Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
1997-09-09
2003-06-17
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S068100, C435S194000, C424S094500
Reexamination Certificate
active
06579691
ABSTRACT:
The present invention relates to a DNA sequence encoding a novel neuronal protein kinase (NPK) which phosphorylates tau proteins as well as other microtubule associated proteins (MAPs) in positions crucial for the binding to microtubules. The invention further relates to Serine or Theorine residues and epitopes comprising said residues phosphorylated by said NPK on said MAPs, to antibodies specifically binding to said protein kinase, pharmaceutical compositions comprising inhibitors to said protein kinase, in particular for the treatment of Alzheimer's disease and cancer, to diagnostic kits and to in vitro diagnostic methods for the detection of Alzheimer's disease and cancer.
Microtubule associated proteins (MAPs) regulate the extensive dynamics and rearrangement of the microtubule network which is thought to drive neurite outgrowth (reviewed recently by Hirokawa, 1994). Several lines of evidence suggest that the phosphodrylation state of MAPs, balanced by protein kinases and phosphatases in a hitherto unknown way, plays a pivotal role in the modulation of these events. Tau protein, a class of MAPs in mammalian brain (Cleveland et al., 1977), is phosphorylated on several sites in vivo (Butler & Shelanski 1986; Watanabe et al., 1993) and is a substrate for many protein kinases in vitro (reviewed by Lee, 1993; Goedert, 1993; Mandelkow & Mandelkow, 1993; Anderton, 1993). During neuronal degeneration in Alzheimer's disease, tau protein aggregates into paired helical filaments (PHFs), the principal fibrous component of the characteristic neurofibrillary lesions (reviewed by Lee & Trojanowski, 1992). Tau isolated from these aggregates displays some biochemical alterations, of which hyperphosphorylation is the most striking (Grundke-Iqbal et al., 1986; Brion et al., 1991; Ksiezak-Reding et al., 1992; Goedert et al., 1992). Most of the reported aberrant phosphorylation sites are Ser/Thr-Pro sequences (Lee et al., 1991; Biernat et al., 1992; Lichtenberg-Kraag et al., 1992; Gustke et al., 1992; Watanabe et al., 1993), suggesting a dysregulation of proline-directed kinases (Drewes et al., 1992; Mandelkow et al., 1992; Hanger et al., 1992; Vulliet et al., 1992; Baumann et al., 1993; Paudel et al., 1993, Kobayashi et al., 1993) or the corresponding phosphatases (Drewes et al., 1993; Gong et al., 1994). Phosphorylation-dependent antibodies, which discriminate between ‘normal’ tau and the hyperphosphorylated, ‘pathological’ forms, were prepared by several laboratories (Kondo et al., 1988; Lee et al., 1991; Mercken et al., 1992; Greenberg et al., 1992). All of these antibodies were shown to be directed against epitopes of the Ser/Thr-Pro type (Lee et al., 1991; Biernat et al., 1992; Lichtenberg-Kraag et al., 1992; Lang et al., 1992; Watanabe et al., 1993).
The microtubule binding region of tau (
FIG. 1
) includes three or four pseudorepeats of 31 residues each depending on isoform type (Lee et al., 1989; Goedert et al., 1989; Himmler et al., 1989). This region probably forms the building block of the paired helical filaments (Kondo et al., 1988; Wischik et al., 1988; Ksiezak-Reding & Yen, 1991; Wille et al., 1992). It does not contain any of the 14-16 Ser/Thr-Pro motifs, which accumulate in the regions flanking the repeats. However, it contains a conserved Serine residue (Ser262) within the sequence KIGS in the first repeat, which was found to be one of the predominant sites phosphorylated by a tissue extract from brain (Gustke et al., 1992). This site is also found to be phosphorylated in Alzheimer PHF-tau, but not in ‘normal’ tau or fetal tau (Hasegawa et al., 1992). So far, it is the only pathological phosphorylation site found within the repeat domain of tau.
Recently, a site-directed mutagenesis approach was used to show that phosphorylation of tau at this site strongly decreases its microtubule binding capacity, whereas the phosphorylation on Ser/Thr-Pro motifs had only a minor effect (Biernat et al., 1993). This initiated a search for protein kinases in neuronal tissue with the ability to phosphorylate tau at Ser262. The technical problem underlying the present invention was to provide a protein kinase which is causative for the onset of Alzheimer's disease by phosphorylating the crucial Serine 262 residue of human tau protein and a corresponding nucleotide sequence.
The solution to this technical problem is achieved by providing the embodiments characterised in the claims.
Thus, the present: invention relates to a DNA sequence encoding a neuronal protein kinase (NPK) or a functional fragment thereof that is capable of phosphorylating a sequence motive of the type KXGS in tau, MAP4, MAP2 and MAP2c characterised by the following features:
(a) it encodes the amino acid sequence depicted as MARK-1 in Table 6;
(b) it encodes the amino acid sequence depicted as MARK-2 in Table 6; or
(c) it hybridises to the DNA of (a) or (b).
The term “DNA sequence” comprises any DNA sequence such as genomic or cDNA, semisynthetic or synthetic DNA.
It was surprisingly found that none of the prior art kinases is mediating the phosphorylation of the four KXGS motifs in the repeat domain of tau to an extent that is sufficient to explain the biological and pathological effects associated with said phosphorylation. This is particularly true for Serine residue 262 which is indicative of the onset of Alzheimer's disease. Instead, the present invention provides a DNA sequence encoding a novel protein kinase with the above identified features which is responsible for the phosphorylation of the amino acid residues crucial for the onset of Alzheimer's disease. Said protein kinase is, also termed NPK, MARK-1 or MARK-2 throughout this application. The numbering of amino acid residues referred to in this application ensues with regard to the sequence of htau 40, the longest of the human tau isoforms (441 residues, Goedert et al., 1989).
In a preferred embodiment, the present invention further relates to a DNA sequence wherein the neuronal protein kinase (NPK) is characterised by the following features:
(a) it has an apparent molecular weight of 110 kD as determined by SDS-PAGE;
(b) it phosphorylates Serine residues 262, 293, 305, 324 and 356 of human tau protein; and
(c) it comprises the following amino acid sequences
KLDTFCGSPPYAAPELFQGK (SEQ ID NO: 1)
DRWMNVGHEEEELKPYAEP (SEQ ID NO: 2)
(K) SSRQNIPRCRNNI (SEQ ID NO: 3)
In a preferred embodiment of the DNA sequence of the present invention, the NPK is further characterised by the following features:
(d) it is deactivated by phosphatase PP-2A; and
(e) it phosphorylates the following Serine or Threonine residues of tau related microtubule-associated proteins (MAPs) MAP2, MAP2c and MAP4
MAP2/MAP2C: S37, S1536, S1676, S1707, S1792, S1796, S1799
MAP4: T829, T873, T874, T876, S899, S903, S928, S941, S1073
(f) it causes the dissociation of tau, MAP4, MAP2 and MAP2c from microtubules.
Another surprising finding that was made in accordance with the present invention is that the NPK by phosphorylating microtubule-associated proteins other than tau causes dissociation of these proteins from microtubules. This in turn results in the destabilisation of said microtubules, an increased dynamic instability thereof, and the ensuing effects on cell proliferation, cell differentiation, or cell degeneration. The NPK of the invention thus has the capacity to regulate the dynamics and rearrangements of microtubules in brain via the phosphorylation of tau or other MAPs. The finding referred to above has important implications for the role in the kinase of the invention in the generation of cancer.
This is because it is believed that cancer essentially is uncontrolled cell proliferation. Many anti-cancer drugs therefore interfere with cellular division and proliferation by poisoning the microtubules. On the other hand, “oncogenes” are often kinases, the cellular regulation of which is impaired. The deregulation of a kinase equal or homologous to the NPK of the invention could have serious effects on the stability of microtubules of various cell types. As microtub
Biernat Jacek
Drewes Gerard
Mandelkow Eckhard
Mandelkow Eva-Marie
Marshall Gerstein & Borun
Max-Planck-Gesellschaft zur Forderung der Wissenschaften E.V.
Prouty Rebecca E.
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