteins possessing these domains.
  • A The alignment of the SH2 domains was ca ⁇ ied out using the Clustal W program. The NCBI accession number for Src is P12931, for PLC- ⁇ l is P19174, for PI 3-kinase p85 ⁇ subunit is P27986, for PTP-2C is Q06124, for APS is BAA 22514 and for Ras GAP is P20936.
  • the NCBI accession numbers for Grpl is AF001871, for PDK1 is AA 0 17995, for PKB is S33364, for pleckstrin is P08567 and for Ras p21 GAP is P20936.
  • the four key residues thought to be required for high affinity interaction with PtdIns(3,4,5)P 3 [4] are indicated with asterisks. Numbering is based on the sequences in the data base.
  • the conserved Arg residue found in all SH2 domains and the conserved Trp residue found in all PH domains is shaded in black, and homologous residues found in these domains are boxed.
  • FIG. 3 Tissue distribution of human DAPPl mRNA expression.
  • Full length DAPPl was amplified by PCR from a cDNA panel derived from the indicated human tissues (see materials and methods). After 30, 34 and 38 cycles of PCR an aliquot of the reaction was removed and analysed by electrophoresis on agarose gel. The PCR product identified as DAPPl was verified from each tissue by DNA sequencing and a Southern blot. As a control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was amplified by PCR from the same cDNA panel. Identical results to those shown were obtained in three separate experiments.
  • GPDH glyceraldehyde 3-phosphate dehydrogenase
  • FIG. 4 Comparison of the phospholipid binding properties of DAPPl with PDK1 and PKB.
  • the ability of the following GST fusion proteins of human DAPPl (hDAPPl) or mouse DAPPl (mDAPPl), as well as PDK1 and PKB to bind a variety of phospholipids (see below) was analysed using a protein-lipid overlay.
  • the indicated, phospholipids (3 pmol) were spotted onto a nitrocellulose membrane which was then incubated with the purified GST fusion proteins.
  • the membranes were washed and the GST-fusion proteins bound to the membrane by virtue of their interaction with lipid was detected using a GST antibody.
  • a representative experiment of three is shown.
  • Figure 5 Nucleotide sequences encoding human and mouse DAPPl.
  • 293 cells were transiently transfected with DNA constructs expressing GST- DAPP1. 24 h post transfection the cells were deprived of serum for 16h and incubated for 10 min in the absence or presence of 100 nM wortmannin. The cells were then immediately stimulated with either 100 nM insulin or 100 ng/ml IGF1 or lOOng/ml EGF for the times indicated. The cells were lysed in lysis buffer (as used in the paper) and the GST-DAPP1 purified from 0.5 mg of lysate by affinity chromatography of the lysate on glutathione-Sepharose beads. The samples were made 1 % SDS, and subjected to SDS/polyacylamide gel electrophoresis and immunoblotted using an anti- phosphotyrosine antibody (termed PY99 from Santa Cruz).
  • FIG. 7 Dephosphorylation of DAPPl by tyrosine phosphatase PTP-1B 293 cells were transiently transfected with DNA constructs expressing GST- DAPP1. 24 h post transfection the cells deprived of serum for 16h and then stimulated with 100 ng/ml IGFl for 10 min to maximally phosphorylate DAPPl. The cells were lysed and the GST-DAPP1 purified from the lysate by affinity chromatography of the lysate on glutathione-Sepharose beads, and the concentration of purified GST-DAPP1 was established.
  • the indicated concentrations of GST-DAPP1 were then incubated for 30 min at 30 °C in the absence or presence of O.lmg/ml of the tyrosine phosphatse termed PTP-1B.
  • the samples were made 1 % SDS, and subjected to SDS/polyacylamide gel electrophoresis and immunoblotted using the PY99 anti-phosphotyrosine antibody.
  • FIG. 8 DAPPl is phosphorylated at Tyrl39 in stimulated cells.
  • A 293 cells were transiently transfected with DNA constructs expressing wild type GST-DAPP1. 24 h post transfection the cells were serum starved for 16 h and stimulated with insulin (100 nM), IGFl (100 ngml) or EGF (100 ng/ml) for the indicated times. The cells were lysed, GST-DAPP1 purified from aliquots of the supernatant by affinity chromatography on glutathione-Sepharose.
  • the purified protein was electrophoresed on a 7.5 % SDS/polyacrylamide gel and immunoblotted using an anti-phosphotyrosine antibody (anti-pTyr) or a FLAG antibody to detect GST-DAPP1 (GST-DAPP1 is also flag epitope tagged [11; Example 4]).
  • anti-pTyr anti-phosphotyrosine antibody
  • FLAG antibody FLAG antibody to detect GST-DAPP1
  • GST-DAPP1 is also flag epitope tagged [11; Example 4].
  • B As in (A), except that the 293 cells were transiently transfected with DNA constructs expressing wild type (WT) GST-DAPP1 or the GST-DAPP1[Y139F] mutant. The cells were stimulated with insulin(10 min) and IGFl (10 min) or EGF (5 min).
  • FIG. 9 Phosphorylation of DAPPl is dependent on the PH domain and requires PI 3-kinase.
  • A 293 cells were transiently transfected with DNA constructs expressing wild type (WT) GST-DAPP1 or mutants of GST- DAPP1 that do not interact with 3- ⁇ hosphoinositides (GST-DAPP1[K173L] or GST-DAPP1[W250L].
  • WT wild type
  • GST-DAPP1[K173L] or GST-DAPP1[W250L 3- ⁇ hosphoinositides
  • the cells were serum starved for 16 h prior to stimulation with 100 nM insulin (10 min), 100 ng/ml IGFl (10 min) and 100 ng/ml EGF (5 min).
  • the cells were lysed, and GST- DAPP1 purified and immunoblotted using an anti-phosphotyrosine antibody (anti-pTyr) or a FLAG antibody to detect GST-DAPP1.
  • anti-pTyr anti-phosphotyrosine antibody
  • FLAG antibody FLAG antibody to detect GST-DAPP1.
  • B As in (A) except 293 cells were transiently co-transfected with DNA constructs expressing GST-DAPP1[WT] or GST-DAPP1[Y139F] together with constructs expressing membrane targetted wild type PI 3-kinase (pi 10*), membrane targetted kinase dead PI 3-kinase (pllO-KD) or a dominant negative PI 3- kinase ( ⁇ p85 ⁇ ) or empty pCMV5 vector (vector).
  • pi 10* membrane targetted wild type PI 3-kinase
  • pllO-KD membrane targetted kinase dead PI 3-kinase
  • FIG. 10 Phosphorylation of DAPPl at Tyrl39 by Src-family kinases in vivo.
  • 293 cells were transiently transfected with DNA constructs expressing wild type GST-DAPP1. 24 h post transfection, the cells were serum starved for 16 h and incubated with DMSO (-) or the indicated concentrations of PP2 and PP3, 20 min prior to stimulation with 100 nM insulin (10 min), 100 ng/ml IGFl (10 min) and 100 ng/ml EGF (5 min). The cells were lysed, and GST-DAPP1 purified and immunoblotted using an anti-phosphotyrosine antibody (anti-pTyr) or a FLAG antibody to detect GST-DAPP1.
  • anti-pTyr anti-phosphotyrosine antibody
  • FLAG antibody FLAG antibody
  • FIG. 11 Phosphorylation of DAPPl at Tyrl39 by Src-family kinases in vitro.
  • Src-family tyrosine kinases were incubated with purified wild type (WT) GST DAPPl or GST-DAPP1 [Y139F] in the presence of ⁇ PATP as described in the materials and methods section. The reactions were analysed by electrophoresis on a 7.5% poly aery lamide gel which was stained with Coomassie Blue and the gels autrodiographed to detect phosphorylated DAPPl.
  • the tryptic peptide map of 32 P-labelled DAPPl is shown and 85% of the radioactivity applied to the column was recovered from the major 32 P-peptide eluting at 18.8% acetonitrile.
  • D A portion of the major 32 P-peptide (5 pmol) was analysed on an Applied Biosystems 476A sequencer, and the phenylthiohydantoin amino acids identified after each cycle of Edman degradation are shown using the single letter code for amino acids. A portion of this peptide (2000 cpm) was also coupled covalentiy to a Sequelon arylamine membrane and analysed on an Applied Biosystems 470A sequencer, and the 32 P radioactivity released was measured after each cycle of Edman degradation.
  • Example 1 DAPPl: A Dual Adaptor for Phosphotyrosine and 3- Phosphoinositides.
  • DAPPl This example describes the identification and initial characterisation of a widely expressed novel protein termed DAPPl which contains both a PH domain that interacts specifically with PtdIns(3,4,5)P 3 /PtdIns(3,4)P 2 as well as a phosphotyrosine-binding SH2 domain.
  • DAPPl a widely expressed novel protein termed DAPPl which contains both a PH domain that interacts specifically with PtdIns(3,4,5)P 3 /PtdIns(3,4)P 2 as well as a phosphotyrosine-binding SH2 domain.
  • This protein is likely to play an important role in triggering signal transduction pathways that lie downstream from receptor tyrosine kinases and PI 3-kinase.
  • GST glutathione-S-transferase
  • PKB Protein kinase B
  • PKC Protein kinase C
  • PDK 3-phosphoinositide-dependent protein kinase- 1
  • PtdIns Phosphatidylinositol
  • PI3 kinase Phosphoinositide 3-kinase
  • PH pleckstrin homology.
  • Hybond-Cextra was from Amersham Pharmacia, Advantage Taq cDNA PCR kit, Marathon-Ready human placenta and mouse lung cDNA libraries, Multiple Tissue Northern Blot, and human cDNA Multiple Tissue cDNA panel were from Clontech, Human Universal cDNA Library was purchased from Strategene, pCR 2.1 Topo vector from InVitrogen.
  • PKB [11] and PDK1 [ 12] were expressed as fusion proteins with glutathione-S-transferase (GST) in 293 cells.
  • PDK1 [13] was also expressed in insect cells with a His tag.
  • Buffer A 50 mM Tris-HCl pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 % (by mass) Triton-X 100, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M sucrose, 1 ⁇ M rnicrocystin-LR, 0. 1 % (by vol) ⁇ -mercaptoethanol and "complete" proteinase inhibitor cocktail (one tablet per 50 ml, Roche).
  • Buffer B 50 mM Tris/HCl pH 7.5, 0.1 mM EGTA, 10 mM ⁇ -mercaptoethanol and 0.27M sucrose.
  • DAPPl A partial human DAPPl (hDAPPl) sequence encoding the C-terminal 156 residues of DAPPl was obtained from DNA sequencing of the EST clones listed in Table 1. The 5' end of the DAPPl cDNA was obtained by two independent procedures. Firstly, by carrying out a 5' -RACE using the Marathon-Ready human placenta cDNA library as a template, and an antisense primer derived from DAPPl (5'-
  • AACGACCGAGATCGGATCGTGCC-3' an adaptor primer, API, provided with the library.
  • the PCR product was cloned into pCR2.1TOPO vector (InVitrogen). Three independent clones possessed identical sequences, and all had the same in frame stop codon 5 * to the predicted initiating ATG codon. We were also able to amplify the predicted full length DAPPl by PCR using primers specific to the 5' (5'CACAGAGCGAGAAGGTGTCAG GAGC3') and 3'(5' CAAGGAGATGGCAACATCATGG 3') untranslated regions of DAPPl.
  • the 5' end of the mouse DAPPl cDNA was obtained by carrying out a 5' -RACE PCR reaction using the Marathon-Ready mouse cDNA placenta library as a template, and nested primers derived from EST 515361 (5'-
  • DAPPl as a GST-fusion protein in 293 cells.
  • DNA constructs expressing full length human DAPPl (residues 2-280), the isolated SH2 domain of DAPPl (hDAPPl- ⁇ PH residues 2 to 166), or the isolated PH domain of DAPPl (hDAPPl- ⁇ SH2 residues 125 to 280) with the FLAG epitope tag (DYKDDDDK) at the N-terminus were obtained by a standard PCR based approach using Marathon Ready human placenta cDNA as template.
  • the PCR products were designed to incorporate Kpnl restriction sites at the termini and were cloned into the eukaryotic expression vector pEBG-2T [14], which codes for the expression of these constructs with an N- terminal GST tag.
  • the construct encoding the isolated mouse PH domain (mDAPPl - ⁇ SH2 residues 125 to 280) was also prepared as above except that EST515361 was used as the template for the PCR with Spel restriction sites designed for the termini.
  • PH domain mutants were generated by site-directed mutagenesis ca ⁇ ied out using the QuikChange Kit (Strategene) following instructions provided by the manufacturer.
  • the suspension was centrifuged for 1 min at 3000 x g, the beads washed three times with 10 ml of Buffer A containing 0.5 M NaCl, and then a further ten times with 10 ml of Buffer B.
  • the protein was eluted from the resin at ambient temperature by incubation with 1.0 ml of Buffer B containing 20 mM glutathione, and the beads removed by centifugation through a 0.44 micron filter. The eluate was divided into aliquots, snap frozen in liquid nitrogen, and stored at -80°C.
  • a kit consisting of single stranded cDNA isolated from different human tissues was obtained from Clontech. These were used as template to obtain the full length hDAPPl by PCR using the sense primer 5' CACAGAGCGAGAAGGTGTCAGGAGC 3' and the antisense primer 5' CAAGGAGATGGCAACATCATGG 3'.
  • the following PCR conditions were employed: the initial denaturing step was ca ⁇ ied out at 94°C for 1 min followed by 30-38 cycles of 94°C for 0.5 min and 68°C for 4 min using Advantage Taq (Clontech). Control reactions to PCR glyceraldehyde 3- phosphate dehydrogenase were ca ⁇ ied out in parallel using the primers provided by Clontech.
  • Protein-Lipid overlay To assess the phospholipid binding properties of each wild type and mutant DAPPl, a Protein Lipid Overlay was performed using the GST fusion protein [16,17]. 1 ⁇ l of lipid solution containing 3 pmol of phospholipids dissolved in a mixture of choroform:methanol:water (1:2:0.8) was spotted onto Hybond-C extra membrane and allowed to dry at room temperature for 1 h. The membrane was blocked in 3% (by mass) fatty acid-free BSA in TBST (10 mM Tris/HCl pH 8.0, 150 mM NaCl and 0.1 % Tween-20 (by vol) for lh.
  • the membrane was then incubated overnight at 4°C in the same solution containing 0.5 ⁇ g/ml of the indicated GST DAPPl fusion protein.
  • the membrane was washed 6 times over 30 min in TBST and then incubated for lh with 1/1000 dilution of anti-GST monoclonal antibody (Sigma).
  • the membrane was washed as before, then incubated for lh with 1/5000 dilution of anti-mouse-HRP conjugate (Pierce).
  • the membrane was washed as above and the GST-fusion protein bound to the membrane, by virtue of its interaction with phospholipid detected by enhanced chemiluminescence.
  • DAPPl binding did not fit well to this model due to the slow dissociation of the protein from the surface. Therefore, the affinity of binding of DAPPl to polyphosphoinositides is likely to be overestimated by this method but are given as apparent equilibrium dissociation constants for comparative purposes.
  • DAPPl is a novel SH2 and PH domain containing protein.
  • the NCBI EST database was inte ⁇ ogated with the DNA sequence encoding the N- terminal region of the PH domain of human PKB ⁇ (residues 8 to 33). This search revealed several EST sequences (see Table 1) encoding the partial sequence of a novel PH domain-containing protein most homologous to the PH domains of PKB, PDK1 and other PH domains known to interact with PtdIns(3,4,5)P 3 . Full length clones were then isolated from human and mouse cDNA libraries as described in the methods.
  • the open reading frame encoded a protein of 280 amino acids with a predicted molecular mass of 32 kDa, and the human and mouse sequences were 93 % identical (Fig 1).
  • a stop codon immediately 5' to the predicted initiating ATG codon in the human sequence indicated that the protein sequence was full length.
  • Both the human and mouse proteins start with a Met-Gly sequence (Fig 1) which indicates that after cleavage of the initiating methionine, DAPPl may be myristoylated at the N-terminal glycine residue [18].
  • Analysis of the sequence revealed the presence of a SH2 domain at the N-terminus of the protein and a PH domain at its C-terminus (Fig 2).
  • the former contains the conserved residues present in all known SH2 domains including the invariant Arg residue (Arg 61, Fig 2A).
  • the PH domain contains the conserved residues found in all PH domains as well as the invariant Tip residue (Trp250, Fig 2B).
  • the PH domain also contains the motif K-X 8 _ 12 -R/K-X-R-Hyd where X is any amino acid and Hyd is a hydrophobic amino acid (Fig 2B), found in all the PH domains that interact with PtdIns(3,4,5)P3 and Ptdlns(3,4)p2 with high affinity [4]. Because of these features this protein was termed DAPPl, for Dual Adaptor for Phosphotyrosine and 3- Phosphoinositides .
  • DAPPl MRNA Tissue distribution of DAPPl MRNA.
  • Phospholipids including phosphoinositides were spotted on to a nitrocellulose membrane, and incubated with the indicated DAPPl GST fusion protein and with GST- PDKI or GST-PKB ⁇ as controls. The membranes were then washed and immunoblotted using a GST antibody to detect the GST fusion proteins bound to the membrane by virtue of their interaction with lipid.
  • DAPPl interacted far less strongly with its stereoisomer SAL-PtdIns(3,4,5)P 3 with an apparent K ⁇ , of 160 nM compared with 330 nM for His-PDKl. Both His-PDKl and GST- hDAPPl interacted weakly with the diC 16 PtdIns(3,4,5)P 3 /PtdIns(3,4)P 2 . However, the magnitude of the binding was too small to give reliable estimates of the apparent affinities of PDKl or DAPPl to these lipids. Nevertheless, the selectivities for these lipids were revealed by the protein lipid overlay assay (Fig 4). Therefore, as observed previously with PKB and PDKl, DAPPl interacts with highest affinity with the physiological PtdIns(3,4,5)P 3 enantiomer.
  • APS is composed solely of a SH2 domain and a PH domain. APS appears to associate with activated B-cell receptors [20], growth factor receptors [21], and insulin receptors [22] and becomes tyrosine phosphorylated in response to agonists that activate these receptors.
  • B-cell receptors [20] activated B-cell receptors [21]
  • insulin receptors [22] becomes tyrosine phosphorylated in response to agonists that activate these receptors.
  • the PH domain of APS is located N-terminal to the SH2 domain, suggesting a different evolutionary origin.
  • APS is unlikely to interact with PtdIns(3,4,5)P 3 /PtdIns(3,4)P 2 with high affinity and specificity because it lacks three out of the four conserved residues in the consensus 3-phosphoinositide binding motif lying in the N- terminal region of the PH domain (see Fig 2B). All proteins that bind with high affinity to PtdIns(3,4,5)P 3 such as DAPPl, PKB, human PDKl, GRPl and BTK possess this motif [4]. Plant PDKl, although possessing a PH domain very similar in sequence to that of human PDKl, lacks two of the four residues.
  • DAPPl could potentially be recruited to the cell membrane by three mechanisms. Firstly, it possesses a putative myristoylation site at the N- terminus which could facilitate DAPPl's interaction with the lipid bilayer. Secondly, DAPPl possesses a SH2 domain which could interact with phosphotyrosine residues on membrane associated proteins such as activated tyrosine kinase receptors. Thirdly, DAPPl contains a PH domain which exhibits a high affinity interaction with the PtdIns(3,4,5)P 3 /PtdIns(3,4)P 2 second messengers produced at the cell membrane following the activation of PI 3-kinases. It is likely that DAPPl functions as an adaptor to recruit other proteins to the plasma membrane in response to extracellular signals. Future work will focus on identifying the proteins with which DAPPl interacts and the roles of the putative myristoylation site, SH2 and PH domains in the function of DAPPl.
  • Figure 6 demonstrates that DAPPl becomes tyrosine phosphorylated when cells are stimulated with insulin, IGFl or EGF. Incubation of cells with the PI 3-kinase inhibitor wortmannin prevents the tyrosine phosphorylation of DAPPl. This establishes that DAPPl is likely to be a downstream of PI 3-kinase in cells.
  • Figure 7 demonstrates that incubation of tyrosine phosphorylatd DAPPl with a tyrosine phosphatase termed PTB-1 or LAR, results in it becoming dephosphorylated. This indicates that DAPPl is tyrosine phosphorylated.
  • Example 3 Identification of a tyrosine kinase capable of phosphorylating DAPPl.
  • Unstimulated or IGFl, Insulin or EGF stimulated cell extracts are fractionated on an anion or cation exchange chromatographic column. Fractions of these columns are then incubated with unphosphorylated GST-DAPP1 (purified from either unstimulated mammalian cells or expressed in bacteria) together with 0.1 mM gamma 32 P-ATP 10 mM magnesium acetate, 10 mM manganese acetate, 50 mM Tris pH 7.5, 0.1 % (by vol) 2-mercaptoethanol in the presence or absence of phospholipid vesicles consisting of 0.1 mM phosphatidylserine, O.lmM phosphatidylcholine and O.OlmM sn-l-stearoyl-2-arachidonoyl-D- PtdIns(3,4,5)P3.
  • the assays are incubated at 30 °C for 30 min, then made 1 % SDS, and subjected to SDS/polyacylamide gel electrophoresis and immunoblotted using either the PY99 anti-phoshotyrosine antibody or a phosphospecific antibody recognizing DAPPl specifically phosphorylated at Tyrl39.
  • the screen identifies a tyrosine kinase activity that is stimulated by the presence of sn-l-stearoyl-2-arachidonoyl-D- PtdIns(3,4,5)P3 or activated by stimulation of cells with insulin, IGFl or EGF.
  • a tyrosine kinase activity which is stimulated by insulin, IGFl or EGF is inhibited by incubating cells with wortmannin.
  • Another method of identifying a tyrosine kinase which phosphorylates DAPPl is to carry out a yeast 2 hybrid screen of DAPPl.
  • known tyrosine kinase such as Src, Lck, Fyn and receptor tyrosine kinases are tested to see if they phosphorylate DAPPl, for example as described in Example 4.
  • Example 4 Phosphoinositide 3-kinase dependent phosphorylation of the Dual Adaptor of Phosphotyrosine and 3-Phosphoinositide, by the Src-family of tyrosine kinase.
  • Tyrl39 following agonist stimulation of cells.
  • Overexpression of a constitutively active form of PI 3-kinase induced the phosphorylation of DAPPl in unstimulated cells.
  • Tyrl39 of DAPPl is likely to be phosphorylated in vivo by a Src-family tyrosine kinase, as the specific Src-family inhibitor PP2 but not an inactive variant of this drug, PP3, prevented the agonist-induced tyrosine phosphorylation of DAPPl in cells.
  • PtdIns(3,4,5)P3 or PtdIns(3,4)P2 bind to DAPPl, recruiting it to the plasma membrane, where it becomes phosphorylated at Tyrl39 by a Src- family tyrosine kinase.
  • Insulin was from Novo-Nordisk; IGFl and EGF from Life Technologies; PP2 and PP3 from Calbiochem; Protease inhibitor tablets from Roche; wortmannin, LY 294002 and anti-FLAG M2 antibody from Sigma.
  • Antibodies against phosphotyrosine (PY99), Lyn and Lck were from Santa Cruz; partially purified Src and Lyn as well as the anti-Src antibody were from Upstate Biotechnology and anti-CD2 antibody was from Plasminogen. Lck was expressed in sf9 cells infected with baculovirus vectors by Dr Andrew Paterson in the MRC Unit.
  • Plasmids The constructs encoding wild type and mutant DAPPl with an N-terminal glutathione-S-transferase (GST) tag followed by a Flag epitope tag, were subcloned into either pEBG2T for expression in 293 cells (as described previously [11]), or pGEX-4T-2 for expression in E.Coli.
  • GST glutathione-S-transferase
  • Constructs expressing Lyn, Itk and Bmx (in the pSR ⁇ vector), JAK2 (in the pSVK3 vector), Btk (in the pRK5 vector) and FAK (in the pCMV5 vector) were provided by Hiroyuki Mano (Jichi Medical School, Tochigi, Japan).
  • the construct expressing Lck (in the pCDNA vector) was provided by Dr Bart Sefton (The Salk Institute, California, USA).
  • the construct expressing c-Src (in the pUSEamp vector) was purchased from UBI. Transfection, cell stimulation and immunoblotting.
  • the cells were lysed in 1 ml of ice- cold lysis buffer (50 mM Tris-HCl pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 % (by mass) Triton-XlOO, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M sucrose, 1 ⁇ M microcystin-LR, 0.1 % (by vol) ⁇ -mercaptoethanol and one tablet of protease inhibitor cocktail per 50 ml of buffer).
  • ice- cold lysis buffer 50 mM Tris-HCl pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 % (by mass) Triton-XlOO, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M sucrose, 1 ⁇ M microcystin-LR, 0.1 % (by
  • the lysates were cleared by centrifugation at 13 000 x g for 10 min at 2 °C, and aliquots of supernatant (50 ⁇ g protein) were incubated for 1 h at 4°C with 10 ⁇ l of glutathione-Sepharose.
  • the beads were washed twice in lysis buffer containing 0.5 M NaCl, followed by two further washes with Buffer B (50mM Tris-HCl pH7.5, O. lmM EGTA and 0.1 % v/v ⁇ - mercaptoethanol).
  • the beads were resuspended in 1 vol of Sample Buffer containing 100 mM Tris/HCl pH 6.8, 4% (by mass) SDS, 20% (by vol) glycerol and 200 mM DTT, and the released protein subjected to SDS polyacrylamide gel electrophoresis.
  • the gels were analysed by immunoblotting with the indicated antibodies. Briefly, membranes were blocked in 50 mM Tris/HCl pH 7.5, 0.15M NaCl, 0.5% (by vol) Tween (TBST) containing either 3 mg/ml bovine serum albumin (for anti- phosphotyrosine blots) or 10% (by mass) skimmed milk (for all other blots performed).
  • nitrocellulose membranes were immunoblotted in the same buffer with anti-phosphotyrosine PY99 antibody (at a dilution of 1:2000), anti-Flag monoclonal antibody (1:10000) and anti-Src, Lck, Lyn, CD2 antibody (1:1000). Detection was performed using horse radish peroxidase conjugated secondary antibodies and the enhanced chemiluminescence reagent (Amersham).
  • the pGEX-4T-2 constructs encoding wild type DAPPl and DAPPl [Y139F] were transformed into BL21 E.coli cells and a 0.5L culture was grown at 37 °C in Luria Broth containing 100 ⁇ g/ml ampicillin, until the absorbance at 600 nm was 0.6. 250 ⁇ M isopropyl- ⁇ -D-galactosidase was added and the cells cultured for a further 16 h at 26 °C. The cells were lysed and the GST-DAPP1 fusion proteins purified by affinity chromatography on glutathione-Sepharose beads as described previously for ERK2 [16].
  • DAPPl is phosphorylated at Tyrl39 in cells.
  • Full length human DAPPl was expressed in human 293 cells as a GST-fusion protein, the cells serum starved for 16h, and then stimulated with either insulin, IGFl or EGF.
  • the cells were lysed, GST-DAPP1 purified by affinity chromatography on glutathione- Sepharose, and the GST-DAPP1 immunoblotted with an anti- phosphotyrosine antibody (Fig 8A).
  • These agonists induced a rapid phosphorylation of DAPPl within 1-2 min, which was sustained for 80 min after stimulation with insulin, but was more transient with IGFl and EGF (Fig 8A).
  • Tyrl39 of DAPPl is located between the SH2 and PH domains of DAPPl and is the only tyrosine residue in the DAPPl protein not located in the SH2 or PH domains. To our knowledge no tyrosine residue in a SH2 or PH domain has previously been shown to be phosphorylated. We therefore, speculated that Tyrl39 was the site of phosphorylation on DAPPl. To investigate this we mutated Tyrl39 to Phe and expressed this mutant in 293 cells as a GST-fusion protein. Stimulation of cells with insulin, IGFl or EGF failed to induce any detectable phosphorylation of the GST-DAPP1[Y139F] mutant (Fig 8B), indicating that Tyrl39 was indeed the site of phosphorylation.
  • Phosphorylation of DAPPl is downstream of PI 3-kinase.
  • PI 3-kinase inhibitors wortmannin (100 nM) or LY 294002 (100 ⁇ M) to the tissue culture medium at concentrations that inhibit the insulin-induced activation of PKB in these cells [18] prior to stimulation of the cells. These inhibitors prevented tyrosine phosphorylation of DAPPl induced by insulin, IGFl and EGF (Fig 8C).
  • a membrane targetted, catalytically inactive mutant of PI 3-kinase failed to induce tyrosine phosphorylation of DAPPl and the overexpression of a dominant negative mutant of PI 3-kinase (termed ⁇ p85 ⁇ ) inhibited the IGFl -induced tyrosine phosphorylation of DAPPl (Fig 9B).
  • overexpression of the constitutively activated form of PI 3-kinase did not induce tyrosine phosphorylation of the mutants of GST-DAPP1 which no longer interact with 3-phosphoinositides (GST- DAPPl [K173L] and GST-DAPP1 [W250L]) (Fig 9C) .
  • PP2 inhibits the Src family kinase members tested with an IC 50 of " 5 nM in vitro and does not significantly inhibit any other class of tyrosine kinase tested [20, 21] or 25 serine/threonine protein kinases tested (S.Davis, H. Reddy & P.Cohen, unpublished work).
  • PP2 potently inhibited the insulin-induced tyrosine phosphorylation of DAPPl (Fig 10A), even at very low concentrations of this drug (0.1 ⁇ M).
  • PP3 even at a concentration of 10 ⁇ M, did not inhibit insulin-mediated tyrosine phosphorylation, significantly (Fig 10A).
  • PP2 also inhibited the tyrosine phosphorylation of DAPPl induced by IGFl and EGF (Fig 10A).
  • DAPPl Phosphorylation was accompanied by a decrease in the electrophoretic mobility of DAPPl and was not increased further by stimulating cells with insulin, IGFl or EGF (data not shown).
  • DAPPl with other tyrosine kinases including the focal adheson kinase, Janus kinase-2 and the Tec tyrosine kinases (BTK, Itk and BMX) failed to induce phosphorylation of wild type GST-DAPP1 (data not shown).
  • the site on DAPPl phosphorylated by Lck was also mapped by phosphorylating DAPPl with Lck to 0.8 mol phosphate per mol protein, digesting the 32p-i a belled DAPPl with trypsin and subjecting the resulting peptides to chromatography on a CJS column at pH 1.9. Only one major 32p_i a b e ii ec i peptide eluting at 18.8 % acetonitrile was observed (Fig 11C). Its sequence commenced at residue 132 of DAPPl and a single burst of radioactivity occu ⁇ ed after the eighth cycle of Edman degradation (Fig 11D).
  • DAPPl becomes phosphorylated at a single tyrosine residue (Tyrl39) following stimulation of 293 cells with agonists such as insulin, IGFl and EGF.
  • the phosphorylation of DAPPl on Tyrl39 is likely to be downstream of PI 3-kinase as inhibitors of PI 3- kinase (Fig 8C) and expression of a dominant negative PI 3-kinase mutant prevented agonist-induced DAPPl tyrosine phosphorylation (Fig 9B).
  • DAPPl also possesses an SH2 domain at its N-terminus and, in order to investigate the role of this domain, we mutated a conserved Arg residue (Arg 61 to a Glu) that in principle should prevent the SH2 domain of DAPPl from binding to tyrosine phosphorylated proteins.
  • Arg 61 to a Glu conserved Arg residue
  • the Src-family of protein kinases contain eight members some which are broadly expressed (e.g. Src) and others whose expression is restricted to certain tissues (such as Lck and Lyn, which are mainly expressed in haematopoietic cells) [24]. These kinases possess overlapping substrate specificities, are known to play key functions in regulating signal transduction pathways and have also been shown to play a role in several human malignancies [25]. Src-family tyrosine kinases are activated by both receptor tyrosine kinases, as well as by receptors that are not protein kinases.
  • DAPPl is likely to be a physiological substrate for the Src-family of tyrosine kinase, as the Src-family inhibitor PP2 prevented agonist induced tyrosine phosphorylation of DAPPl in cells.
  • 293 cells express detectable levels of Src and Lyn (data not shown) which may be the kinases responsible for the phosphorylation of transfected GST-DAPP1 at Tyrl39 observed in our experiments.
  • Src, Lyn or Lck phosphorylated DAPPl on Tyrl39 in vitro. Tyrl39 is conserved in human, mouse and sea squirt DAPPl (NCBI accession number AV383515), which would be predicted if phosphorylation of this residue is of physiological importance.
  • Src-family tyrosine kinase are located at the plasma membrane by virtue of myristylation and palmitylation of their N-termini [26], it is likely that the role of Ptdlns(3, 4,5) ⁇ 3 is to recruit DAPPl to the membrane of cells where it can be phosphorylated with Src-family tyrosine kinases.
  • PtdIns(3,4,5)P3/PtdIns(3,4)P2 functions in cells, is to recruit PH domain-containing proteins to the plasma membrane, enabling them to become phosphorylated and hence activated (see introduction). This also appears to be the case for DAPPl which, like BTK, Vav, and Gabl, becomes phosphorylated at tyrosine residues following its interaction with PtdIns(3,4,5)P3/PtdIns(3,4)P2 in cells.
  • DAPPl like BTK and Vav, is phosphorylated by Src-family kinases in vitro at a similar rate in the presence or absence of PtdTns(3,4,5)P3 (Fig 11B) suggesting that the interaction of DAPPl with 3-phosphoinositides does not induce a conformational change that converts it into a substrate for the Src-family of tyrosine kinase. This is in contrast to PKB which is converted into a substrate for PDKl through its interaction with PtdIns(3,4,5)P3 (Fig 11B) [27].
  • results presented in this paper indicate that DAPPl is likely to be a direct cellular target for both PI 3-kinase and Src-family tyrosine kinases, and that the phosphorylation of DAPPl at Tyrl39 may play an important role in triggering signal transduction pathways that lie downstream of both PI 3-kinase and Src-family of tyrosine kinases.
  • Physiological processes regulated by phosphorylation of DAPPl may therefore be inhibited by either PI 3-kinase or Src inhibitors.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un polypeptide sensiblement pure comprenant la séquence d'acide aminé (I) ou (II) ou une variante, un fragment, une fusion ou un dérivé de celle-ci, ou une fusion de cette variante ou de ce fragment ou de ce dérivé. Le polypeptide a un domaine PH et un domaine SH2 et peut interagir avec des protéines tyrosine-phosphorylées et 3-phosphoinositides. Le polypeptide est utile dans les criblages de médicaments.
PCT/GB2000/002599 1999-08-09 2000-07-05 Dapp1, adaptateur double pour phosphotyrosine et 3-phosphoinositides WO2001011042A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU28070/01A AU2807001A (en) 1999-08-09 2000-07-05 DAPP1, a dual adaptor for phosphotyrosine and 3-phosphoinositides

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9918680.1 1999-08-09
GBGB9918680.1A GB9918680D0 (en) 1999-08-09 1999-08-09 Polypeptide

Publications (1)

Publication Number Publication Date
WO2001011042A1 true WO2001011042A1 (fr) 2001-02-15

Family

ID=10858788

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2000/002599 WO2001011042A1 (fr) 1999-08-09 2000-07-05 Dapp1, adaptateur double pour phosphotyrosine et 3-phosphoinositides

Country Status (3)

Country Link
AU (1) AU2807001A (fr)
GB (1) GB9918680D0 (fr)
WO (1) WO2001011042A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023225572A3 (fr) * 2022-05-17 2024-04-11 Nvelop Therapeutics, Inc. Compositions et méthodes pour administration in vivo efficace

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999003994A1 (fr) * 1997-07-18 1999-01-28 Schering Corporation Suppresseurs de signalement de cytokine; socs16 et reactifs correspondants

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999003994A1 (fr) * 1997-07-18 1999-01-28 Schering Corporation Suppresseurs de signalement de cytokine; socs16 et reactifs correspondants

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
S. DOWLER ET AL.: "DAPP1: a dual adaptor for phosphotyrosine and 3-phosphoinositides", BIOCHEMICAL JOURNAL, vol. 342, no. 1, 15 August 1999 (1999-08-15), ISSN:0264-6021, pages 7 - 12, XP002153290 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023225572A3 (fr) * 2022-05-17 2024-04-11 Nvelop Therapeutics, Inc. Compositions et méthodes pour administration in vivo efficace

Also Published As

Publication number Publication date
AU2807001A (en) 2001-03-05
GB9918680D0 (en) 1999-10-13

Similar Documents

Publication Publication Date Title
Dowler et al. DAPP1: a dual adaptor for phosphotyrosine and 3-phosphoinositides
JP4173544B2 (ja) 治療剤としての、または診断におけるｒａｃ−プロテインキナーゼ
US20070196883A1 (en) Enzyme
EP1141003B9 (fr) Methode d'activation de la SGK par phosphorylation.
AU2001263952B2 (en) Tumour suppressor and uses thereof
Dowler et al. Phosphoinositide 3-kinase-dependent phosphorylation of the dual adaptor for phosphotyrosine and 3-phosphoinositides by the Src family of tyrosine kinase
AU2001263952A1 (en) Tumour suppressor and uses thereof
US20100047823A1 (en) Polypeptides
US20020127214A1 (en) RAC-protein kinase as therapeutic agent or in diagnostics
US6482605B1 (en) Protein tyrosine phosphatase PTP20 and related products and methods
WO2001011042A1 (fr) Dapp1, adaptateur double pour phosphotyrosine et 3-phosphoinositides
EP0803571B1 (fr) Protéine TAB1 et ADN codant pour celle-ci
AU771307B2 (en) Identification and functional characterization of a novel ribosomal S6 protein kinase
US6753413B1 (en) P35NCK5A binding proteins
US6297019B1 (en) Recombinant polynucleotides encoding CYP7 promoter-binding factors
US6770467B2 (en) Screening method for ligands with class II P13 kinase modulating activity
EP1200591A1 (fr) Homologue humain de la proteine secretoire neuroendocrine bovine, nesp55, polynucleotides et utilisations associees liees a l'obesite
US7083511B1 (en) Methods
AU734736B2 (en) Nucleic acid sequences that encode a cell growth regulatory protein and uses thereof
AU735512B2 (en) Modulators of BRAC1 activity
US20020001823A1 (en) Novel compounds
US6608025B1 (en) Human NESP55 polypeptides, polynucleotides and uses thereof
US20070020723A1 (en) Ceramide kinase-like proteins
CA2403434A1 (fr) Facteur de transcription humain gata-5

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 10071914

Country of ref document: US

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP