U.S. patent application number 08/817832 was filed with the patent office on 2003-06-05 for protein kinase npk-110.
Invention is credited to BIERNAT, JACEK, DREWES, GERARD, MANDELKOW, ECKHARD, MANDELKOW, EVA-MARIE.
Application Number | 20030104516 08/817832 |
Document ID | / |
Family ID | 8216419 |
Filed Date | 2003-06-05 |
United States Patent
Application |
20030104516 |
Kind Code |
A1 |
MANDELKOW, ECKHARD ; et
al. |
June 5, 2003 |
PROTEIN KINASE NPK-110
Abstract
The present invention relates to a DNA sequence encoding a
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
Inventors: |
MANDELKOW, ECKHARD;
(HAMBURG, DE) ; MANDELKOW, EVA-MARIE; (HAMBURG,
DE) ; BIERNAT, JACEK; (HAMBURG, DE) ; DREWES,
GERARD; (HAMBURG, DE) |
Correspondence
Address: |
JOSEPH A. WILLIAMS, JR.
MARSHALL O'TOOLE GERSTEIN
MURRAY & BORUN
233 S WACKER DRIVE, 6300 SEARS TOWER
CHICAGO
IL
606066402
|
Family ID: |
8216419 |
Appl. No.: |
08/817832 |
Filed: |
September 9, 1997 |
PCT Filed: |
October 30, 1995 |
PCT NO: |
PCT/EP95/04258 |
Current U.S.
Class: |
435/69.1 ;
435/194; 435/320.1; 435/325; 435/455; 536/23.2 |
Current CPC
Class: |
A61P 25/28 20180101;
C12N 9/1205 20130101; C07K 14/4711 20130101; A61P 35/00 20180101;
A61K 38/00 20130101 |
Class at
Publication: |
435/69.1 ;
435/194; 435/320.1; 435/325; 435/455; 536/23.2 |
International
Class: |
C12P 021/02; C12N
005/06; C07H 021/04; C12N 009/12; C12N 015/85 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 28, 1994 |
EP |
94117122.5 |
Claims
1. 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).
2. The DNA sequence according to claim 1, wherein the NPK is
further 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
7 KLDTFCGSPPYAAPELFQGK DRWMNVGHEEEELKPYAEP (K)SSRQNIPRCRNNI
3. The DNA sequence according to claim 1 or 2, wherein the NPK is
further characterised by the following features: (d) it is
deactivated by phosphatases 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, S92B, S941, S1073 (f) it causes the dissociation
of tau, MAP4, MAP2 and MAP2c from microtubules
4. The DNA sequence according to any one of claims 1 to 3, wherein
the NPK is obtainable from brain tissue by the following steps: (a)
homogenisation of brain extract and subsequent centrifugation
thereof; (b) chromatography of the supernatant obtained in step (a)
on cellulosephosphate wherein the NPK active fractions elute
between 200 to 400 mM NaCl; (c) ammonium sulfate precipitation of
active fractions obtained in step (b) and dialyses of the
precipitate; (d) anion exchange chromatography of the dialysate
obtained in step (c) on Q-Sepharose (Pharmacia) and elution of the
NPK active fractions wherein said NPK active fractions elute as a
single peak at about 0.2 M NaCl, with subsequent dialyses of the
active fractions; (e) cation exchange chromatography on Mono S HR
10/10 (Pharmacia); (f) chromatography on Mono Q HR 5/5, wherein the
NPK active fractions elute at about 250 mM NaCl; (g) gel filtration
chromatography on Superdex G-200, wherein the NPK activity elutes
with an apparent molecular weight of 100 kD; and (h) affinity
chromatography on ATP-cellulose, wherein the NPK active fractions
elute with an apparent molecular weight of about 110 kD as
determined by SDS-PAGE; wherein the NPK activity is measured by
incubating a peptide comprising amino acid residues 255 to 267 of
human adult tau in the presence of radioactively labelled ATP and
determining the radioactivity incorporated into said peptide.
5. The DNA sequence according to any one of claims 1 to 4, wherein
the NPK is an NPK from a mammalian brain.
6. The DNA according to claim 5, wherein said mammalian brain is
human or porcine brain.
7. A polypeptide encoded by the DNA sequence of any one of claims 1
to 6, or a functional fragment thereof.
8. A Serine or Threonine residue phosphorylated by the polypeptide
of claim 7, said Serine or Threonine residue being located in the
following amino acid position 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
9. An epitope comprising the Serine or Threonine residue of claim
8.
10. An antibody specifically binding to the polypeptide or fragment
thereof according to claim 7.
11. An antibody specifically binding to the epitope of claim 9.
12. The antibody according to claim 10 or 11, which is a monoclonal
antibody or a derivative or fragment thereof.
13. The antibody according to claim 10 or 11, which is a polyclonal
antibody or a derivative or fragment thereof.
14. A pharmaceutical composition which is containing a specific
inhibitor for the polypeptide or fragment thereof according to
claim 7, optionally in combination with a pharmaceutically
acceptable carrier and/or diluent.
15. The pharmaceutical composition according to claim 14 for the
treatment of Alzheimer's disease.
16. The pharmaceutical composition according to claim 14 for the
treatment of cancer.
17. The pharmaceutical composition according to any one of claims
11 to 13 wherein said inhibitor is the antibody according to any
one of claims 10 to 13, a phosphatase capable of dephosphorylating
the polypeptide or fragment thereof according to claim 7,
preferably phosphatase PP-2A, an inhibitor of the activating kinase
of the polypeptide of claim 7, a tau derived peptide comprising the
Ser262 residue, or a MAP4 or MAP2/MAP2c derived peptide comprising
at least one of Serine or Threonine residues mentioned in claim
8.
18. A diagnostic composition comprising: (a) the polypeptide
according to claim 7; (b) the antibody according to any one of
claims 10 to 13; and/or (c) a peptide comprising the serine residue
according to claim 2(e).
19. A method for the in vitro diagnosis and/or monitoring of
Alzheimer's disease comprising assaying a cerebrospinal fluid
isolate of patient or carrying out a biopsy of nerve tissue (for
example, olfactory epithilium) and testing said tissue for the
presence of the polypeptide or fragment thereof according to claim
7.
20. A method for the in vitro diagnosis and/or monitoring of
Alzheimer's disease comprising assaying a cerebrospinal fluid
isolate of a patient or carrying out a biopsy of nerve tissue and
testing said tissue for the presence of unphysiological amounts or
activity of the polypeptide or fragment thereof according to claim
7.
21. The method according to claim 19 or 20, wherein the NPK is
detected by the antibody according to any one of claims 10, 12 or
13.
22. A method for the in vitro diagnosis for cancer or the onset of
cancer comprising assaying a suitable tissue or body fluid for the
presence of phosphorylated Serine or Threonine residues of tau
related microtubule associated proteins (MAPs) MAP2, MAP2c and MAP4
in the positions: MAP2/MAP2c: S37, S1536, S1676, S1707, S1792,
S1796, S1799 MAP4: T829, T873, T874, T876, S899, S903, S928, S941,
S1073 or for the presence of unphysiological amounts of the
polypeptide or fragment of claim 7 or an specific phosphatase for
said polypeptide or fragment.
23. A method for the in vitro conversion of normal MAP2, MAP2c or
MAP4 by the treatment with the polypeptide or fragment of claim 7
into proteins phosphorylated at positions: MAP2/MAP2c: S37, S1536,
S1676, S1707, S1792, S1796, S1799 MAP4: T829, T873, T874, T876,
S899, S903, S928, S941, S1073 said phosphorylation status being
indicative of cancer or the onset of cancer.
24. Use of the phosphorylated Serine or Threonine residue(s) of the
MAP of claim 8 or the epitope comprising said residue(s) of claim 9
for the generation of specific antibodies indicative of cancer or
the onset of cancer.
25. An RNA sequence complementary to the DNA sequence of any one of
claims 1 to 6.
Description
[0001] 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.
[0002] 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
phosphorylation 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).
[0003] 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.
[0004] 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.
[0005] The solution to this technical problem is achieved by
providing the embodiments characterised in the claims.
[0006] 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:
[0007] (a) it encodes the amino acid sequence depicted as MARK-1 in
Table 6;
[0008] (b) it encodes the amino acid sequence depicted as MARK-2 in
Table 6; or
[0009] (c) it hybridises to the DNA of (a) or (b).
[0010] The term "DNA sequence" comprises any DNA sequence such as
genomic or cDNA, semisynthetic or synthetic DNA.
[0011] 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).
[0012] 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:
[0013] (a) it has an apparent molecular weight of 110 kD as
determined by SDS-PAGE;
[0014] (b) it phosphorylates Serine residues 262, 293, 305, 324 and
356 of human tau protein; and
[0015] (c) it comprises the following amino acid sequences
1 KLDTFCGSPPYAAPELFQGK DRWMNVGHEEEELKPYAEP (K) SSRQNIPRCRNNI
[0016] In a preferred embodiment of the DNA sequence of the present
invention, the NPK is further characterised by the following
features:
[0017] (d) it is deactivated by phosphatase PP-2A; and
[0018] (e) it phosphorylates the following Serine or Threonine
residues of tau related microtubule-associated proteins (MAPs)
MAP2, MAP2c and MAP4
[0019] MAP2/MAP2c: S37, S1536, S1676, S1707, S1792, S1796,
S1799
[0020] MAP4: T829, T873, T874, T876, S899, S903, S928, S941,
S1073
[0021] (f) it causes the dissociation of tau, MAP4, MAP2 and MAP2c
from microtubules.
[0022] 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.
[0023] 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 microtubules play an important role in cell division,
deregulation of said NPK can in turn lead to an uncontrolled
cellular division and the transformation of normal cells to cancer
cells. Alternatively, the deregulation of said NPK could provide
postmitotic terminally differentiated cells such as neurons (which
do not divide) with a stimulus to divide. This "unnormal" stimulus
would lead the neurons directly into apoptosis (and thus, an
Alzheimer's like state) because due to their differentiation status
they are unable to divide.
[0024] In a further preferred embodiment of the DNA sequence of the
present invention, the NPK is obtainable from brain tissue by the
following steps:
[0025] (a) homogenisation of brain extract and subsequent
centrifugation thereof;
[0026] (b) chromatography of the supernatant obtained in step (a)
on cellulosephosphate, wherein the NPK active fractions elute
between 200 to 400 mM NaCl;
[0027] (c) ammonium sulfate precipitation of active fractions
obtained in step (b) and dialysis of the precipitate;
[0028] (d) anion exchange chromatography of the dialysate obtained
in step (c) on Q-Sepharose (Pharmacia) and elution of the NPK
active fractions, wherein said NPK active fractions elute as a
single peak at about 0.2 M NaCl, with subsequent dialysis of the
active fractions;
[0029] (e) cation exchange chromatography on Mono S HR 10/10
(Pharmacia);
[0030] (f) chromatography on Mono Q HR 5/5, wherein the NPK active
fractions elute at about 250 mM NaCl;
[0031] (g) gel filtration chromatography on Superdex G-200, wherein
the NPK activity elutes with an apparent molecular weight of 100
kD; and
[0032] (h) affinity chromatography on ATP-cellulose, wherein the
NPK active fractions elute with an apparent molecular weight of
about 110 kD as determined by SDS-PAGE;
[0033] wherein the NPK activity is measured by incubating a peptide
comprising amino acid residues 255 to 267 of human adult tau in the
presence of radioactively labelled ATP and determining the
radioactivity incorporated into said peptide.
[0034] Further details as to how this NPK of the invention which in
one embodiment has an apparent molecular weight of 110 kD (NPK-110)
can be isolated are provided in Example 1. However, the person
skilled in the art would know from the technical teaching given
above how to supplement said details.
[0035] The NPK of the invention may be derived from any vertebrate
brain. In a preferred embodiment, the NPK is derived from a
mammalian brain.
[0036] The invention also relates to a RNA sequence complementary
to the DNA sequence of the invention.
[0037] In a particularly preferred embodiment, said mammalian brain
is human or porcine brain.
[0038] The invention further relates to a polypeptide encoded by
the DNA sequence or a functional fragment or derivative thereof.
Said polypeptide, fragment or derivative may be posttranslationally
or chemically modified. Throughout this specification, the term NPK
or, alternatively, MARK (1 or 2) may also comprise such fragments
or derivatives, even if this is not specifically indicated.
[0039] The present invention further relates to the following
Serine or Threonine residues phosphorylated by NPK-110of tau
related microtubule-associated proteins (MAPs) MAP2, MAP2c and
MAP4:
[0040] MAP2/MAP2c: S37, S1536,S1676, S1707, S1792, S1796, S1799
[0041] MAP4: T829, T873, T874, T876, S899, S903, S928, S941,
S1073
[0042] and to epitopes comprising said phosphorylated Serine or
Threonine residues.
[0043] The invention relates further to an antibody specifically
binding to the NPK of the invention.
[0044] Said antibody may be a serum derived or a monoclonal
antibody. The production of both monoclonal and polyclonal
antibodies to a desired epitope is well known in the art (see, for
example, Harlow and Lane, Antibodies, A Laboratory Manual, Cold
Spring Harbor Laboratory, Cold Spring Harbor, 1988). Furthermore,
said antibody may be a natural or an antibody derived by genetic
engineering, such as a chimeric antibody derived by techniques
which are well understood in the art. Moreover, the term antibody
as used herein also refers to a fragment of an antibody which has
retained its capacity to bind the specific epitope, such as a Fab,
F(ab).sub.2 or an Fv fragment.
[0045] Additionally, the present invention relates to an antibody
specifically binding to epitopes comprising the phosphorylated
Serine or Threonine residues of MAP2, MAP2c and MAP4:
[0046] MAP2/MAP2C: S37, S1536, S1676, S1707, S1792, S1796,
S1799
[0047] MAP4: T829, T873, T874, T876, S899, S903, S928, S941,
S1073
[0048] Again, said antibody may be a polyclonal or a monoclonal
antibody, or a fragment thereof retaining its binding
specificity.
[0049] In a preferred embodiment, the antibody of the invention is
a monoclonal antibody or a fragment or derivative thereof.
[0050] In a further preferred embodiment of the invention, said
antibody is a polyclonal antibody or a fragment or a derivative
thereof.
[0051] The invention furthermore relates to a pharmaceutical
composition which contains a specific inhibitor of the NPK of the
invention, optionally in combination with a pharmaceutically
acceptable carrier and/or diluent.
[0052] The term "specific inhibitor of the NPK of the invention"
refers to substances which specifically inhibit the enzymatic
action of the protein kinase of the present invention. Inhibitors
to enzymes such as protein kinases and their mode of action are
well known in the art. For example, such an inhibitor may bind to
the catalytic domain of the enzyme, thus rendering it incapable of
converting its substrate.
[0053] Said pharmaceutical composition may be administered to a
patient in need thereof by a route and in a dosage which is deemed
appropriate by the physician familiar with the case.
Pharmaceutically acceptable carriers and/or diluents are well known
in the art, and may be formulated according to the route of
administration or the special disease status of the patient.
[0054] In a preferred embodiment, the present invention relates to
a pharmaceutical composition for the treatment of Alzheimer's
disease.
[0055] Again, said pharmaceutical composition may be administered
to a patient in need thereof by a route and in a dosage which is
deemed appropriate by the physician handling the case.
[0056] In a further preferred embodiment, the pharmaceutical
composition of the present invention is used for the treatment of
cancer.
[0057] As has been pointed out above, the deregulation of the NPK
of the invention can lead a variety of cell types expressing
microtubule associated proteins into a pathway that eventually
results in the neoplastic transformation of said cells.
Accordingly, a pharmaceutically effective amount of an NPK
inhibitor will halt and/or reverse the transformation process. The
amount of inhibitor to be administered will be determined by-the
physician handling the respective cases.
[0058] In a further preferred embodiment of the pharmaceutical
composition of the invention, said inhibitor is the antibody of the
invention, a phosphatase capable of dephosphorylating the NPK of
the invention, preferably phosphatase PP-2A, an inhibitor of the
activating kinase of said NPK, a tau derived peptide comprising the
Ser262 residue or a MAP2, 2c or MAP4 derived peptide comprising at
least one of the Serine or Threonine residues of MAP2, MAP2c or
MAP4:
[0059] MAP2/MAP2c: S37, S1536, S1676, S1707, S1792, S1796,
S1799
[0060] MAP4: T829, T873, T874, T876, S899, S903, S928, S941,
S1073
[0061] The terms "tau derived peptide comprising the Ser262 residue
and a MAP2, 2c or MAP4 derived peptide comprising at least one of
the Serine or Threonine residues of MAP2, MAP2c and MAP4:
[0062] MAP2/MAP2c: S37, S1536, S1676, S1707, S1792, S1796,
S1799
[0063] MAP4: T829, T873, T874, T876, S899, S903, S928, S941,
S1073"
[0064] as used herein refers to a peptide which in its three
dimensional structure reconstitutes the natural conformation of the
tau protein or the MAP2, 2c or 4 proteins with regard to the
epitope comprising Serine residue 262 (tau) or the other residues
referred to above (MAP) MAP2, MAP2c and MAP4. These peptides will
mimic the natural substrate (i.e. tau or tau related MAPs) of the
NPK of the invention, but will not display any NPK associated
biological effect. The synthesis of said peptides which solely may
consist of the epitopes, or may comprise additional flanking amino
acids, is well known in the art.
[0065] The present invention further relates to a diagnostic
composition comprising:
[0066] (a) the NPK of the invention;
[0067] (b) the antibody or fragment or derivative of the invention;
and/or
[0068] (c) a peptide comprising the phosphorylatable Serines or
Threonines of tau, MAP2, MAP2c or MAP4 indicated above.
[0069] Said diagnostic composition may, for example, be used for
the detection of Alzheimer's disease or cancer or the onset
thereof. The antibody of the invention may be used to detect
abnormal, in particular higher concentrations or levels, of the NPK
of the invention, a higher degree of activation of said NPK, which
are indicative of said diseases. The NPK delivered with the
composition could be used as an internal control. On the other
hand, the above defined peptides may be used as substrates to
detect an abnormal activity of the NPK of the invention. Again, the
activity of the NPK comprised in the diagnostic composition may
serve as an internal control.
[0070] The antibody specifically binding to the phosphorylated
Serine residues enumerated above and comprised in MAP4, MAP2 or
MAP2c may be used to detect an abnormal phosphorylation status or
pattern of these microtubule associated proteins which is
indicative of cancer.
[0071] Further applications of the diagnostic composition are as
follows. Thus, in one embodiment, said diagnostic composition may
comprise an antibody of the invention directed to one of the
epitopes referred to above. For example, an Alzheimer's or cancer
correlated disease state of a sample may be detected by treating
said sample with an antibody recognising one or more of said
epitopes. The antibody-epitope (hapten) complex may be visualised
using a second antibody directed to the antibody of the invention
and being labelled according to methods known in the art (see, for
example, Harlow and Lane, ibid.).
[0072] In still another embodiment of the present invention, said
diagnostic composition may consist of an epitope referred to above
and an antibody of the invention. Treatment of a sample with said
antibody may give rise to conclusions with regard to the disease
state of the corresponding patent, if the binding of said antibody
to said sample is brought in relation to binding of said antibody
to said epitope referred to above used as a reference sample.
[0073] In still another embodiment, the diagnostic composition may
comprise an epitope referred to above, the NPK of the invention and
an antibody of the invention. Kinase activity may be monitored with
respect to phosphorylation of the sample as compared to the
phosphorylation of the epitope of the invention. From the
quantitated NPK activity the phosporylation state of the tau
protein or the MAP2, 2c or 4 contained in said sample and therefore
the disease state of the patient may be deduced. The kinase
activity may, for example, be deduced by including a substrate
analog in the same reaction, which is visually detectable upon
enzymatic conversion. Such substrate analogs are widely used in the
art. Alternatively, the amount of a phosphorylated tau protein or
MAP2, 2c or 4 in the sample may be detected after treatment with
the kinase of the invention by employing an antibody of the
invention directed to the phosphorylated epitope and using the
amount of antibody-epitope complex provided by the diagnostic
composition as an internal standard, or by determining the amount
of phosphate incorporated into tau protein or MAP2, 2c or 4 by the
NPK, for example, by radioactive tracer methods which are well
known in the art.
[0074] It should be kept in mind, however, that the person skilled
in the art, being familiar with diagnostic principles, can easily
combine the above mentioned compound in a different manner or
supplement the composition with secondary or tertiary, labelled or
unlabelled antibodies, or with enzymes and substrates. These
embodiments are also covered by the present invention.
[0075] In still another embodiment, the invention relates to a
method for the in vitro diagnosis and/or monitoring of Alzheimer's
disease comprising assaying a cerebrospinal fluid isolate of
patient or carrying out a biopsy of nerve tissue (for example,
olfactory epithilium) and testing said tissue for the presence of
the NPK of the invention.
[0076] The invention further relates to a method for the in vitro
diagnosis and/or monitoring of Alzheimer's disease comprising
assaying a cerebrospinal fluid isolate of a patient or carrying out
a biopsy of nerve tissue and testing said tissue for the presence
of unphysiological amounts of the NPK of the invention, or for
unphysiological activity of said NPK.
[0077] An example of a nerve tissue suitable for said biopsy is the
olfactory epithelium.
[0078] The method of the invention may, for example, be carried out
by using the diagnostic composition of the invention, in particular
the antibody directed to said NPK. Therefore, in a preferred
embodiment of the invention, the NPK of the invention is detected
by the antibody of the invention specifically binding to said
NPK.
[0079] Additionally, the invention relates to a method for the in
vitro diagnosis for cancer or the onset of cancer comprising
assaying a suitable tissue or body fluid for the presence of
phosphorylated Serine or Threonine residues of tau related
microtubule associated proteins (MAPs) MAP2, MAP2c and MAP4 in the
positions:
[0080] MAP2/MAP2c: S37, S1536, S1676, S1707, S1792, S1796,
S1799
[0081] MAP4: T829, T873, T874, T876, S899, S903, S928, S941,
S1073"
[0082] or for the presence of unphysiological amounts of the NPK of
the invention or an NPK specific phosphatase. It is understood that
the phosphorylation status of the Serine or Threonine residues has
to be an unphysiological one. Methods for determining such a
phosphorylation status have been described in detail in PCT/EP 92
02 829, which is incorporated herein by reference.
[0083] The assay for said phosphorylated Serine or Threonine
residues may, for example, be carried out using an antibody
specifically detecting said phosphorylated residues or the epitopes
comprising said residues.
[0084] The amount of the NPK in the sample may be measured by using
antibodies specifically directed thereto or by measuring their
activity using a suitable substrate, for example, a peptide
comprising the above referenced Serine or Threonine in a
non-phosphorylated state or any of MAP2, MAP2c and MAP4 in
unphosphorylated state. Methods for measuring the phosphorylation
status of proteins have been described in detail in PCT/EP 92 02
829. The activity of the phosphatases, for example PP-2A, PPI or
calcineurin may be tested by providing the substrate, NPK of the
invention, for example, comprised in the diagnostic composition of
the invention.
[0085] A suitable tissue or body fluid for carrying out this in
vitro method of the invention is cerebrospinal fluid, blood,
biopsies of tissue (for example, liver or skin).
[0086] Still another object of the invention is to provide a method
for the in vitro conversion of normal MAP2, MAP2c or MAP4 by the
treatment with the NPK of the invention into proteins
phosphorylated at positions:
[0087] MAP2/MAP2c: S37, S1S36, S1676, S1707, S1792, S1796,
S1799
[0088] MAP4: T829, T673, T874, T876, S899, S903, S928, S941,
S1073"
[0089] said phosphorylation status being indicative of cancer or
the onset of cancer. The conditions allowing the phosphorylation of
said MAPs can be determined by following the general teachings
provided by the present application. The phosphorylated MAPs can
then be recognised by specific antibodies. The results of said in
vitro method will allow further insights into the generation of
cancer.
[0090] Moreover, inhibitors may be tested which prevent the
conversion of normal to MAP protein phosphorylated in the positions
indicated above. These "inhibitors" may be specific for the epitope
to be phosphorylated by, for example, blocking the epitope, or may
be directed to various domains on the protein kinase of the
invention, NPK, as long as they prevent or disturb its biological
activity. Another type of inhibition is the antagonistic action of
phosphatases on said MAPs or said NPK, or the inhibition of the
activating kinase of said NPK. Furthermore, the MAP generated by
the method of the present invention may be employed in binding
studies to microtubule structures in vitro and in vivo, thus
contributing to the elucidation of the molecular basis underlying
cancer.
[0091] The present invention relates, moreover, to the use of the
phosphorylated Serine or Threonine residue(s) of the MAP of the
invention or the epitope comprising said residue(s) for the
generation of specific antibodies indicative of cancer or the onset
of cancer.
[0092] The methods for obtaining said antibodies are well known in
the art; thus, the generation of polyclonal or monoclonal
antibodies may be conducted using standard methods (see, for
example, Harlow and Lane, ibid.). If an oligo- or polypeptide is
used for the generation of antibodies, it is desirable to couple
the peptide comprising the epitope to a suitable carrier molecule
capable of inducing or enhancing the immune response to said
epitope, such as bovine serum albumin or keyhole limpet hemocyanin.
The methods of coupling hapten (comprising or being identical to
the epitope) and carrier are also well known in the art (Harlow and
Lane, ibid.). It is also to be understood that any animal suitable
to generate the desired antibodies may be used therefor.
THE FIGURES SHOW
[0093] FIG. 1: Bar diagram of tau (isoform htau40, the largest one
in central nervous tissue, Goedert et al., 1989), construct K18
containing the four repeats, and several sites phosphorylated by
the kinase activity from brain (Gustke et al., 1992). The hatched
boxes near the N-terminus are inserts which may be absent because
of differential splicing, the boxes labelled 1-4 represent the four
repeats, of which repeat 2 may be absent. Most phosphorylated sites
are in Ser-Pro or Thr-Pro motifs outside the repeats, but the brain
kinase activity also phosphorylates two sites within the repeats,
Ser262 and Ser356.
[0094] FIG. 2 Isolation of NPK110 from porcine brain. (A) The
tissue extract was loaded onto phosphocellulose and eluted stepwise
with 0.15-1 M NaCl. The filled bars show the total protein
concentration of the eluted material, open bars show the activity
as measured with tau construct K18 as substrate. (B) The material
eluted with 0.35-0.5 M NaCl was submitted to ammonium sulfate
precipitation and the precipitate dialysed and loaded onto a
Q-Sepharose column. The closed symbols show the protein
concentration, open symbols the activity profile. The gradient
composition is indicated on the right axis. (C) Fractions 8-15 from
Q-Sepharose were dialysed and loaded onto a SP-Sepharose column.
(D) Fractions 12-16 from SP-Sepharose were dialysed and loaded onto
a Mono Q HR 5/5 column. (E) Fractions 9-11 from Mono Q were loaded
onto a Superdex 200 gel filtration column. The elution positions of
molecular weight markers are indicated on the right axis.
[0095] FIG. 3: Final purification of NPK110 by affinity
chromatography on ATP-Sepharose (SDS PAGE, lanes 1-3) and
characterisation by in-gel phosphorylation (autoradiography, lanes
4-6). The most active fractions from the gel filtration column
(lane 1) were loaded onto an ATP affinity column. The kinase was
eluted specifically with 5 mM ATP (lanes 2, 3). The silver stained
gel shows a fuzzy band with an apparent molecular weight of
approximately 110 kDal and a second, sharp band with 95 kDal. Lanes
4-6 show autoradiograms of the in-gel phosphorylation of the
samples in lanes 1-3. As a substrate, tau (5 .mu.M) was polymerised
into the gel matrix. After renaturation and incubation with
g-.sup.32P ATP, it is clearly shown that only the 110 kDal band
displays kinase activity towards tau.
[0096] FIG. 4: Phosphorylation of wild type tau and construct K18
(microtubule binding domain) by NPK110. Htau40 (10 .mu.M, lanes 1,
2) and K18 (20 .mu.M, lanes 3, 4) were phosphorylated with 5
.mu.U/ml of NPK110 and 2 mM g-.sup.32P-ATP at 37.degree. C. for 2
hours. Aliquots were electrophoresed on a 7-20% SDS gradient gel:
Lanes 1, 2, htau40 before and after phosphorylation, lanes 3, 4,
K18 before and after phosphorylation. Note the small molecular
weight shift upon phosphorylation in lanes 2 and 4. The right side
shows an autoradiograph of the same gel; phosphorylated htau40 and
K18 are seen in lanes 2 and 4.
[0097] FIG. 5: Tryptic phosphopeptide maps of wild type tau
(htau40) and construct K18 phosphorylated with NPK110. 30 .mu.g of
tau were phosphorylated with 0.5 .mu.U NPK110 for 2 h at 37.degree.
C. (A) full length 4-repeat tau (htau40), (B) construct K18 (MT
binding region, residues 244-372 of full length tau), (C) diagram
of the more prominent spots: Spot 1 on upper left contains Ser262,
spot 2 on upper right Ser356, spot 3 (below 1) Ser305, spot 4
(always part of an overlapping doublet) contained Ser324, spot 5
Ser293 (this tryptic peptide CGSK was not recovered from the HPLC
column, presumably because of its small size, but the spot could be
identified by site-directed mutagenesis). (D) Mixture of identical
amounts of counts (10,000 cpm) derived from phosphopeptides shown
in (A) and (B). The identification of phosphorylation sites shown
in (C) was performed by two dimensional analysis of the
HPLC-purified and sequenced peptides (listed in Table 1). 10,000
cpm of the purified peptides each were analysed alone and in
combination with a 5000 cpm aliquot of the phosphopeptides shown in
(A) in order to allow unambiguous identification.
[0098] FIG. 6: Phosphorylation of Ser262 abolishes the binding of
tau to microtubules. (A) Binding of tau to taxol-stabilised
microtubules (30 .mu.M) was measured in a cosedimentation assay as
described below in Example 2. Full length wild-type tau (`wt`,
htau40) and a Ser262 to Ala mutant (A262) (10 .mu.M) were
previously phosphorylated with NPK110(final concentration 8.5
.mu.U/ml) for 2 hours at 37.degree. C. Curves were obtained by
non-linear regression (Biernat et al., 1993).
[0099] The binding of wild-type tau is completely abolished by
phosphorylation (closed circles), whereas the A262 mutant still
binds, although with lower affinity (triangles). For comparison,
the binding of unphosphorylated tau is also shown (open
circles).
[0100] (B) Microtubule-bound tau comes off during phosphorylation
by NPK110. htau 40 (10 .mu.M) was incubated with taxol-stabilised
microtubules (30 .mu.M). At t=0, NPK110was added to a final
concentration of 10 .mu.U/ml, and aliquots were withdrawn at time
intervals from one to 20 hours and pelleted. Tau was measured in
the pellets and supernatants by densitometry of the SDS gels -
(closed circles). Incorporated phosphate was measured by Cerenkov
counting of gel pieces (open circles) and is indicated on the right
axis. Phosphate incorporation in tau without microtubules is shown
to proceed faster (squares).
[0101] FIG. 7: Dark field video microscopy of microtubules and
effect of phosphorylation of Ser262 on tau. Microtubules (5 .mu.M
tubulin) were nucleated on sea urchin sperm axonemes in the
presence of 2.5 .mu.M tau (isoform htau40) and 10 .mu.U/ml of
NPK110. A, 20 min without ATP, B, with ATP. In A the microtubules
grow continuously, in B Ser262 can be phosphorylated, leading to a
destabilisation and shortening of microtubules. Bar=10 .mu.m.
[0102] FIG. 8: Effect of the unphosphorylated and
NPK110-phosphorylated tau on the length of axoneme nucleated
microtubules measured by darkfield microscopy. For each condition
500 to 600 microtubule plus ends were measured; the mean length was
plotted against time. Tubulin concentration was 5 .mu.M; note that
without added tau, no microtubules are observed at this
concentration. Tau was 2.5 .mu.M in all cases. In control
experiments, ATP was omitted (`- ATP`).
[0103] (A) Tau pre-phosphorylated by NPK110 does not promote
microtubule growth (filled circles) but the pre-phosphorylated
point mutant A262 does (triangles, in accordance with time resolved
binding assay in FIG. 6B).
[0104] (B) Tubulin and tau were mixed at 4.degree. C. with 10
.mu.U/ml of NPK110 (final concentration) in the presence (closed
circles) or absence (open circles) of 2 mM Mg-ATP. At t=0, the
temperature was raised to 37.degree. C. With wild type tau and no
ATP, microtubules grow continuously (open circles); the same result
is obtained with the mutant Ser262-Ala (triangles). However, wild
type tau plus ATP leads to initial growth but subsequent shrinkage
(closed circles). (C-E) Microtubule length histograms at 5 min and
30 min of the corresponding curves in B. Each sample shows a
pronounced peak around 20 .mu.m after 5 min (empty circles). If
Mg-ATP was absent (C) or Ser262 was mutated into Ala (E) the
distribution became broader and shifted to greater lengths at 30
min. By contrast, phosphorylation of tau successfully decreased the
mean microtubule length within 30 min of incubation (D).
[0105] FIG. 9: Tryptic phosphopeptide maps of wild type tau
(htau40) and construct K18 phosphorylated with (A) brain extract,
(B) NPK110, (C) PKC, or (D) PKA, respectively. The numbering of the
spots is analogous to FIG. 5 (spot 1:Ser262, spot 2:Ser356, spot
3:Ser3O5, spot 4:Ser324, spot 5:Ser293). The panels on the right
show the corresponding two-dimensional phosphoamino acid analysis
of full length tau for each kinase.
[0106] Fig. 10: Diagram representing the influence of different
phosphorylation sites on tau-microtubule interactions. The majority
of Ser/Thr-Pro motifs are in the flanking regions of the repeat
domain, they have only a small influence on the binding of tau. The
repeat domain contains several phosphorylatable non-Ser-Pro sites,
especially the four KXGS motifs. Of these, Ser262 in the first KIGS
motif has by far the greatest influence on microtubule binding.
[0107] FIG. 11: Phosphopeptide map of recombinant MAP2c
phosphorylated by NPK-110. The peptides contain the following
phosphorylated residues: I=Ser1707, II=Ser1676, III=Ser37 and
Ser1536, IV=Ser1792, Ser1796 and Ser1799
[0108] (numbering of residues following Albala et al., 1993).
[0109] FIG. 12: Phosphopeptide map of MAP4 fusion protein
phosphorylated by NPK-110. The peptides contain the following
phosphorylated residues: I=Thr829, II=Ser941, III=Ser928,
IV=Thr873, Thr874 and Thr876, V=Ser899 and Ser903, VI=Ser1073,
VII=Ser928
[0110] (numbering of residues following West et al., 1991).
[0111] FIG. 13: Effect of the unphosphorylated and
NPK-110-phosphorylated MAP4, MAP2 and MAP2c on the length of
axoneme nucleated microtubules measured by darkfield microscopy.
For each condition 500 to 600 microtubule plus ends were measured;
the mean length was plotted against time. Tubulin concentration was
5 .mu.M; MAPs were 1 .mu.M. Note that without added MAPs, no
microtubules were observed at this concentration.
[0112] (a) Tubulin and MAP4 were mixed at 4.degree. C. with 10
.mu.U/ml of NPK-110 (final concentration in the presence (closed
circles) or absence (open circles) of 2 mM Mg-ATP. At t=0, the
temperature was raised to 37.degree. C. With MAP4 and no ATP,
microtubules grow continuously (open circles). However, MAP4 plus
ATP leads to initial growth but subsequent shrinkage (closed
circles) because MAP4 becomes phosphorylated, detaches from
microtubules, and microtubules are destabilised.
[0113] (b) Same experiment as in (a) but using MAP2, with similar
results.
[0114] (c) Same experiment as in (a) but using MAP2c, with similar
results.
[0115] FIG. 14: Northern Blot of adult and fetal human tissues with
a MARK cDNA probe.
[0116] left: adult tissue
[0117] lane 1: Pancreas (Pa)
[0118] lane 2: Kidney (Ki)
[0119] lane 3: Muscle (Mu)
[0120] lane 4: Liver (Li)
[0121] lane 5: Lung (Lu)
[0122] lane 6: Placenta (P1)
[0123] lane 7: Brain (Br)
[0124] lane 8: Heart (H)
[0125] Right: fetal tissue
[0126] lane 9: Kidney (Ki)
[0127] lane 10: Liver (Li)
[0128] lane 11: Lung (Lu)
[0129] lane 12: Brain (Br)
[0130] FIG. 15: Binding of recombinant wild type MAP2c and MAP2c
point mutants to taxol stabilized microtubules (30 .mu.M tubulin
dimers) under the influence of phosphorylation by p110MARK. Open
circles: wild-type MAP2c, non-phosphorylated. The binding is tight
(Kd about 0.25 .mu.M) and saturates around 17 .mu.M ligand, or
about 1 MAP2c molecule per 2 tubulin dimers. Closed circles:
wild-type MAP2c, phosphorylated previously with p110MARK (2.5
milliUnits/ml; 1 Unit corresponds to 1 .mu.mol of phosphate
transferred to MAP2c per minute at 30.degree. C.) for 2 h. Note
that there is essentially no binding. Closed and open squares:
MAP2cA319 and MAP2cA350, phosphorylated previously with p110MARK
(2.5 milliUnits/ml) for 2 h. In these mutants the serines 319 or
350 in the KXGS motifs in the first or second repeat were point
mutated to alanines. The affinity to microtubules decreases
markedly (Kd .apprxeq.7 .mu.M) although the stoichiometry remains
similar to the wildtype MAP2c. Triangles: MAP2cA319/A350,
phosphorylated previously with p110mark (2.5 milliUnits/ml) for 2
h. In this mutant both serines 319 and 350 are mutated to alanines.
The binding is similar to the unphosphorylated protein, showing
that the sensitivity to phosphorylation has disappeared because the
two KXGS motifs are no longer phosphorylatable.
[0131] FIG. 16: Effects of unphosphorylated and
p110MARK-phosphorylated MAP4 (A), MAP2 (B), MAP2c (C) and MAP2c
point mutants (D) on the length of self- nucleated microtubules
measured by darkf ield microscopy. For each condition 500-800
microtubules were analyzed, and the mean length were plotted
against time. Tubulin concentration was 10 .mu.M in all cases, the
concentration of MAP4 and MAP2 was 1 .mu.M, that of MAP2c 2 .mu.M.
In control experiments, ATP was omitted (`-ATP`).
[0132] Open circles in A, B and C: The MAPs were preincubated for
30 min with 2.5 mUnits/ml p110MARK (final concentration), but
without ATP. By adding 10 .mu.M tubulin, microtubules were
nucleated and the mean microtubule length increased up to about 20
.mu.m within 30 min. By contrast, if ATP was present no
self-nucleation occurred, showing that the phosphorylation of the
MAPs prevented microtubule formation. Short microtubules of about 2
um length could only be observed by adding axonemes (10-100 fM) to
promote seeded nucleation (open triangles in A, B, C).
[0133] Closed circles in A, B, and C: Tubulin and MAP were mixed at
4.degree. C. with 2.5 mUnits/ml of p110MARK (final concentration),
and the temperature was shifted immediately to 37.degree. C. (so
that initially the MAPs were unphosphorylated). Microtubule growth
was promoted in all three cases, but the final mean microtubule
length was only about half of that observed for the
unphosphorylated MAPs (compare open circles).
[0134] D: The effect of phosphorylation site point mutations of
MAP2c. All proteins were phosporylated as described above (with 30
min preincubation). Triangles; wildtype MAP2c, closed circles;
MAP2cA319 (KXGS in first repeat mutated to KXGA), squares;
MAP2cA350 (KXGS in second repeat mutated to KXGA), closed squares;
MAP2cA319/A350 (KXGS in both repeats mutated to KXGA).
[0135] The Examples illustrate the invention.
[0136] Regarding the tau proteins described in the examples, only
recombinant human tau proteins expressed in E. coli were used. cDNA
clones were prepared as described by M. Goedert (Goedert et al.,
1989) and were expressed using variants of the pET expression
vector (Studier et al., 1990). The proteins were purified making
use of the heat stability of tau and Mono S FPLC (Hagestedt et al.,
1989). Construct K18 is derived from the 4-repeat tau isoform and
comprises the microtubule binding region, residues 244 to 372
(Biernat et al., 1993). Mutant `A262` is based on the longest human
isoform. A single residue, Ser262, was changed into alanine using
conventional technology. Phosphocellulose-purified tubulin
(PC-tubulin) was prepared from porcine brain following Mandelkow et
al., 1985. Protein kinase A catalytic subunit (isolated from bovine
heart, activity 27 catalytic subunit (isolated from bovine heart,
activity 27 nU/.mu.l based on kemptide, 100 pU/.mu.l based on
casein) was obtained from Promega, Protein kinase C (isolated from
rat brain, activity 80 pU/.mu.l based on histone H1) was from
Boehringer Mannheim.
EXAMPLE 1
[0137] Purification and characterisation of the protein kinase
NPK110.
[0138] All operations were performed at 4.degree. C. Fresh porcine
brains (approx. 1 kg) were obtained at the local slaughterhouse and
homogenised into 1 litre of buffer A (50 mM Tris, pH 8.5,
containing 5 mM EGTA, 100 mM NaF, 1 mM PMSF, 1 mM benzamidine, 1 mM
Na.sub.3VO.sub.4, 1 mM DTT, 0.1% Brij-35). The homogenate was
transported to the laboratory on ice and centrifuged at 30,000 g
for 1 h. The supernatant was cleared by ultracentrifugation (50,000
g, 30 min), the pH adjusted to 6.8 and loaded onto a Buchner funnel
containing 150 ml Whatman P11 equilibrated with buffer B (50 mM MES
pH 6.8, 2 mM EGTA, 50 mM NaF, 1 mM PMSF, 1 mM benzamidine, 1 mM
Na.sub.3VO4, 1 mM DTT, 0.1% Brij-35), by applying a slight vacuum.
The phosphocellulose was washed with 500 ml of buffer B and eluted
stepwise with 150 ml each of buffer B containing 0.15 M -1 M NaCl
(FIG. 2A). Fractions were screened for activity by phosphorylation
of a tau construct (K18) consisting of the four microtubule binding
repeats, essentially as described (Drewes et al., 1992). Active
fractions were fractionated by ammonium sulfate precipitation. The
precipitate obtained between 30 and 50 % saturation was dialysed
against buffer A overnight on ice. The dialysate (approx. 50 ml)
was cleared by ultracentrifugation and loaded onto an anion
exchange column (Q-Sepharose HR, Pharmacia, 80.times.16 mm) using a
Superloop (Pharmacia) After washing the column with 100 ml of
buffer A and elution with a stepwise gradient from 0-0.5 M NaCl
(FIG. 2B, flow rate 5 ml/min, fraction size 7 ml), active fractions
(approx. 40 ml) were dialysed against buffer B and loaded onto a
cation exchange column (SP-Sepharose HR, Pharmacia, 60.times.16 mm)
(FIG. 2C, flow rate 4 ml/min, fraction size 7 ml). After elution
with 0-0.5 M NaCl, active fractions (approx 40 ml) were pooled, the
buffer was changed for buffer A on a Sephadex G25 column
(300.times.26 mm) and loaded onto a Mono Q HR 5/5 column
(Pharmacia) and eluted with a steep NaCl gradient (FIG. 2D, flow
rate 0.5 ml/min, fraction size 1 ml). Active fractions (2-3 ml)
were concentrated twofold in a Centricon 30 microconcentrator
(Amicon) and loaded onto a gel filtration column (Superdex 200,
Pharmacia, 300.times.16 mm) equilibrated and eluted with buffer A
(pH 7.8, containing 150 mM NaCl and 10% glycerol).
[0139] The flow rate was 0.2 ml/min, fraction size was 2 ml. The
column had previously been calibrated with a marker protein kit
(Pharmacia). Active fractions were pooled, and the buffer was
changed to buffer C (40 mM .beta.-glycerophosphate, 10 mM
MgCl.sub.2, 2 mM EGTA, 1 mM Benzamidine, 0.2 mM DTT, 0.1% Brij-35)
on a Sephadex G25 column (100.times.16 mm). The protein pool from
the G25 column (10-15 ml) was loaded at 0.1 ml/min onto an
ATP-Sepharose column (Upstate Biotechnology Inc., Lake Placid, USA,
15.times.5 mm). The column was washed with 5 ml of buffer C and
eluted with 2 ml of buffer C containing 5 mM MgATP. The eluate was
concentrated and freed from ATP and buffer substances on a Mono Q
PC 1.6/5 column (`Smart` system, Pharmacia), eluted with 25 mM
Tris-HCl, pH 7.4, containing 250 mM NaCl, 1 mM EGTA, 0.2 mM DTT, 1
mM benzamidine and 0.03% Brij-35. Active fractions were mixed with
50% (v/v) glycerol and stored at -20.degree. C. Under these
conditions, activity was preserved for at least one month.
[0140] With these six chromatographic steps used a .apprxeq.10,000
fold purification of a Ser262-phosphorylating activity from a
porcine brain tissue extract was achieved. As shown in detail in
FIG. 2, phosphocellulose (A), ion exchange chromatography on Q- and
SP-Sepharose and Mono Q (B,C,D), gel filtration (E) and, finally,
affinity chromatography using immobilised ATP were employed. The
activity of this kinase in the tissue extract was .apprxeq.0.2
mU/mg, the activity of the affinity-purified kinase .apprxeq.2 U/mg
(1 unit transfers 1 Amol of phosphate per minute). The molecular
weight of the enzyme was around 90-100 kDal by gel filtration, but
the activity peak was broad and often showed pronounced tailing
(FIG. 2E). On SDS gels, the apparent molecular weight was
.apprxeq.110 kDal (FIG. 3). The enzyme could be renatured in the
gel; if tau was polymerised into the gel matrix as a substrate and
the gel was incubated with .gamma.-.sup.32P-ATP, the 110 kDal band
became prominent upon autoradiography (FIG. 3, lane 4-6), whereas
some minor contaminations observed in the silver stained gel had no
detectable activity. After phosphorylation with NPK-110, both whole
tau and construct K18 showed small but distinct mobility change in
SDS PAGE (FIG. 4, lanes 1-4). The final amount of incorporated
phosphate is .apprxeq.1.8- 2.5 mol per mole of tau, depending
somewhat on enzyme concentration and activity; this level of
phosphorylation could be achieved after .apprxeq.2 hours.
Phosphorylation reactions were carried out as follows:
[0141] Phosphorylation reactions were carried out in 40 mM Hepes,
pH 7.2, containing 2 mM ATP, 5 mM MgCl.sub.2, 2 mM EGTA; 1 mM DTT,
0.1 mM PMSF, 0.03% Brij-35. When extracts or crude fractions of
kinase preparations were screened, 50 mM NaF or 1 .mu.M okadaic
acid (LC Services, Woburn, Mass., USA) was included. Reactions were
terminated by heating to 95.degree. C. Phosphorylation was assayed
in SDS gels (Steiner et al., 1990) or on phosphocellulose paper
discs (Gibco) (Casnellie, 1991). In-gel phosphorylation assays were
performed according to the method of Geahlen et al., 1986.
[0142] The specificity of NPK110 for tau was examined by tryptic
digestion of phosphorylated protein and subsequent two-dimensional
thin layer electrophoresis and chromatography
[0143] (FIG. 5). If one compares the phosphorylation patterns
obtained from recombinant full-length 4-repeat tau (FIG. 5A) and
the 4-repeat fragment K18 (FIG. 5B), it is apparent that most
phosphorylated peptides are generated from the repeat domain. This
was confirmed by analysis of a mixture of both samples (FIG. 5D).
In a second approach, the tryptic digest was resolved by HPLC (not
shown). In more detail, these approaches were carried out as
follows:
[0144] Following phosphorylation reactions, the kinases were
removed by boiling of the samples in 0.5 M NaCl/10 mM DTT and
centrifugation. Tau protein remains in the supernatant and was
precipitated by 15% TCA. Cysteine residues were modified by
performic acid treatment (Hirs, 1967). The protein was digested
overnight with trypsin (Promega, sequencing grade) in the presence
of 0.1 mM CaCl2, using two additions of the enzyme in a ratio of
1:10-1:20 (w/w) Two-dimensional phosphopeptide mapping on thin
layer cellulose plates (Macherey & Nagel, Duren, FRG) was
performed according to Boyle et al., 1991. In brief, first
dimension electrophoresis was carried out at pH 1.9 in formic acid
(88%)/acetic acid/water (50/156/1794), second dimension
chromatography in n-butanol/pyridine/acetic acid/water
(150/100/30/120). For the mapping of phosphorylation sites by
sequencing, recombinant human tau (200 .mu.g, clone htau 40) was
phosphorylated with NPK110 and .sup.32P-ATP (100 Ci/mol) for 2
hours. The phosphorylation was terminated by a brief heat
treatment. The protein was incubated with 6 M urea and 2 mM DTT,
and cysteines were blocked with vinylpyridine (Tarr et al., 1983)
or performic acid treatment. After dialysis against 10 mM ammonium
bicarbonate, the protein was lyophilised and digested with trypsin
(1:20) in the presence of 0.1 mM CaCl.sub.2. Separation of peptides
was performed by two successive HPLC runs on a .mu.RPC C2/C18 SC
2/10 column (`Smart` system, Pharmacia). The digest was acidified
with acetic acid (5% v/v) and fractionated by HPLC using a gradient
of acetonitrile in 10 mM ammoniumacetate (flow rate 0.1 ml/min,
0-25% in 120 min, 25-50% in 20 min). Peptides were detected by UV
absorption at 214, 254 and 280 nm and incorporated phosphate was
measured as Cerenkov radiation in a scintillation counter
(Hewlett-Packard TriCarb 1900 CA). Flowthrough fractions and
radioactive peaks from this gradient were further purified using a
gradient of acetonitrile in TFA (flow rate 0.1 ml/min, 0%
acetonitrile/0.075% TFA to 66% acetonitrile/0.05% TFA in 60 min).
Sequence analysis of peptides was performed using a 477A pulsed
liquid phase sequencer and a 120A online PTH amino acid analyser
(Applied Biosystems). Phosphoserines were identified as the
dithiothreitol adduct of dehydroalanine by gas phase sequencing
(Meyer et al., 1991).
[0145] This yielded several labelled peptides which were analysed
by direct phosphopeptide sequencing and by phosphoamino acid
analysis. Phosphoamino acid analysis: Aliquots of digestion samples
were partially hydrolysed in 6N HCl (110.degree. C., 60 min) and
analysed by two dimensional electrophoresis at pH 1.9 and pH 3.5
(Boyle et al., 1991). The results of the phosphopeptide sequencing
are compiled in Table 1.
2TABLE 1 Tryptic phosphopeptides from htau40 phosphorylated with
NPK110, obtained by HPLC. The sequences are those of the main
radioactive peaks. Listed are the number of counts obtained after
the second purification run, the amount of material, the sequence
with the phosphorylated residue (identified as S-ethylcysteine)
starred, the phosphorylation site (numbering according to htau40).
Note that the tryptic phosphopeptide CGSK from the second repeat
was not detected by HPLC (presumably because of its small size and
hydrophilicity) and thus had to be indentified by phosphopeptide
mapping and site-directed mutagenesis. pmoles cpm in peptide found
Sequence found Phosph. sites 400.00 1000 IGS*TENLK Ser-262 150.000
350 IGS*LDNIPHVPGGGNHK Ser-356 150.000 300 CGS*LGNIHHK Ser-324
60,000 200 HVPGGGS*VQIVYK Ser-305
[0146] Most of the radioactivity was found in a peptide containing
phosphorylated Ser262. Ser356 (in the KIGS motif of the fourth
repeat) and Ser324 (from the KCGS motif of the third repeat) were
also found radioactively labelled. Two dimensional analysis of
these purified peptides lead to the identification of spots shown
in FIG. 5C. This clearly shows that Ser262 (spot 1) is the main
target site of NPK110 on tau, followed by Ser356 (spot 2). Spot 3
was identified as the peptide containing Ser305, spot 4 as Ser324
(in the KCGS motif of the third repeat), spot 5 as Ser293 (in the
KCGS motif of the second repeat). The corresponding tryptic peptide
(.sup.291CGSK) could not be isolated directly by reverse phase HPLC
chromatography, presumably because of its shortness and
hydrophilicity. It was therefore identified by site directed
mutagenesis, using point mutants of K18 where the serines in all
four KXGS motifs (Ser262, 293, 324, 356) were converted into
alanines. After phosphorylation with NPK110 only spot 3 (Ser305)
was visible, while spots 1, 2, 4 and 5 were gone, thus identifying
spot 5 with Ser293 (data not shown).
EXAMPLE 2
[0147] Tau-microtubule binding and dynamic instability.
[0148] Previously it was shown that the phosphorylation of Ser262
strongly decreased the interaction between tau and microtubules;
that is, not only the dissociation constant increased but also the
stoichiometry decreased. Confirming these observations, a similar
result was obtained after phosphorylation of tau by NPK110. In
fact, FIG. GA shows that the reduction in binding is even more
pronounced: NPK110 completely abolishes microtubule binding within
the concentration range accessible. Because the binding became so
weak it was also no longer possible to estimate values for the
dissociation constant and the stoichiometry. In other words, NPK110
efficiently causes the loss of binding of tau to microtubules.
Binding studies were carried out as follows:
[0149] Binding studies were performed by measuring co-sedimentation
of taxol-stabilised microtubules (30 .mu.M) and tau by
ultracentrifugation (Beckman TL 100) of 30 .mu.l-samples. Aliquots
of the pellet and supernatant were assayed using SDS-PAGE and
Coomassie blue staining. Scanner densitometry of dried gels was
used for quantification of protein (for details see Gustke et al.,
1992).
[0150] In order to verify this result a point mutation (Ser262 to
Ala) was introduced into tau so that this site could no longer be
phosphorylated. In this case, incubation of the mutant with NPK110
left the microtubule binding capacity largely intact, although
there was some decrease in affinity and stoichiometry
(.apprxeq.25%, FIG. 6A). This confirms two points of prior art
studies, (i) phosphorylation of Ser262 is the major switch
controlling tau's affinity for microtubules, (ii) the other sites
phosphorylated by the kinase have a small but measurable effect on
the binding (i.e. mainly the equivalent serines in the KXGS motifs
of repeats 2, 3, and 4).
[0151] The next question was: Do microtubules protect tau from
being phosphorylated by NPK110? If this were the case, then tau
--once bound to microtubules-- might retain its high affinity for
microtubules. To answer this point, taxol-stabilised stabilised
microtubules were first saturated with tau, and then incubated with
NPK110. As illustrated in FIG. 6B, tau gradually dissociates from
microtubules, concomitant with phosphorylation. Thus microtubules
retard phosphorylation of tau by the kinase but cannot prevent
it.
[0152] One important function of tau is to stabilise microtubules
and suppress their dynamic instability (Drechsel et al., 1992).
Thus, if tau loses its binding to microtubules one would expect
stable microtubules to become dynamic. This effect can be
illustrated by video dark field microscopy of individual
microtubules seeded onto flagellar axonemes (FIG. 7). The
experiment was carried out as follows:
[0153] Video microscopy of microtubules nucleated on axonemes was
done essentially as described (Trinczek et al., 1993). Briefly, 5
.mu.M PC-tubulin, 2.5 .mu.M tau (unphosphorylated or
phosphorylated) and low amounts of sea urchin sperm axonemes
(10-100 fM) were mixed in 50 m M Na-Pipes, pH 6.9, containing 3 mM
MgCl.sub.2, 2 mM EGTA, 1 mM GTP and 1 mM DTT. 1.0 .mu.l of the
samples was put on a slide, covered with 18 .times.18 mm
coverslips, sealed, and warmed up to 37.degree. C. in a
temperature-controlled air flow within 5 s. A constant temperature
of 37.degree. C. was maintained by the air flow. The axoneme
nucleated microtubules were recorded at time 2.5, 5, 10, 15, 20,
25, and 30 min after the temperature shift. For each condition and
time three to five axonemes of a sample and 10-20 experiments were
analysed, and the lengths of 500-600 microtubule plus ends were
measured. Only those microtubules which were clearly located within
the focal plane were taken into account. The depth of solution was
3-4 .mu.m, and the focal depth was 1-2 .mu.m.
[0154] In the experiment of FIG. 8A the concentration of tubulin
(5.mu.M) was chosen such that microtubules would not assemble by
themselves but would grow upon addition of (unphosphorylated) tau.
Tau phosphorylated with NPK110 did not support growth whereas the
mutant Ser262-Ala did. In other words, tau phosphorylated at Ser262
behaved as "no tau" because it did not interact with microtubules,
in contrast to the mutant which did. Even more dramatic is the
conversion of microtubules from undynamic to dynamic behaviour
under the influence of the kinase. In the experiment of FIG. 8B
microtubules were allowed to grow off axonemes in the presence of
tau and their mean length which increased to .apprxeq.50 .mu.m over
20 min was recorded. In a parallel experiment NPK110 with ATP was
added (or without ATP as a control). In the control experiment
(without ATP) microtubules were able to grow continuously and
showed little dynamic instability (FIG. 8B, open circles). With ATP
added, the mean length increased only to 20.mu.m and then dropped
again, due to the gradual phosphorylation of tau and concomitant
increase in microtubule dynamics (filled circles). When the mutant
Ser262-Ala was used, microtubules grew normally even when the
kinase and ATP were present (triangles). These results are
summarised in the length histograms of FIG. 8C-D. At early times
after initiation of assembly microtubules are short and rather
homogeneous in length (peaks of open circles at 5 min), at later
times of uninterrupted growth the microtubules become long and show
a broad length distribution (filled circles in FIGS. 8C and 8E).
However, when the kinase is allowed to phosphorylate Ser262 (i.e.
the kinase, ATP, and wild type tau with Ser262 are present),
microtubules remain short (open circles in FIG. 8D).
EXAMPLE 3
[0155] Other kinases phosphorylating the repeat domain of tau. Tau
can be phosphorylated in vitro by many kinases which can be
classified by several criteria, depending on function, targets, or
others. Certain proline-directed kinases that are of diagnostic
interest for Alzheimer's disease (because of the antibody reactions
induced by them) phosphorylate the regions flanking the repeats but
appear to have little influence on tau-microtubule binding.
Conversely, one would expect that kinases phosphorylating the
repeat region have an influence on microtubule binding because the
repeats of tau are thought to be involved in this function, and
this is in fact borne out by the results with NPK110 described so
far. The question therefore arises how this kinase compares with
other kinases phosphorylating tau in the repeat domain. Several of
these have been reported so far (Table 2).
3TABLE 2 Summary of phosphorylation sites and kinases affecting the
repeats and nearby regions of tau (only non-proline directed
kinases and sites are listed). Major sites are denoted by X, minor
ones by (x). Note that the results were obtained by different
methods: (1) phosphorylation of tau followed by proteolytic
digestion, separation of peptides, and phosphopeptide sequencing
(Steiner et al., 1990, Steiner, 1993, Gustke et al., 1992; Scott et
al., 1993). (2) Mass spectrometry of phosphopeptides combined with
sequencing (Hasegawa et al., 1992; Watanabe et al., 1993). (3)
Phosphorylation of a synthetic peptide (Correas et al., 1992). (4)
2D mapping of phosphopeptides combined with sequencing (this
report). Since these data are derived from the repeat domain K18
they do not contain information on possible phosphorylation sites
outside the repeats. kinase or reference S262 S293 S324 S356
activity S214 KIGS KCGS S305 KCGS KIGS S377 S409 S416 PKA Scott et
al., (x) (x) (x) X X 1993 PKA Steiner, 1993 X (x) (x) (x) X X PKA
this report ND (x) (x) (x) X X ND ND PKC Correas et al., X 1992 PKC
Steiner, 1993 X (x) (x) (x) X PKC this report ND (x) (x) (x) ND
CaMK Steiner et al., X 1990 brain ex. Gustke et al., X X 1992 brain
ex. this report X X PK 35/41 Biernat et al., X (x) (x) X 1993
NPK110 this report X (x) (x) (x) X brain in vivo: Alzheimer:
Hasegawa et al., X 1992 adult: Watanabe et al., - - - no sites in
repeat region - - - 1993 fetal: Watanabe et al., - - - no sites in
repeat region - - - 1993
[0156] For example, PKA phosphorylates mainly Ser214, Ser409 and
Ser416 outside the repeats, but minor sites include Ser324 and
Ser356 within the repeats (Scott et al., 1993; Steiner, 1993).
Since Ser262 is not one of the sites one would not expect a major
effect on microtule binding, in agreement with our observations.
PKC sites include the KCGS motif in repeat 3 (Correas et al., 1992;
Steiner, 1993), again with no major effect on microtubule binding
in our hands. The partially purified kinase activity described
previously (Biernat et al., 1993) phosphorylated all four KXGS
motifs, and finally, the kinase activities from brain extract
phosphorylated both the Ser/Thr-Pro motifs as well as Ser262 and
Ser356 (Gustke et al., 1992), with the reported strong effects on
microtubule binding due to Ser262. The strategy employed in these
studies was to generate proteolytic fragments from phosphorylated
tau which were then separated by HPLC and identified by sequencing.
This usually generates a multitude of peptides whose recovery is
not always linear, making it difficult to judge the relative amount
of phosphorylation at different sites.
[0157] Because of these uncertainties it was decided to
re-investigate the phosphorylation sites by a different approach.
The phosphopeptides were analysed not only by HPLC and sequencing,
but also by two-dimensional mapping on thin layer cellulose plates
which gives a clearer representation of the relative contributions.
Full length 4-repeat tau and the repeat domain (K18) were
phosphorylated with brain extract, NPK110, PKC, and PKA. This
enabled the comparison of the phosphorylation sites in the repeat
domain of tau and showed the extent of this phosphorylation in
htau40 by each of the kinases. The results are shown in FIG. 9
where the phosphopeptides derived from K18 are labelled according
to FIG. 5. Phosphopeptide spots generated by the other kinases were
identified by running each sample along with the K18 sample
phosphorylated with NPK110 (data not shown).
[0158] The patterns shown in FIG. 9A were obtained by
phosphorylating full length tau and K18 with brain extract. With
full length tau only spot 1 (Ser262) is clearly seen, spot 2
(Ser356) is barely visible. This is even more prominent in the
phosphorylation pattern of K18.
[0159] When the phosphorylation of K18 by NPK110were examined, a
peptide pattern similar to that of the brain extract (compare FIGS.
9A and 9B) is formed; the most prominent spots are 1 and 2,
containing Ser262 and Ser356, while Ser 305 (spot 3), Ser324 (spot
4), and Ser293 (spot 5) represent minor components. This confirms
the role of NPK110 as the major Ser262 kinase. By contrast,
re-investigation of the earlier kinase activity (Biernat et al.,
1993) has so far yielded inhomogeneous results. Although it
phosphorylates the same serines as NPK110 the weighting is
different, and the activity of the kinase in brain extract is at
least 10-fold lower. This explains why even long incubations of tau
with this kinase activity lead to only partial suppression of tau's
binding to microtubules, as described earlier.
[0160] As seen in FIG. 9C, PKC only phosphorylated Ser305 (spot 3),
Ser324 (spot 4) and Ser293 (spot 5) to a significant extent in K18.
The smear and the outermost spot to the left (arrow) are not
phosphopeptides derived from tau since they also occurred in
control experiments where no tau construct had been added (not
shown). The remaining two spots could not be identified; the spot
on the upper right (starred) did not colocalise with either Ser262
(spot 1) or Ser356 (spot 2). Comparison of this pattern with the
one obtained from full length tau revealed that the major
phosphorylation sites of PKC are outside the repeat domain. Only
Ser305 (spot 3) was faintly visible in this pattern (note that the
spot on the upper right does not correspond to the upper right spot
from K18 (starred), as confirmed by control experiments (not
shown)).
[0161] When using purified PKA to phosphorylate full length tau and
construct K18 (FIG. 9D) mainly Ser356 (spot 2), Ser305 (spot 3),
Ser 324 (spot 4) and Ser293 (spot 5) are found. Ser262 (spot 1) is
only a minor phosphorylation site. Phosphorylation of full length
tau (FIG. 9D, left panel) yielded similar spots, plus additional
sites outside the repeat region of tau. These result are in general
agreement with earlier data (Scott et al., 1993; Steiner, 1993).
Some of these sites had also been seen with the "35/41 kDal" kinase
activity described previously (Biernat et al., 1993). In subsequent
experiments it was determined that the 41 kD component is the
catalytic subunit of PKA (using an antibody against PKA obtained
from H. Hilz, Hamburg, data not shown); this explains in part the
overlap in the data. PKC phosphorylates mainly Ser305, Ser293 and
Ser324 (the latter in agreement with Correas et al., 1992), but not
Ser262 (FIG. 9C).
EXAMPLE 4
[0162] Sites of MAP2 and MAP4 phosphorylated by the kinase NPK110.
MAP2 and MAP4 are two microtubule-associated proteins which belong
to the same MAP-family as tau because they show high homology in
the region of the 3 or 4 internal repeats where the proteins bind
to microtubules (for review see Chapin & Bulinski, 1992). MAP2
occurs preferentially in brain, mostly in the somatodendritic
compartment of neurons. Like tau, MAP2 can be expressed in
different forms due to alternative splicing (Kindler et al., 1990):
The second repeat may be absent (this is the "classical" MAP2); in
addition the region of residues 152-1514 (i.e. 1363 out of 1830
residues) may be absent (generating a protein with 467 residues;
this form is commonly called MAP2c). The phosphorylation
experiments described here have been performed with recombinant
MAP2c expressed in E. coli (Table 3).
4TABLE 3 Peaks from Peptide second no. extinction peptide phosphor.
col. (FIG. 11) cpm (214 nm) sequence residue 1 I 300,000 0.05
1705.CGS*LK Ser1707 (in 2nd repeat) 2 II 200,000 0.3 1674:IGS*
Ser1676 TDNIK (in 1st repeat) 3 III 100,000 0.8 33:DQGGS Ser 37
GEGLSR Ser1536 1535:SS*LPP 4 IV 100,000 0.6 1791:LS*NVSS* Ser1792
SGS*IN Ser1796 Ser1799 Note: Asterisks follow the phosphorylated
residue. The numbering of residues follows that of Albala et al.,
1993.
[0163] MAP4 is a ubiquitous MAP which is probably involved in
mitosis, it also occurs as several splicing isoforms (West et al.,
1991). The phosphorylation experiments have been done with a
recombinant MAP4 construct comprising the C-terminal 496 residues
(including the repeat domain) and expresssed in E. coli (Table
4).
5TABLE 4 Peaks from Peptide second no. extinction peptide phosphor.
col. (FIG. 12) cpm (214 nm) sequence residue 1 I 135,000 0.8
825:SPATT*LP Thr829 2 II 150,000 0.35 939:VGS* Ser941 TENIK (in 1st
repeat) 3 III 120,000 0.35 923:LATTVS* Ser928 APDLK 4 IV 100,000
0.8 872:NT*T*PT* Thr873 GAAPP Thr874 Thr876 5 V 55,000 0.3
898:SS*GALS* Ser899 VDK Ser903 6 VI 100,000 0.8 1071:VGS*LD Ser1073
(in 4th repeat) 7 VII 33,000 0.04 923:LATTVS* Ser928 APDLK Note:
Asterisks follow the phosphorylated residue. The numbering of
residues follows that of West et al., 1991.
[0164] The Phosphorylation methods are identical to the ones
described in Example 2. MAP2 and MAP4 were phosphorylated with
NPK110 using radioactive ATP, the phosphorylated protein was
digested with trypsin and analysed by two-dimensional
phosphopeptide mapping (FIG. 11 for MAP2c, FIG. 12 for MAP4
construct). The peptides were then purified by two HPLC gradient
columns. The purified radioactive peptides were sequenced (for
identification of the phoshorylated residues) and identified by
two-dimensional phosphopeptide mapping.
[0165] Effects of phosphorylation on interactions with
microtubules:
[0166] The effects of phosphorylation of MAP2 and MAP4 by NPK110
were the same as for tau, that is, the affinity to microtubules
decreased several-fold, and the dynamic instability of microtubules
became much greater. This can be demonstrated, for example, by the
decrease in the mean length of microtubules in the presence of the
MAP in question, the kinase NPK110, and ATP (required for
phosphorylation). FIG. 13 shows examples for the cases of MAP4,
MAP2, and MAP2c. Microtubule assembly starts at time 0. Hollow
circles show the increase of mean length in the absence of ATP (no
phosphorylation). Filled circles show that in the presence of ATP
(and therefore with phosphorylated MAPs) the mean length is only
about half of the control.
[0167] The biological significance of the novel NPK-110 can be
summarised as followed:
[0168] NPK-110 is an efficient kinase for the repeat domain of tau,
MAP2, MAP2c and MAP4. It phosphorylates all four KXGS motifs in
tau, the first and fourth (Ser262 and Ser356) being the most
pronounced sites. In this regard the kinase reproduces earlier
observations with the kinase activity from the brain extract
(Gustke et al., 1992, and see FIG. 9). The most dramatic effects of
the kinase are that it virtually eliminates tau's binding to
microtubules (FIG. 6B), it causes the release of tau from
microtubules, and it turns stable microtubules into dynamically
unstable ones, as seen by video microscopy. These effects are
mainly dependent on the phosphorylation of Ser262, as shown by the
point mutant Ser262-Ala. These features make NPK110 a candidate
enzyme for controlling the state of assembly of microtubules in
neurons. They are also consistent with the "Tau Hypothesis of
Alzheimer's Disease" which assumes that tau's failure to bind to
and stabilise microtubules leads to their breakdown and cessation
of axonal transport. This could occur either by the detachment of
tau from microtubules, or by the inhibition of newly synthesised
tau to bind to microtubules, in both cases resulting from
phosphorylation. According to this scheme, an intervention that
would slow down NPK110 or turn off its potential activating cascade
would be suitable for a treatment of Alzheimer's disease.
[0169] It is furthermore noted that the motif KXGS is conserved not
only within the tau repeats, but also within other MAPs such as the
neuronal MAP2 and the ubiquitous MAP4 (for review see Chapin &
Bulinski, 1992). It is therefore possible that NPK-110 has a more
general role, affecting different MAPs and perhaps other proteins.
One role which might be envisaged is the involvement of NPK-110 in
the generation of cancer.
EXAMPLE 5
Further Characterization of the NPK of the Invention
Description of the cDNA Clones
[0170] A screening of a rat brain cDNA library with degenerate
oligonucleotides derived from the brain-p110MARK peptide sequences
yielded nine clones which were sequenced. They code for at least
two different kinases from at least two different genes, with a 70%
mutual homology. The peptide sequences fit completely with the
larger clone, termed MARK-1 (corresponding to NPK-110), whose
5'-prime end is missing (mol. wt. of the encoded protein approx. 90
kDal). The smaller cDNA MARK-2 encodes a protein of 81 kDal.
Peptides suitable for the design of oligonucleotides for screening
said cDNA libraries is provided in Table 5. The amino acid
sequences of the identified clones are provided in Table 6.
Homologies
[0171] A database search for homologous sequences obtained two
related but no identical sequences:
[0172] MMKEM (X70764), a mouse CDNA encoding a putative protein
kinase of unknown function (Inglis et al., 1993), shows 73%
homology to MARK-1 and 96% homology to MARK2.
[0173] HUMP78A (M80359), an unpublished human cDNA sequence, shows
73% homology to MARK-1 and 69% homology to MARK-2. All kinases show
a low homology (about 25%) to the KIN1 and KIN2 proteins from
Saccharomyces cerevisiae (Levin et al., 1987, 1990).
Tissue Distribution
[0174] As judged by Northern blotting (FIG. 14), MARK-1 and MARK-2
mRNAs are ubiquitously expressed in fetal and adult tissues.
Expression is highest in muscle, brain and fetal (but not adult)
kidney.
Activation
[0175] p110/MARK prepared from brain is at least 100-fold more
active than MARK expressed in E. Coli. The activity is dependent on
phosporylation of MARK itself on Ser and/or Thr residues, since,
after dephosphorylation with phosphatase 2A, all activity is
lost.
[0176] The phosphorylation of pllO/MARK reveals an apparent
molecular weight of 110kD on SDS gels, whereas the predicted
molecular weight from cDNA sequencing is 90 kD. This shift in
apparent molecular weight is often observed -with
phosphoproteins.
Targets
[0177] p110MARK phosphorylates not only tau protein, but also
related MAPs such as MAP2 or MAP2c (neuronal MAPs largely confined
to the somatodendritic compartment) and MAP4 (a ubiquitous MAP),
indicating a widespread function of the enzyme. The major
phosphorylation sites are similar in these MAPs, namely the serines
in the KXGS motifs in the repeat domain. The effect of
phosphorylation is also comparable, namely a strong reduction in
the microtubule-binding capacity of the MAPs, and hence a loss of
microtubule stability (see FIGS. 15, 16 for examples).
6TABLE 5 Peptide sequences obtained from a porcine brain MARK
preparation by lysC digestion. Fraction Sequence: 1 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18 33-12 D R W M N V G H E E E E L K P Y A
E 19 20 21 P E P 41 I A N E L K 47-16 A E N L L L D A D M N I K
71-09* X S S R Q N I P R C R N N I I 85 I L N H P N I V K 87-24* L
D T F C G S P P Y A A P E L F Q G K 120 L F V L N P I K 121 L F R E
V R I X 130-13 Y R I P F Y M S T D C E N 140-9 F R Q I V S A V Q Y
C H Q K 140-20 R I E I M V T M G F L
[0178]
* * * * *