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WO1993003177A1 - Nouvelles proteines kinases a neurofilament et tau - Google Patents

Nouvelles proteines kinases a neurofilament et tau Download PDF

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Publication number
WO1993003177A1
WO1993003177A1 PCT/US1991/005698 US9105698W WO9303177A1 WO 1993003177 A1 WO1993003177 A1 WO 1993003177A1 US 9105698 W US9105698 W US 9105698W WO 9303177 A1 WO9303177 A1 WO 9303177A1
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Prior art keywords
antibody
dephoεphorylated
kinase
completely
activity
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PCT/US1991/005698
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English (en)
Inventor
Vernon M. Ingram
Hanno M. Roder
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Massachusetts Institute Of Technology
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Priority to PCT/US1991/005698 priority Critical patent/WO1993003177A1/fr
Publication of WO1993003177A1 publication Critical patent/WO1993003177A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes

Definitions

  • This invention relates to novel TAU/neurofilament protein kinases, DNA sequences therefor and cell lines relating thereto, a ⁇ well as inhibitors of the kinases and immunoassays relating to the kinases.
  • Neurofilaments (NF) the intermediate filaments (IF) ⁇ pecific for neurons, are an assembly of three subunit ⁇ of -apparent Mr on SDS-PAGE of 68 kD, 160 kD and 200 kD, termed NF-L, NF-M and NF-H, re ⁇ pectively. All three subunits contain a highly con ⁇ erved helical rod domain. The two heavier ⁇ ubunits also have extended C-terminal tail domains which are heavily pho ⁇ phorylated.
  • the cDNA-derived sequences of the two heavy NF-subunit ⁇ have revealed the presence of 5, 12 and 40 Ly ⁇ -Ser-Pro (Val,Ala,X) repeat ⁇ in the C-terminal domains of rat NF-M, human NF-M and human NF-H, re ⁇ pectively (Napolitano et al . , 1987; Myer ⁇ et al., 1987 and Lee ⁇ et al., 1988).
  • the ⁇ e ⁇ eguences form the epitope ⁇ of several pho ⁇ phoepitope-specific anti-NF- Ab ⁇ (Lee et al., 1988).
  • the invention provides preparations containing e ⁇ sentially pure, non ⁇ keletal-a ⁇ ociated kina ⁇ es, the kinase ⁇ capable of pho ⁇ phorylating depho ⁇ phorylated NF-M to an extent sufficient to cause a ⁇ hift on SDS-PAGE of the apparent M r of depho ⁇ phorylated NF-M toward that of native NF-M.
  • the kinases further are capable of phosphorylating Tau and are capable of phosphorylating and reconstituting SMI epitopes on completely dephosphorylated NF-triplet or purified dephosphorylated NF-M.
  • the kinases also are inhibited by excess ATP.
  • PK40 has an apparent molecular weight of 40 kD and i ⁇ capable of pho ⁇ phorylating completely dephosphorylated NF-M to an extent sufficient to cause a complete ⁇ hift of apparent molecular weight from that of completely dephosphorylated NF-M to that of native NF-M.
  • This kinase also i ⁇ capable of causing a complete shift for TAU and a partial shift for NF-H.
  • PK36 ha ⁇ an apparent molecular weight of 36 kD and is capable of pho ⁇ phorylating completely dephosphorylated NF-M to an extent sufficient to cause at least a partial shift of apparent molecular weight from that of completely dephosphorylated NF-M toward that of native NF-M.
  • novel assays are provided.
  • One as ⁇ ay involve ⁇ a method for detecting a mammalian kina ⁇ e.
  • a fraction of biological material derived from a mammal i ⁇ prepared, it being unknown whether the fraction contain ⁇ the kina ⁇ e of intere ⁇ t.
  • the fraction i ⁇ sub ⁇ tantially free of epitope ⁇ characteri ⁇ tic of a pho ⁇ phorylated neuroprotein and reactive with a te ⁇ t antibody.
  • the fraction is contacted with a depho ⁇ phorylated neuroprotein free of the epitope under condition ⁇ so a ⁇ to permit the pho ⁇ phorylation of the neuroprotein if the kina ⁇ e i ⁇ pre ⁇ ent.
  • the fraction then i ⁇ te ⁇ ted for the presence of the epitope u ⁇ ing the te ⁇ t antibody.
  • the fraction is contacted with a completely dephosphorylated neuroprotein.
  • the presence of the epitope can be detected u ⁇ ing an antibody reactive with an epitope correlated with pho ⁇ phorylated neuroproteins such as SMI-31 antibody or SMI-34 antibody, and reagents may be employed to produce a color in the presence of an immunoprecipitate complex between the antibodies and the epitope.
  • the color produced then may be measured as a quantitative measure of the pre ⁇ ence of the complex.
  • Fibrobla ⁇ t ⁇ are grown in vitro from normal and f om a te ⁇ t mammal. Aliguots of the fibroblasts then are contacted with varying concentrations of an uncoupler of oxidative phosphorylation. Then, the aliguots of fibroblast ⁇ are mea ⁇ ured for the pre ⁇ ence of an immunological epitope correlated with pho ⁇ phorylation of neuroproteins, and the extent to which the normal fibrobla ⁇ t ⁇ and the te ⁇ t fibrobla ⁇ t ⁇ exhibit the immunological epitope at the varying concentration ⁇ i ⁇ determined.
  • Yet another novel immunoa ⁇ ay according to the invention employ ⁇ depho ⁇ phorylated NF.
  • Preferred embodiments involve a ⁇ ay ⁇ utilizing completely dephosphorylated NF-triplet, completely dephosphorylated NF-M and completely dephosphorylated NF-H.
  • antibodies to the novel kinase of the invention are provided.
  • Monoclonal and polyclonal antibodies selectively specific for PK40 and PK36 are provided.
  • the antibodies are capable of inhibiting the kina ⁇ e activity of either PK40 or PK36.
  • the antibodie ⁇ may be used among other thing ⁇ for detecting the pre ⁇ ence of PK40 or PK36.
  • the invention al ⁇ o provide ⁇ method ⁇ for inhibiting neuroprotein pho ⁇ phorylation activity in a cell by introducing into a cell an inhibitor of PK40 or PK36 in an amount ⁇ ufficient to inhibit the pho ⁇ phorylation activity of the PK40 or PK36.
  • Preferred inhibitor ⁇ include fragment ⁇ of substrate ⁇ of PK40 or PK36, antibodie ⁇ ⁇ electively ⁇ pecific for PK40 or PK36 and ATP or analog ⁇ of ATP.
  • Mo ⁇ t preferably the inhibitor i ⁇ admini ⁇ tered in an amount sufficient to prevent the formation of neurofilament tangles.
  • vectors are provided containing oligonucleotides encoding PK40 or unique fragments thereof and PK36 or unique fragments thereof.
  • cell line ⁇ are provided that are transformed or transfected with an oligonucleotide encoding PK40 or a unique fragment thereof or PK 36 or a unique fragment thereof. Products of the cell line also are provided.
  • Fig. 1 shows the results of immuno-dotblot as ⁇ ays of ammonium ⁇ ulfate fractions from whole brain supernatant using SMI-31 and SMI-34 antibodie ⁇ .
  • Fig. 2 show ⁇ the re ⁇ ult ⁇ of immuno-dotblot a ⁇ ay ⁇ wherein time, pH and ionic ⁇ trength were varied to determine optimal condition ⁇ for the
  • Fig. 3 hows the results of immuno-dotblot a ⁇ ays wherein Mg 2+, ATP and crude enzyme concentration ⁇ were varied to determine optimal condition ⁇ for the SMI a ⁇ ays.
  • Fig. 5 is an autoradiograph of a
  • Fig. 6 i ⁇ a photograph of a ⁇ tained gel (12% SDS-PAGE) including lane ⁇ corre ⁇ ponding to the fraction ⁇ identified in Fig. 4.
  • Fig. 7 show ⁇ the re ⁇ ult ⁇ of a Mono Q FPLC separation of PK40, u ⁇ ing immuno-dotblot assay ⁇ , a 32P-a ⁇ say and non-denaturing 12% SDS-PAGE with staining.
  • Fig. 8 how ⁇ the re ⁇ ult ⁇ of a Mono Q FPLC separation of PK36, using a 32P-a ⁇ ay and 12%
  • Fig. 9 is a photograph of a ⁇ tained gel representing the ⁇ eparation of a PK36/40 mixture by non-denaturing 7.5% PAGE, a ⁇ well a ⁇ an autoradiograph of a 32P-assay correlating stained positions on the gel with kina ⁇ e activity.
  • Fig. 10 i ⁇ a photograph of a ⁇ tained gel repre ⁇ enting the ⁇ eparation of a PK36/40 mixture by 10% PAGE containing SDS, a ⁇ well a ⁇ an autoradiograph of a 32P-a ⁇ ay correlating ⁇ tained po ⁇ itions on the gel with kina ⁇ e activity.
  • Fig. 11 i ⁇ a photograph of a ⁇ tained gel repre ⁇ enting the ⁇ eparation of variou ⁇ PK36/40 mixture ⁇ by SDS-PAGE.
  • Fig. 12 i ⁇ a graph depicting the relative ⁇ pecificity of PK40 and PK36 for variou ⁇ kina ⁇ e substrate ⁇ .
  • Fig. 13 i ⁇ a graph depicting the dependence of PK40 activity on ATP concentration.
  • Fig. 14 i ⁇ a graph depicting the dependence of PK36 activity on ATP concentration.
  • Fig. 15 i ⁇ a graph containing Hane ⁇ -Woolf plots for PK40.
  • Fig. 17 ⁇ how ⁇ the re ⁇ ult ⁇ of te ⁇ t ⁇ de ⁇ igned to determine the ability of PK36 and PK40 to pho ⁇ phorylate depho ⁇ phorylated NF-M, a ⁇ mea ⁇ ured by immuno-dotblot a ⁇ ay ⁇ , gel mobility ⁇ hift ⁇ and 32P-mcorporation.
  • Fig. 18 show ⁇ the re ⁇ ult ⁇ of te ⁇ t ⁇ de ⁇ igned to determine the ability of PK36 and PK40 to pho ⁇ phorylate depho ⁇ phorylated NF-H, a ⁇ measured by immuno-dotblot a ⁇ say ⁇ , gel mobility shifts and 32P-mcorporation.
  • Fig. 19 shows the result ⁇ of te ⁇ t ⁇ de ⁇ igned to determine the ability of a mixture of PK36 and PK40 to pho ⁇ phorylate depho ⁇ phorylated NF-M, as measured by immuno-dotblot assays, gel mobility shift ⁇ and 32P-incorporation.
  • Fig. 20 is a photograph of a ⁇ tained gel representing the ability of PK40 to phosphorylate native and dephosphorylated TAU.
  • Fig. 21 is an autoradiograph of lanes a and b of Fig. 20, showing the presence of
  • the invention in one aspect involves the identification of novel kinase ⁇ , PK40 and PK36.
  • PK40 and PK36 have been i ⁇ olated from bovine brain a ⁇ de ⁇ cribed in Example 4 and are es ⁇ entially pure.
  • e ⁇ entially pure it i ⁇ meant that at lea ⁇ t 40% of the material in a preparation i ⁇ the kinase of interest.
  • the preparation ⁇ of the invention are ⁇ ufficiently pure so a ⁇ to permit amino acid ⁇ equencing by conventional method ⁇ , and further, can be made sufficiently pure to permit the generation and identification of antibodies to the kinases of interest.
  • PK40 and PK36 have apparent molecular weights ( r ) of 40 kD and 36 kD on SDS-PAGE, respectively.
  • the kinases are noncytoskeletal-a ⁇ sociated. "By noncyto ⁇ keletal-a ⁇ ociated it i ⁇ meant that the kina ⁇ e doe ⁇ not co-purify with the NF-cyto ⁇ keleton under high- ⁇ alt extraction conditions. "
  • the kina ⁇ es are capable of pho ⁇ phorylating a variety of dephosphorylated native sub ⁇ trate ⁇ .
  • the native ⁇ ub ⁇ trates have characteristic mobilities on SDS-PAGE which change when the substrates are depho ⁇ phorylated.
  • Treatment of the ⁇ e depho ⁇ phorylated ⁇ ubstrates with the kina ⁇ es of the invention under conditions permitting phosphorylation of the sub ⁇ trate ⁇ may re ⁇ ult in a mobility ⁇ hift on SDS-PAGE of the apparent of the depho ⁇ phorylated ⁇ ub ⁇ trate toward that of the native ⁇ ub ⁇ trate, depending upon the particular ⁇ ubstrate and kinase selected, and the conditions applied.
  • a “shift” is any detectable change in mobility.
  • complete ⁇ hift it i ⁇ meant that the mobility of the previou ⁇ ly depho ⁇ phorylated ⁇ ub ⁇ trate, after treatment with the kinase of the invention, is the same as that of the native sub ⁇ trate.
  • partial shift means that the mobility ha ⁇ moved between that of the depho ⁇ phorylated substrate and that of the native substrate.
  • No ⁇ hift mean ⁇ no detectable change in mobility after treatment of the depho ⁇ phorylated ⁇ ubstrate with the kinase of the invention.
  • PK40 i ⁇ capable of pho ⁇ phorylating completely-depho ⁇ phorylated NF-M (cdNF-M) ⁇ o a ⁇ to cau ⁇ e a complete ⁇ hift on SDS-PAGE of the apparent M of the cdNF-M to that of native NF-M.
  • PK40 also is capable of causing a complete shift of completely-dephosphorylated native bovine TAU or pure human TAU isoform expressed in E.coli from the clone Htau 40 (Goedert et al. 1989); In addition, PK40 causes a partial shift of completely-dephosphorylated NF-H (cdNF-H) .
  • PK36 is capable of phosphorylating cdNF-M ⁇ o a ⁇ to cau ⁇ e at lea ⁇ t a partial shift on SDS-PAGE of the apparent of the cdNF-M to that of native NF-M.
  • kina ⁇ e i ⁇ activated by the u ⁇ ual ⁇ econd me ⁇ enger ⁇ i.e. , small molecules (such as cAMP, cGMP, Calcium, Ca + Pho ⁇ phatidyl Serine and Ca/CAM) that are produced in ⁇ ide the cell when the out ⁇ ide of the cell membrane receive ⁇ a ⁇ ignal or ⁇ timulu ⁇ , ⁇ uch a ⁇ a peptide hormone.
  • ATP dependence and inhibition of the activities of PK40 and PK36 were determined as de ⁇ cribed in Example 6.
  • the ⁇ e values reflect a requirement for relatively high ATP concentrations. Both kinases, however, are strongly inhibited by an exce ⁇ of ATP, i.e. , when ATP i ⁇ in con ⁇ iderable
  • PK36 but not PK40, is inhibited by the Walsh inhibitor.
  • Thi ⁇ immunoa ⁇ say required NF proteins devoid of immunoreactivity with mAbs SMI-31 and SMI-34, in that the assay mea ⁇ ure ⁇ kinase activity specific for epitopes recognized by the ⁇ e antibodie ⁇ , i.e. , the repeated pho ⁇ phorylated KSP sequences.
  • the NF proteins were completely dephosphorylated as described in Example 1.
  • completely dephosphorylated it is meant nonreactive with SMI-31 and SMI-34 antibodies.
  • a colorimetric immunoa ⁇ ay al ⁇ o de ⁇ cribed in Example 2 can be used to quantitatively measure levels of pho ⁇ phorylating activity.
  • the amount of color produced i ⁇ determined, thus providing a quantitative measurement of the amount of complex formed.
  • Such a measurement correlates with the KSP- ⁇ pecific pho ⁇ phorylating activity pre ⁇ ent in the sample tested.
  • completely-dephosphorylated neuroprotein can be used as a substrate, although there are instances that do not necessarily require completely-dephosphorylated material as a sub ⁇ trate.
  • the invention also pertains to the nucleic acids encoding the human kinases corresponding to bovine PK40 and PK36, and to a method for cloning DNA seguence ⁇ encoding the human kina ⁇ es.
  • the purified bovine kinase ⁇ are ⁇ equenced a ⁇ de ⁇ cribed in Example 11. With this ⁇ equence information, oligonucleotide probe ⁇ are con ⁇ tructed and u ⁇ ed to identify the gene encoding the human kina ⁇ e in a cDNA library. Due to degeneracy of the genetic code, mo ⁇ t amino acid ⁇ are repre ⁇ ented by more than one codon.
  • the amino acid ⁇ equence cho ⁇ en from the bovine kina ⁇ e that i ⁇ u ⁇ ed to ⁇ ynthe ⁇ ize the corre ⁇ ponding oligonucleotide probe will be from a region that ha ⁇ a minimal amount of degeneracy.
  • a radiolabeled ⁇ ynthetic oligonucleotide hybridization probe corre ⁇ ponding to the least degenerate codon sequence of the peptide sequence for each of kinase PK40 and PK36 i ⁇ prepared and used to screen a cDNA library from human cell ⁇ a ⁇ de ⁇ cribed in Example 12.
  • Clone ⁇ are obtained who ⁇ e codon order matche ⁇ the amino acid sequence of each of the kina ⁇ e ⁇ .
  • a full-length cDNA sequence for each of the human kina ⁇ e ⁇ which corre ⁇ pond to bovine PK40 and PK36, i ⁇ thu ⁇ identified, and recombinant vector molecule ⁇ containing the total cDNA ⁇ equences are obtained.
  • Such a cloning method can be utilized because each of the corresponding human kinases is encoded by an oligonucleotide with sub ⁇ tantial homology to either bovine PK40 or PK36. Thu ⁇ , there i ⁇ ⁇ ufficient homology ⁇ uch that the human cDNA i ⁇ capable of being identified by the hybridization technology de ⁇ cribed herein.
  • a vector containing an oligonucleotide means a vector containing the cDNA sequence, but not necessarily expres ⁇ ing it.
  • expre ⁇ ion of the cDNA ⁇ e uence it must be operably linked to a eukaryotic or prokaryotic expre ⁇ ion control DNA ⁇ equence.
  • Such recombinant molecules are easily prepared and identified by one of ordinary skill in the art using routine skill and without undue experimentation.
  • Cell ⁇ transformed or transfected with the ⁇ e recombinant vector molecule ⁇ are capable of expre ⁇ ing the human kina ⁇ e, or fragment ⁇ thereof.
  • the human kina ⁇ e ⁇ can be i ⁇ olated according to the method ⁇ de ⁇ cribed in the Example ⁇ that were u ⁇ ed for i ⁇ olating bovine PK40 and PK36.
  • Human PK40 and PK36 kina ⁇ e ⁇ are inhibited by exce ⁇ ATP, .pho ⁇ phorylate depho ⁇ phorylated neurofilament and TAU protein ⁇ , and in particular, phosphorylate KSP sequences in these proteins.
  • Human kinase ⁇ herein mean those nonskeletal-a ⁇ ociated kina ⁇ e ⁇ identified a ⁇ de ⁇ cribed in thi ⁇ invention, including human PK40 and human PK36. Except for the Example ⁇ , a ⁇ u ⁇ ed herein and in the claim ⁇ , PK40 and PK36 mean mammalian PK40 and PK36, naturally occurring and cloned. In the Examples, unless specifically referred to otherwise, PK40 and PK36 mean bovine PK40 and PK36. By human PK40 and PK36, is meant the human kinase ⁇ corresponding to bovine PK40 and PK36.
  • antibodie ⁇ both polyclonal and monoclonal
  • Monoclonal antibodie ⁇ are obtained by the method described by Milstein and Kohler. Such a procedure involves injecting an animal with an immunogen, removing cells from the animal's spleen and fusing them with myeloma cells to form a hybrid cell, called a hybridoma, that reproduces m vitro.
  • the population of hybridomas is screened and individual clones are isolated, each of which secretes a single antibody specie ⁇ to a ⁇ pecific antigenic site on the immunogen.
  • the monoclonal antibodies are u ⁇ eful for detecting the pre ⁇ ence or ab ⁇ ence of the PK40 or PK36 kina ⁇ e ⁇ .
  • the monoclonal or polyclonal antibodie ⁇ are u ⁇ eful a ⁇ inhibitors of the PK40 and PK36 kinases.
  • the invention also involves the identification of inhibitors of PK40 and/or PK36.
  • An inhibitor of PK40 or PK36 is a molecule that is capable of binding to PK40 or PK36 in a manner so as to inhibit the pho ⁇ phorylating activity of PK40 or PK36.
  • Thi ⁇ invention disclo ⁇ e ⁇ that PK40 and PK36 are strongly inhibited by an excess of ATP.
  • Other inhibitor ⁇ may be identified by those of ordinary skill in the art using the as ⁇ ays a ⁇ de ⁇ cribed herein, e.g. , adding the putative inhibitor to the kinase and subjecting the mixture to the quantitative colorimetric immunoa ⁇ ay described in Example 2.
  • variou ⁇ analog ⁇ and conjugate ⁇ of ATP may be ⁇ creened for their ability to inhibit the pho ⁇ phorylating activity of PK40 or PK36.
  • analog ⁇ are readily available in the literature and can be acce ⁇ ed u ⁇ ing variou ⁇ data-ba ⁇ e ⁇ , including full-text patent data-ba ⁇ e ⁇ .
  • the inhibitor ⁇ thu ⁇ can re ⁇ emble the molecular ⁇ tructure of ATP, especially in the distribution of charged groups.
  • the inhibitors can be modified to enable them to enter neurons in a variety of ways.
  • the charged group ⁇ of ATP analog ⁇ can be modified by e ⁇ terification by analogy with dibutyryl-cyclic-AMP.
  • inhibitor ⁇ Ten ⁇ of thousands of putative inhibitor ⁇ may be screened, first in mixtures containing, for example, 1000 candidates, and then, after inhibition by a mixture is establi ⁇ hed, in ⁇ ubmixture ⁇ containing, for example, 100, then 10, and then one inhibitor.
  • Other inhibitor ⁇ may include, but are not limited to, KSP binding site protein ⁇ , or protein ⁇ which bind to one of the kina ⁇ e ⁇ of thi ⁇ invention, e.g. , substrate ⁇ , fragment ⁇ of substrate, antibodies, frag ent ⁇ of antibodie ⁇ , and peptide ⁇ ⁇ uch a ⁇ ⁇ ingle chain antibody con ⁇ truct ⁇ or ⁇ tructural analogs of any of these.
  • An inhibiting fragment of a substrate of PK40 and/or PK36 a ⁇ used herein is a peptide that is a " structural analog of at least a portion of the substrate and that i ⁇ capable of binding to PK40 and/or PK36, ⁇ o a ⁇ to titrate out the pho ⁇ phorylating activity of PK40 and/or PK36 for the native substrate.
  • Such fragment ⁇ may be identified and prepared by cleaving sub ⁇ trate ⁇ of PK40 and/or PK36, e.g.
  • structural analog ⁇ of the PK40 or PK36 ⁇ ub ⁇ trate ⁇ may be prepared which contain at least one KSP site and are re ⁇ i ⁇ tant to degradation by cytopla ⁇ mic, proteolytic enzyme ⁇ .
  • fragment ⁇ are ea ⁇ ily prepared and identified by one of ordinary ⁇ kill in the art u ⁇ ing routine skill and without undue experimentation. For example, they can be prepared from known sequence information of substrate ⁇ of the kina ⁇ e ⁇ of the invention.
  • a use of this invention i ⁇ to admini ⁇ ter to a cell an inhibitor of one of the kinases of the invention.
  • This can act -to reduce the pho ⁇ phorylation activity in the cell and also to reduce or prevent the formation of paired helical filaments or tangles.
  • This permits the analysis, for example, of the contribution of such pho ⁇ phorylation activity to cell maintenance a ⁇ well a ⁇ to neurocellular states characteristic of neurodegene ative disea ⁇ e and aging.
  • a therapeutic u ⁇ e of thi ⁇ invention i ⁇ to admini ⁇ ter to a ⁇ ubject in need of ⁇ uch treatment an inhibitor of one of the kina ⁇ e ⁇ of thi ⁇ invention in order to treat neurodegenerative condition ⁇ ⁇ uch as Alzheimer ' s disea ⁇ e and normal aging.
  • Such an inhibitor can reduce the formation of paired helical filaments.
  • the inhibitor i ⁇ admini ⁇ tered to a ⁇ ubject in a therapeutically acceptable amount is administered to a ⁇ ubject in a therapeutically acceptable amount.
  • ⁇ ubject i ⁇ intended to include mammal ⁇ .
  • therapeutically acceptable amount i ⁇ that amount which i ⁇ capable of ameliorating or delaying progression of the diseased or degenerative condition in the subject.
  • a therapeutically acceptable amount can be determined on an individual ba ⁇ i ⁇ and will be based, at least in part, on consideration of the subject's size, severity of symptoms to be treated, result ⁇ ⁇ ought, and the ⁇ pecific inhibitor used.
  • a therapeutically acceptable amount can be determined by one of ordinary skill in the art employing such factors and u ⁇ ing no more than routine experimentation.
  • inhibitor ⁇ include, but are not limited to, ATP, analog ⁇ of ATP, KSP binding site protein ⁇ , or protein ⁇ which bind to one of the kina ⁇ e ⁇ of thi ⁇ invention, e.g., sub ⁇ trate ⁇ , fragment ⁇ of substrate, antibodies, fragments of antibodies, and peptides such a ⁇ single chain antibody constructs.
  • Administration of the inhibitor of this invention may be made by any method which allows the inhibitor to reach the target cells. Typical methods include oral, rectal, peritoneal, subcutaneous, intravenous and topical administration of the inhibitor.
  • Other delivery sy ⁇ tem ⁇ can include ⁇ u ⁇ tained relea ⁇ e delivery systems.
  • Preferred sustained release delivery systems are those which can provide for release of the inhibitor of the invention in su ⁇ tained release pellets or capsule ⁇ .
  • Many type ⁇ of sustained release delivery ⁇ y ⁇ tems are available. These include, but are not limited to: (a) erosional ⁇ y ⁇ tems in which the inhibitor is contained in a form within a matrix, found in U.S. Patent Nos.
  • a pump-ba ⁇ ed hardware delivery ⁇ y ⁇ tem can be u ⁇ ed, some of which are adapted for implantation directly into the brain.
  • a particular problem which must be overcome for those sy ⁇ tems which deliver inhibitor via the bloodstream is to cross the blood-brain barrier, which controls the exchange of materials between the plasma and the central nervous system. Many substances are unable to pass through this barrier.
  • One way to accomplish transport of the inhibitor across the blood-brain barrier is to couple the inhibitor to a secondary molecule, a carrier, which is either a peptide or a non-proteinaceous moiety. The carrier is selected such that it is able to penetrate the blood-brain barrier.
  • the carrier can be a compound which enters the brain through a specific transport sy ⁇ tem in brain endothelial cell ⁇ , ⁇ uch a ⁇ tran ⁇ port ⁇ y ⁇ tems for tran ⁇ ferring in ⁇ ulin, or in ⁇ ulin-like growth factor ⁇ I and II.
  • Thi ⁇ combination of inhibitor and carrier i ⁇ called a prodrug.
  • the prodrug may remain intact or the chemical linkage between the carrier and inhibitor may be hydrolyzed, thereby separating the carrier from the inhibitor.
  • Lipo ⁇ omes are single or multi-compart ented bodies obtained when lipids are di ⁇ per ⁇ ed in aqueou ⁇ ⁇ u ⁇ pen ⁇ ion.
  • the wall ⁇ or membrane ⁇ are compo ⁇ ed of a continuou ⁇ lipid bilayer which enclo ⁇ e an inner aqueou ⁇ ⁇ pace.
  • Such ve ⁇ icle ⁇ can be u ⁇ ed to encapsulate and deliver therapeutic agents.
  • a lipo ⁇ ome delivery ⁇ y ⁇ tem in which the therapeutic agent i ⁇ encap ⁇ ulated within the lipo ⁇ ome, and the out ⁇ ide layer of the liposome has added to it molecules that normally are transported acro ⁇ the blood-brain barrier.
  • Such lipo ⁇ ome ⁇ can target endogenous brain transport sy ⁇ tem ⁇ that tran ⁇ port ⁇ pecific ligand ⁇ across the blood-brain barrier, including but not limited to, transferring insulin, and insulin-like growth factors I and II.
  • antibodies to brain endothelial cell receptors for ⁇ uch ligand ⁇ can be added to the outer lipo ⁇ ome layer.
  • U.S. Patent No. 4,704,355 (Bernstein) also describes methods for coupling antibodie ⁇ to lipo ⁇ ome ⁇ .
  • Patent No. 4,704,355 de ⁇ cribe ⁇ preparing lipo ⁇ omes which encapsulate ATP
  • the invention also describe ⁇ a novel a ⁇ ay that can be u ⁇ ed a ⁇ a diagno ⁇ tic te ⁇ t for early Alzheimer' ⁇ di ⁇ ea ⁇ e.
  • the a ⁇ ay mea ⁇ ure ⁇ the level of neuroprotein pho ⁇ phorylation activity in a human cell by human kina ⁇ es corre ⁇ ponding to PK40 and PK36.
  • Skin fibrobla ⁇ t ⁇ are grown in vitro from a normal and from a te ⁇ t ⁇ ubject.
  • Varying concentrations of an uncoupler of oxidative pho ⁇ phorylation from ATP production are added to the ⁇ kin fibrobla ⁇ t ⁇ and the pre ⁇ ence of immunological epitope ⁇ that are correlated with pho ⁇ phorylated neuroprotein ⁇ are determined.
  • Fibrobla ⁇ t ⁇ from Alzheimer's patient ⁇ how thi ⁇ effect at lower concentration ⁇ of uncoupling agent than fibrobla ⁇ t ⁇ from normal ⁇ ubject ⁇ .
  • the appearance of ⁇ uch epitopes will indicate the release from inhibition of kina ⁇ e ⁇ PK40 and PK36.
  • the novel kina ⁇ e immunoa ⁇ ay ⁇ required NF protein ⁇ devoid of immunoreactivity with mAb ⁇ SMI-31 and SMI-34.
  • the ⁇ e immunoa ⁇ says measure kinase activity ⁇ pecific for these epitopes, i.e. , the repeated KSP sequences. Such specificity was required becau ⁇ e crude brain extract ⁇ contain a very large number of protein kinases.
  • the NF protein ⁇ mu ⁇ t be completely depho ⁇ phorylated.
  • NF-triplet protein and individual NF- ⁇ ubunit ⁇ were prepared, and ⁇ ub ⁇ equently depho ⁇ phorylated, a ⁇ follow ⁇ .
  • NF-triplet protein wa ⁇ prepared by one of two method ⁇ : “native” or “recon ⁇ tituted. " The preparation of "native” NF-triplet was a modification of previously described procedures (Tokutake et al. , 1983; Lee et al., 1987). A freshly obtained bovine spinal cord (100-l50g, Arena & Son ⁇ , Hopkinton, MA) wa ⁇ de ⁇ heathed, minced with a razor blade and left for 2 hour ⁇ in 3 1 of 10 mM Tri ⁇ , pH 7.0, 50 M NaCl, 2 mM EGTA, 1 M DTT, 0.1 mM PMSF at 4°C for ⁇ welling.
  • the adsorbent was sedimented for 10 minutes at 15,000xg and wa ⁇ hed (10 minutes each) ⁇ ub ⁇ equently with 100 ml ad ⁇ orption buffer, 3 x 85 ml 130 mM KPO., pH 7.0, 8 M urea, 0.5% ⁇ -ME and once each with 50 ml 300 mM and 250 mM KP0 4 , pH 7.0, 8 M Urea, 0.5% ⁇ -ME.
  • the ⁇ upernatant ⁇ of the latter two wa ⁇ he ⁇ contained the bulk amount of NF-L, NF-M and NF-H and were combined for recon ⁇ titution of the NF-triplet by dialy ⁇ i ⁇ into 3 change ⁇ of 1 liter of 10 mM MES, pH 6.8, 100 mM NaCl, 1 mM MgCl 2 and 1 mM EGTA. After 30 minute ⁇ of incubation at 37°C and centrifugation for 6 hour ⁇ at 120,000xg, 40-60 mg of NF-triplet proteins were obtained. The gelatinous precipitate was rehomogenized in 40% glycerol with a glas ⁇ -teflon homogenizer to form suspen ⁇ ion ⁇ of 2.5-3 mg/ml and stored at -20°C.
  • NF- ⁇ ubunit ⁇ For separation of the individual NF- ⁇ ubunit ⁇ a previou ⁇ ly de ⁇ cribed procedure (Tokutake, 1984) wa ⁇ modified.
  • the native NF-triplet precipitate wa ⁇ taken up (0.5-1 ml/mg NF protein) in 10 mM ⁇ odiu pho ⁇ phate, pH 6.8, 6 M urea, 0.5% ⁇ -ME (starting buffer), centrifuged at 100,000xg for 1 hour and loaded onto a 40 x 1.5 cm DEAE-Sephacel column (Pharmacia).
  • NF subunits were eluted at room temperature with 600 ml of a linear gradient formed by starting buffer and 400 mM sodium phosphate, pH 6.8, 6 M urea, 0.5% ⁇ -ME at 10-15 ml/hour. Fractions were collected (120 fractions, 5 ml each) and fractions 41-48, 71-80 and 85-94 were pooled; these contained pure NF-H, NF-M, and NF-L, re ⁇ pectively, according to analy ⁇ i ⁇ by SDS-PAGE. The three fraction ⁇ were concentrated to 2-3 ml by vacuum dialy ⁇ i ⁇ and dialy ⁇ ed into water.
  • NF-L was obtained as a clear gelatinou ⁇ precipitate after centrifugation for 1 hour at 100,000xg; NF-M and NF-H were precipitated by ammonium ⁇ ulfate.
  • the pure subunits were homogenized (NF-L) or dis ⁇ olved (NF-M, NF-H) in 40% glycerol to form ⁇ tock concentrations of about 1 mg/ml of protein.
  • NF-subunit ⁇ were ⁇ eparated by FPLC on a Mono Q 5/55 column (Karl ⁇ on et al . , 1987) .
  • Depho ⁇ phorylated NF-triplet tended to aggregate over ⁇ everal week ⁇ of ⁇ torage.
  • Depho ⁇ phorylation of subunit NF-M was accomplished by incubating NF-M (0.5g) with 2 units (80 ⁇ g) E. coli alkaline pho ⁇ phata ⁇ e for 5 day ⁇ in a total volume of 1 ml under the ⁇ ame buffer condition ⁇ a ⁇ used for the NF-triplet.
  • the phosphatase was removed by gel filtration of the mixture on a 50 x 1.5 cm Sephadex G200 column (50-120 ⁇ m, 10 l/hr flow rate), equilibrated with 10 mM BisTri ⁇ , pH 7.0, 100 mM NaCl. Fraction ⁇ were analyzed by SDS-PAGE.
  • NF-M containing fraction ⁇ around the exclu ⁇ ion volume were pooled (4 ml), dialyzed into water, concentrated in a SpeedVac and ⁇ tored at -20°C a ⁇ a 0.3 mg/ml ⁇ tock ⁇ olution containing 40% glycerol.
  • the yield wa ⁇ 270 ⁇ g (54%).
  • the dephosphorylation reaction ⁇ for both NF-subunits were monitored by spotting 1-1.5 ⁇ g of NF-protein on nitrocellulose. Blocking, staining with SMI-31 and SMI-34 and development of the blot ⁇ were performed as de ⁇ cribed for Immuno-dotblot assays.
  • FPLC-purified NF-M 'but not FPLC-purified NF-H, wa ⁇ dephosphorylated with E. coli alkaline phosphata ⁇ e so a ⁇ to be unreactive to SMI-31 and SMI-34 under condition ⁇ ⁇ imilar to tho ⁇ e u ⁇ ed for the NF-triplet.
  • the shift of apparent M r on SDS-PAGE and the removal of SMI-31 and SMI-34 immunoreactivity remained incomplete even after five day ⁇ of incubation with high concentrations of E. coli alkaline phosphatase.
  • the phosphata ⁇ e ⁇ were removed by gel filtration. Heat treatment and freezing of the NF were avoided becau ⁇ e the protein ⁇ tended to aggregate.
  • EXAMPLE 2 The preferred method for the immunoa ⁇ ay for detecting KSP-pho ⁇ phorylating kina ⁇ e ⁇ i ⁇ a ⁇ follow ⁇ ' .
  • Immuno-dotblot assays were performed in 50 mM HEPES, pH 7.0, 2 M MgCl 2 , 1 M ATP, 2 mM DTT in a total volume of 30 ⁇ l with 5 ⁇ g of dephosphorylated native NF-triplet or 1.2 ⁇ g of dephosphorylated pure subunits NF-M or NF-H as sub ⁇ trate ⁇ together with a control a ⁇ ay lacking NF ⁇ . After incubation at 37°C for 18 hours, as ⁇ ay ⁇ were diluted to 100 ⁇ l with 10 mM PBS, pH 7.2, and aliquot ⁇ of 50 ⁇ l were spotted on nitrocellulose (0.22 ⁇ m, Schleicher and Sch ⁇ ll).
  • Blots were blocked by 1 hour incubation with 3% BSA in lOmM PBS, pH 7.2, and washed once in 0.5% Triton-X100/10 mM PBS. Antibodies were diluted in sterile 10 mM PBS, pH 7.2, 0.5% Triton-XlOO, 10% fetal calf serum. Blots were incubated with SMI mAbs for at least 2 hours. The blots were then wa ⁇ hed five time ⁇ .
  • Mou ⁇ e mAb ⁇ were detected by reaction with hor ⁇ eradish-peroxidease-1inked goat-anti-mouse antibody (Cappel Co.) in 1:200 dilution and by staining with 0.05% 4-chloro-l-naphthol (Sigma) and 0.05% H 2 0 2 in 50 mM TBS, pH 7.5, 33% ethanol for 5-20 minutes. All incubations and washe ⁇ were at room temperature. Incubation ⁇ were ⁇ ealed in pla ⁇ tic bag ⁇ with 50 ⁇ l of ⁇ olution/cm 2 membrane.
  • the SMI-31 and SMI-34 epitopes were reconstituted.
  • the activity wa ⁇ NF- ⁇ pecific, ⁇ ince control immunoa ⁇ ay ⁇ lacking depho ⁇ phorylated NF-triplet were negative.
  • the soluble nature of the kina ⁇ e ⁇ in the 35-45% fraction wa ⁇ confirmed when the activity did not co ⁇ ediment under low ⁇ alt condition ⁇ (lOmM HEPES Buffer pH7) after 15 minute ⁇ of incubation with the pho ⁇ phorylated native NF-triplet at 37°C ora ⁇ embled cold ⁇ olubilized microtubules according to the method of Shelanski et al (1973) [4M glycerol, 1 M GTP, 37°C, 30 minute ⁇ ] , in the ab ⁇ ence or pre ⁇ ence of 5m Mg/ATP.
  • FIG. 2 ⁇ how ⁇ the determination of the optimal incubation time, pH and NaCl concentration for conducting the a ⁇ ay u ⁇ ing the 35-45% fraction (left panel, central panel and right panel, re ⁇ pectively).
  • the a ⁇ ay ⁇ were performed with 0.5mM Mg 2+ (unle ⁇ indicated otherwi ⁇ e) and the a ⁇ ay ⁇ for pH and NaCl were performed at 18 hour incubation time.
  • the a ⁇ ay re ⁇ ponses were optimal at pH 7.0, low ⁇ alt condition ⁇ and ImM ATP.
  • FIG. 3 repre ⁇ ent ⁇ immuno-dotblot-a ⁇ ay ⁇ conducted to determine the optimal Mg 2+ and
  • FIG: 3 illustrates the immuno-dotblot-assays using the SMI-31 antibodies (identical results were obtained with
  • Lanes a-g contained 0.04, 0.09, 0.13,
  • microgram ⁇ of crude enzyme protein per a ⁇ say 0.18, 0.22, 0.33 and 0.44 microgram ⁇ of crude enzyme protein per a ⁇ say.
  • the control assays were without NFs using 0.4 micrograms crude enzyme protein per assay (lane h) .
  • the Mg 2+ and ATP concentrations were at 1.0, 2.0 and 5.0 mM.
  • the optimal Mg2+ and ATP concentrations were found to be 2mM and ImM, re ⁇ pectively. ATP
  • GTP could not sub ⁇ titute for ATP.
  • a quantitative colorimetric immunoas ⁇ ay of PK40 and PK36 can be u ⁇ ed.
  • PK40 and PK36 alone or together, were a ⁇ ayed by a quantitative ELISA-type a ⁇ ay based on the mAbs SMI-31, SMI-34 (Sternberger-Meyer
  • PK36 were incubated with 6 ⁇ l 250 mM HEPES buffer, pH 7.0/10 mM MgS04, 1.2 ⁇ l 25 mM ATP and 40-160 ug of depho ⁇ phorylated bovine neurofilament triplet protein in a total volume of 30 ⁇ l. Incubation wa ⁇ for 18 hour ⁇ at 37°C and was followed by dilution to 150 ⁇ l with 10 mM PBS. Each as ⁇ ay (20 ⁇ l) was applied to a nitrocellulose membrane a ⁇ a dot, the membrane was blocked with bovine serum albumin and individual dots were punched out. They were next incubated with SMI-31 mAb (1:500, 100 ⁇ l) for 6 hour ⁇ at 25°C.
  • Radioactive a ⁇ ay ⁇ in the ⁇ ame buffer ⁇ y ⁇ tem as for immunoassay ⁇ contained 5 ⁇ g of HTP-purified native NF-triplet a ⁇ ⁇ ubstrate (3 ⁇ g of sub ⁇ trate protein ⁇ other than NF ⁇ ) and 150-250 cpm/pmole gamma- 32P-ATP. Incubation times were 15 minutes at 37°C for activities up to about 1 pmole/min/assay, since the a ⁇ ay re ⁇ pon ⁇ e ⁇ were linear within the ⁇ e time interval ⁇ .
  • a ⁇ ay ⁇ to be analyzed on SDS-PAGE were ⁇ topped with an equivalent amount of ⁇ a ple buffer, boiled for 3 minute ⁇ and run on 7.5% gel ⁇ . After ⁇ taining with Cooma ⁇ ie Blue, de ⁇ taining and drying on Whatman 3MM paper, autoradiography was performed with a DuPont Cronex screen intensifier at -70°C. For quantitative mea ⁇ urement ⁇ , radioactive band ⁇ of individual NF- ⁇ ubunit ⁇ were cut out, placed in an Eppendorf vial immer ⁇ ed in 20 ml water and the Cerenkov radiation of the sample was counted. Counting efficiency was about 30%.
  • EXAMPLE 4 The method for purifying the kina ⁇ e ⁇ wa ⁇ optimized by expo ⁇ ing the 35-45% AS-fraction to a variety of chromatography media at 4°C.
  • the preferred method for purifying the KSP-phosphorylating kina ⁇ e ⁇ is as follows.
  • the turbid ⁇ upernatant ⁇ were clarified by centrifugation at 100,000xg for 4 hours.
  • Solid ammonium sulfate wa ⁇ added ⁇ lowly over about 4 hour ⁇ while keeping the pH at 8.0-8.5 with ammonia.
  • the precipitate obtained between 35% and 45% ⁇ aturation wa ⁇ collected by centrifugation at 20,000xg for 20 minute ⁇ , redissolved in 20 ml 10 mM HEPES, pH 7.0, 1 mM MgCl 2 , 1 mM EGTA and 1 mM DTT, and dialyzed exten ⁇ ively again ⁇ t thi ⁇ 'buffer to form a "crude enzyme" stock solution of about 20 mg/ml protein, which could be stored for several weeks at 4°C with little loss of activity.
  • CM-Sepharose starting buffer 5 mM magnesium acetate, 5 mM ATP, 1 mM DTT, 10% glycerol, 0.02% sodium azide, adjusted to pH 6.0 with BisTris
  • CM-Sepharose starting buffer 5 mM magnesium acetate, 5 mM ATP, 1 mM DTT, 10% glycerol, 0.02% sodium azide, adjusted to pH 6.0 with BisTris
  • the column was washed with 60 ml ⁇ tarting buffer at about 50 ml/hr, then the kina ⁇ e ⁇ were eluted in one step with 85 mM magnesium acetate, 5 mM ATP, 1 mM DTT, 10% glycerol, 0.02% sodium azide, pH 6.0 as a fraction of 15 ml volume.
  • the collected fraction ⁇ (numbered 10-28) were te ⁇ ted in immuno-dotblot-a ⁇ ays using SMI-31 and SMI-34 antibodie ⁇ after 18 hour ⁇ of incubation with NF-triplet.
  • the fraction ⁇ also were subjected to 32P-as ⁇ ay ⁇ (30 minute ⁇ of incubation with NF-triplet) to te ⁇ t for the pre ⁇ ence of kina ⁇ e activity (FIG.5).
  • the kina ⁇ e activity eluded a ⁇ a very broad peak.
  • Method A Gel filtration fractions containing significant amounts of PK40 (# 17-19) and PK36 (# 21-22), according to SDS-PAGE analy ⁇ i ⁇ , were pooled, dialyzed into Mono Q ⁇ tarting buffer (20 mM Tri ⁇ , pH 8.0, 20 mM MgCl 2 , 5mM ATP, 1 mM DTT, 0.02% ⁇ odium azide) and loaded on an HR 5/5 Mono Q FPLC-column (Pharmacia) equilibrated with ⁇ tarting buffer.
  • Mono Q ⁇ tarting buffer (20 mM Tri ⁇ , pH 8.0, 20 mM MgCl 2 , 5mM ATP, 1 mM DTT, 0.02% ⁇ odium azide
  • Elution of PK40 at a flow rate of 1 ml/min started with 5 ml starting buffer followed by a linear gradient of 7 ml up to 65 mM MgCl 2 , 7 ml isocratic elution at 65 mM MgCl 2 and finally a linear gradient up to 110 mM MgCi formed with elution buffer (20 mM Tris, pH 8.0, 110 mM MgCl 2 , 5 mM ATP, 1 mM DTT, 0.02% ⁇ odiumazide) .
  • the variou ⁇ fraction ⁇ were ⁇ ubjected to a variety of a ⁇ says, ⁇ hown in FIG. 7.
  • the top panel is a dotblot as ⁇ ay, the central panel is a
  • 32 P-as ⁇ ay and the bottom panel is a 12% SDS-PAGE gel electrophoresi ⁇ .
  • the various panels are aligned such that the activity within the fractions is demonstrated by the immuno-dotblot-a ⁇ ay and 32P-as ⁇ ay and can be correlated with the particular fraction run on the SDS-PAGE gel and, ⁇ importantly, with the particular band revealed by the SDS-PAGE.
  • the activity of the dotblot as ⁇ ay and 32P-a ⁇ ay correlated with the pre ⁇ ence of the 40kD band (arrow).
  • the as ⁇ ay ⁇ revealed relatively prominent pho ⁇ phorylation of NF-M and NF-H by PK40.
  • the weaker activity in fraction 8-10 may be correlated with a band that ha ⁇ a slightly higher apparent M on 12% SDS-PAGE and PK40, and therefore, was not combined with PK40. Pooling of the PK40 and PK36 in the gel filtration and FPLC steps precluded contamination with this uncharacterized kinase activity.
  • the various fractions were subjected to a variety of as ⁇ ay ⁇ , ⁇ hown in FIG. 8.
  • the top panel is a dotblot as ⁇ ay, the central panel i ⁇ a
  • the variou ⁇ panel ⁇ are aligned ⁇ uch that the activity within the fraction ⁇ i ⁇ demon ⁇ trated by the 32P-a ⁇ ay and can be correlated with the particular fraction run on the SDS-PAGE gel and, importantly, with the particular band revealed by the SDS-PAGE.
  • a ⁇ can be ⁇ een, the activity of the 32P-a ⁇ ay correlated with the pre ⁇ ence of the 40kD band (arrow).
  • the a ⁇ ay ⁇ revealed relatively prominent pho ⁇ phorylation of NF-M and NF-H by PK36.
  • the weaker activity in fraction 8-10 may be correlated with a band that ha ⁇ a slightly higher apparent M on 12% SDS-PAGE and PK36, and therefore, was not combined with PK40. Pooling of the PK36 in the gel filtration and FPLC ⁇ teps precluded contamination with this uncharacterized kina ⁇ e activity.
  • Peak fraction ⁇ of the NF-kina ⁇ e ⁇ (PK40: #11-12; PK36: # 12-13) were pooled, dialyzed into ⁇ torage buffer (20 mM Bi ⁇ Tris, pH 7.0, 2 mM MgCl 2 , 2 mM ATP, 1 mM DTT, 0.02% sodium azide) and concentrated about 10-fold in microconcentrators (Amicon 10) for ⁇ torage purpo ⁇ e ⁇ .
  • the enzyme i ⁇ ⁇ table in the Mg/ATP-containing ⁇ torage buffer.
  • Activity wa ⁇ retained for ⁇ everal day ⁇ at 4°C and after 5 cycles of freeze-thawing with little los ⁇ .
  • the ⁇ e pooled fraction ⁇ of PK40 and PK36 " re ⁇ ulted in the preparation ⁇ c and d of FIG. 11, below.
  • Method B All gel filtration fraction ⁇ containing SMI-epitope reconstituting activity (# 14-23) were pooled and PK40 was separated from PK36 by Mono Q FPLC with a gradient profile similar to Method A for PK40. Fractions #9-10 and ll-12 contained almost exclusively PK36 and PK40, respectively, although in much lower purity than in Method A.
  • PK40 wa ⁇ substantially improved by preparative gel electrophoresis on non-denaturing 7.5% PAGE- of 110 mm length containing Mm ATP.
  • gel slice ⁇ were partially eluted by leaving them overnight in 30 ⁇ l of 20 mM BisTris pH 7.0, 2 mM Mg-ATP, 1 mM DTT; aliquot ⁇ of supernatants were used in standard 32P and immunoassays.
  • FIG. 9 shows the stained 7.5%
  • the PK36/40 mixture (No. 14-23) al ⁇ o wa ⁇ ⁇ eparated on a 10% PAGE containing SDS and MgATP. Slices (2mm) were analyzed a ⁇ in connection with the material ⁇ of FIG. 9. In this case, only the 36kD protein retained NF-kina ⁇ e activity, while the activity of the 40kD protein could not be recon ⁇ tituted after SDS-expo ⁇ ure (FIG. 10).
  • FIG. 10 A ⁇ in FIG. 9, FIG. 10 ⁇ how ⁇ the ⁇ tained 10% SDS-PAGE and the
  • PK40 was eluted preparatively from gel slice ⁇ of a 7.5% PAGE in an electro-eluter (model UEA, International Biotechnologie ⁇ , New Haven, CT) in two con ⁇ ecutive 30 minute run ⁇ at 120 V and 4°C into a trapping buffer con ⁇ i ⁇ ting of 7.5 M ammonium acetate, 10 mM Mg-ATP, 2 mM DTT and a trace of bromophenol blue.
  • the elution buffer contained 25 mM Tri ⁇ pH 8.3, 192 mM glycine, 2 mM Mg-ATP and 1 mM DTT.
  • FIG. 11 illustrates a comparison of several NF-kinase preparation ⁇ u ⁇ ing SDS-PAGE and ⁇ taining with Cooma ⁇ ie blue.
  • Lane a contain ⁇ a fraction obtained by pooling of PK40 and PK36 after gel filtration and elution from Mono Q with an uninterrupted linear radiant.
  • Lane b contain ⁇ a fraction of PK40 obtained by preparative gel electrophore ⁇ is in 7.5% pages described in connection with FIG. 9.
  • Lanes c and d contain a fraction of PK40 and PK36, re ⁇ pectively, after pooling of gel filtration fraction ⁇ and Mono Q elution (protocol according to Method A) .
  • Preparative gel electrophore ⁇ i ⁇ of the fraction contained in Lane c did not improve the purity beyond that of the fraction contained in Lane b.
  • Table 1 below, details the enrichment of PK40 and PK36 using various step ⁇ of methods A and B. Table 1. Enrichment of PK40 and PK36 through various chro atographic steps by alternative methods A (standard) and B a
  • Method A Gel filtration fractions analyzed by SDS-PAGE were pooled to contain either the 36kD or the 40kD band as described above.
  • Method B All fractions containing SMI-3l/SMI-34-epitope reconstituing activity were pooled after gel filtration and PK36 and PK40 were separated in the subsequent step, not determined. NF-specific activity too low against background. - 41/2 -
  • Electroelution of PK36 from a preparative SDS-gel was unsucce ⁇ ful. nmoles 32P-P04 transferred/min/ g protein. e nmole ⁇ 32P-P04 transferred/min.
  • EXAMPLE 5 The sub ⁇ trate ⁇ pecificity of PK40 and PK36 was determined as follows. Among neuronal proteins tested, the specificity of PK40 for dephosphorylated NF-M was most striking. Other
  • ⁇ ub ⁇ trate ⁇ were le ⁇ efficient.
  • the order of ⁇ pecificity wa ⁇ : depho ⁇ phorylated NF-M >> TAU > NF-M NF-L > depho ⁇ phorylated NF-H > NF-H.
  • TAU-I AND TAU-II Two chromatographically ⁇ eparable TAU preparation ⁇ (TAU-I AND TAU-II) were i ⁇ olated and te ⁇ ted a ⁇ ⁇ ub ⁇ trate ⁇ for the kina ⁇ e ⁇ .
  • the TAU protein wa ⁇ i ⁇ olated in the cour ⁇ e of the kina ⁇ e purification procedure a ⁇ a by-product from gel filtration fractions preceding the fractions containing kinase activity.
  • TAU was obtained after HC10 4 treatment and ammonium sulfate fractionation of the ⁇ upernatant a ⁇ de ⁇ cribed (Ueda et al., 1990).
  • Two TAU fraction ⁇ were obtained by ⁇ ub ⁇ equent FPLC on Mono S with a linear gradient Of 0-200 mM NaCl, 20 mM HEPES pH 6.9, 1 mM EDTA, 1 mM DTT (Hage ⁇ tedt et al. , 1989), which were di ⁇ tingui ⁇ hed only by the relative amount ⁇ of the 3 major i ⁇ oform ⁇ re ⁇ olved by SDS-PAGE.
  • MAP2 in a crude microtubule-prepara ion was a ⁇ ub ⁇ trate for PK40 and PK36 comparable to, or better than, TAU protein ⁇ , e ⁇ pecially for PK40.
  • MAP#2 i ⁇ pho ⁇ phorylated by both kina ⁇ e ⁇ above background level (PK40:2.5x; PK36:1.5x; determined by CERENKOV-counting) .
  • Ly ⁇ ine-rich hi ⁇ tone type III (calf thymus, Sigma Chemical ⁇ ) wa ⁇ the most preferred sub ⁇ trate for both PK36 and PK40.
  • the acidic protein phosvitin (Sigma Chemical ⁇ ) and tubulin (from calf brain, gift of Dr. F. Solomon, Dept. of Biology, MIT) were very poor substrates for either PK36 or PK40.
  • FIG. 12 graphically depicts the specificity of PK40 and PK36 for various sub ⁇ trate ⁇ , a ⁇ mea ⁇ ured by the 32P-a ⁇ ay relative to NF-L.
  • the graph plot ⁇ the ⁇ ub ⁇ trate at a concentration of O.lmg/ml again ⁇ t the % relative activity, the indicated values being means (+ or - S.D.) of triplicate assay ⁇ .
  • EXAMPLE 6 The ATP dependence and inhibition of the activitie ⁇ of PK40 and PK36 were determined at 2 mM Mg 2+ with ⁇ oluble depho ⁇ phorylated NF-M a ⁇ ⁇ econd substrate to avoid uncertainties arising from the aggregation state of NF-triplet in suspension. The optima were at 0.5 - 1 mM ATP for both kinases. Apparent K values for ATP of both kinase ⁇ were e ⁇ timated from Woolf-Hane ⁇ plot ⁇ (Dixon and Webb, 1979) for a range of ATP concentration ⁇ sufficiently below the onset of -44-
  • FIGS. 13 and 14 are graph ⁇ ⁇ howing the dependence of PK40 and PK36, re ⁇ pectively, on ATP concentration at a Mg 2+ concentration of 2 mM.
  • FIGS. 15 and 16 are Hane ⁇ -Woolf plot ⁇ for
  • PK40 and PK36 re ⁇ pectively, with NF-M a ⁇ a ⁇ ub ⁇ trate.
  • PK36 and particularly PK40 were ⁇ trongly inhibited to 14% and 7%, re ⁇ pectively, of the control level in the pre ⁇ ence of 5 mM
  • PK36 see Table 1, Method A, step IV and Fig,
  • EXAMPLE 7 A comparison of the phosphorylating activity of PK40 and PK36 with other kinases was performed. Phosphorylation of the KSP sequence in dephosphorylated NF-triplet and dephosphorylated NF-M, using the SMI-31 immunoassay, i.e., measuring reconstitution of the SMI-epitopes, was achieved with a mixture of PK40 and PK36, but not with calcium/calmodulin dependent kinase II, protein kinase C, cAMP-dependent kinase or second messenger-independent microtubule-associated kinase.
  • Ca 2+/calmodulin-dependent kinase II and protein kinase C were performed at 37°C in 30 microliters of 50 mM HEPES, pH 7.5, 10 mM Mg 2+ , 5 mM Ca 2+ , 1 M EGTA, 2mM DTT, 1 mM ATP and 50 micrograms/microliter calmodulin and phosphatidyl ⁇ erine, respectively, and 5 micrograms NF-triplet protein.
  • EXAMPLE 8 PK40 and PK36 induced mobility shifts of the heavy NF- ⁇ ubunits on SDS-PAGE and incorporated pho ⁇ phate in high molar ratios.
  • the purified activitie ⁇ of FIG. 11, lane ⁇ b and d were incubated in increa ⁇ ing concentration ⁇ with dephosphorylated NF-M and depho ⁇ phorylated NF-H.
  • FIG. 17 depict ⁇ the ⁇ aturation phosphorylation of completely dephosphorylated NF-M by PK40 and PK36. Increasing amounts of enzyme activity are (measured against extent of phosphorylation in
  • FIG. 19 repre ⁇ ent ⁇ data relating to the ⁇ aturation pho ⁇ phorylation of depho ⁇ phorylated NF-M by a mixture of PK36 and 40, again according to the ⁇ ame condition ⁇ as set forth above in connection with FIG. 17, except that the mobilities of the dephosphorylated and native NF-subunit ⁇ are shown in the last two lanes in FIG. 19.
  • PK40 incorporated up to 15 phosphate groups into NF-M which corre ⁇ ponds well to the number of pho ⁇ phate ⁇ found in i ⁇ olated bovine NF-M (Wong et al. , 1984) and induced a complete shift of the NF-M band on SDS-PAGE to the higher apparent of native NF-M. In contrast, only a partial shift of NF-H was achieved with a maximum of 7 pho ⁇ phates introduced into a molecule with presumably about 40 KSP-site ⁇ .
  • NF-M NF-M.
  • SMI-immuno ssay responses were correlated with the gel mobility shift, but did not respond at lower levels of phosphorylation, i.e. , ⁇ 5 moles P0 4 /mole NF-M.
  • the SMI-34 immuno-a ⁇ ay required a higher level of pho ⁇ phorylation than the SMI-31 a ⁇ ay.
  • EXAMPLE 9 PK40 induced a ⁇ ub ⁇ tantial ⁇ hift of apparent of TAU protein ⁇ on SDS-PAGE (10% acrylamide) .
  • Depho ⁇ phorylation of TAU protein wa ⁇ achieved as follows. TAU-I and TAU-II (165 icrogram ⁇ total of 1:2 mix) were incubated overnight with 4.8 microgram ⁇ E. coli alkaline pho ⁇ phata ⁇ e and 6.5 microgram ⁇ calf inte ⁇ tinal pho ⁇ phata ⁇ e in 0.2 ml 50 mM Tri ⁇ pH 8.5, 0.5 M MgS0 4 , 0.5 mM ZnS0 4 , 0.5 mM PMSF at 37°C.
  • the pho ⁇ phatase ⁇ were quantitatively removed by precipitation with 6 microliters 70% HC10 4 and centrifugation for 15 minute ⁇ at 12,000xg; a con ⁇ iderable amount of the TAU-I and TAU-II protein ⁇ al ⁇ o precipitated in thi ⁇ ⁇ tep.
  • Depho ⁇ phorylated TAU-I + TAU-II 25 micrograms were recovered from the supernatant after neutralization and dialysi ⁇ into water.
  • FIG. 20 i ⁇ a Coomas ⁇ ie blue stained gel depicting the effects of PK40 treatment on native and dephosphorylated bovine TAU under saturation conditions.
  • Lane a contains PK40-treated native TAU;
  • lane b contains PK40-treated dephosphorylated TAU;
  • lane c contain ⁇ native TAU;
  • lane d contain ⁇ -49-
  • FIG. 21 is an autoradiogram of lanes a and b of FIG. 20 showing the pre ⁇ ence of 32P-label in both the PK40 treated native and dephosphorylated TAU, respectively.
  • the phosphatase-treated native bovine TAU converted the pattern of three distinguishable isoform ⁇ on SDS-PAGE into a four band pattern as expected (Lindwall et al. , 1984), accompanied by a ⁇ hift of about 15 kD to a lower apparent M . Thi ⁇ ⁇ hift could be completely reversed and the original three band pattern restored after phosphorylation with PK40.
  • the kinase al ⁇ o incorporated ⁇ ubstantial amounts of pho ⁇ phate into native bovine TAU but induced only a ⁇ mall additional mobility ⁇ hift.
  • Uncoupling of oxidative phosphorylation from ATP production by chemical means causes the appearance of immunological epitopes in fibroblast cells from healthy patients, cultured under special conditions. (Bla ⁇ et al., 1990). This observation is used for the diagnosi ⁇ of early Al heimer's disea ⁇ e by linking the appearance of the ⁇ e epitopes to the activity of kina ⁇ es PK40 and PK36, which are released from inhibition when ATP levels fall, as i ⁇ the case when oxidative pho ⁇ phorylation i ⁇ uncoupled from ATP production. Uncoupling i ⁇ achieved by the u ⁇ e of an uncoupling reagent, e.g.
  • the diagno ⁇ tic te ⁇ t for early neuronal degeneration is applicable for various conditions where neurons degenerate, e.g. , Alzheimer's disease, Parkinson' ⁇ di ⁇ ea ⁇ e, Huntington' ⁇ chorea, normal aging, and brain infarct ⁇ .
  • a diagnostic test for early Alzheimer's disea ⁇ e i ⁇ de ⁇ cribed u ⁇ ing kina ⁇ e ⁇ PK40 and PK36 Primary culture ⁇ of ⁇ kin fibrobla ⁇ t ⁇ are obtained from the patient to be te ⁇ ted. The ⁇ e are grown in Dulbecco' ⁇ modified Eagle's medium containing 0.1 mM dibutyryl cyclic-AMP, 0.1 ug/ml 7S nerve growth factor, 10 ug/ml mixed bovine ganglioside ⁇ and 5% chick embryo extract.
  • an uncoupler of oxidative pho ⁇ phorylation from ATP production e.g. , CCCP (carbonyl cyanide m-chlorophenyl hydrazone)
  • CCCP carbonyl cyanide m-chlorophenyl hydrazone
  • the cells show immunological epitopes (Alz-50, PHF-epitopes,SMI-31/SMI-34-po ⁇ itive TAU/neurofilament epitopes), indicating the relea ⁇ e from inhibition of kina ⁇ es PK40 and PK36.
  • Cell ⁇ from Alzheimer patients show this effect at lower concentrations of uncoupler, compared to normal cell ⁇ . Thu ⁇ , cell ⁇ from patient ⁇ to be tested are "titrated” with increasing concentrations of uncoupling agent or with decrea ⁇ ing oxygen concentration ⁇ . They are distingui ⁇ hed from cell ⁇ from normal individual ⁇ by their lower re ⁇ i ⁇ tance to the effects of decrea ⁇ ing the ATP concentration.
  • EXAMPLE 11 A protein sequencing procedure for the PK40 and PK36 kina ⁇ es is described. The purified kinase ⁇ are reduced with dithiothreitol and free thiol ⁇ blocked with [ 14C]iodoacetamide in 6 M guanidine HC1 (Steiner et al. , 1979). Protein i ⁇ recovered without prior dilution by organic precipitation, wa ⁇ hed with methanol, and di ⁇ olved in 70% (v/v) formic acid containing 50 mg CNBr/ml, which cleave ⁇ the protein at methionine re ⁇ idues. The ⁇ olution i ⁇ ⁇ tirred at room temperature for 24 hour ⁇ under N 2 ga ⁇ .
  • the cleavage product ⁇ are ⁇ ubjected to preliminary separation on a 1.5 cm x 50 cm
  • EXAMPLE 12 A cloning procedure for cDNAs encoding kinases PK40 and PK36 is described.
  • a radiolabeled synthetic oligonucleotide hybridization probe corresponding to the lea ⁇ t degenerate codons of the peptide sequence for each of the PK40 and PK36 kinase ⁇ i ⁇ prepared.
  • the oligonucleotide for PK40 and the oligonucleotide for PK36 are u ⁇ ed to ⁇ creen lambda gtll cDNA librarie ⁇ prepared from poly(A) RNA from human fetal brain cell ⁇ , commercially available from a variety of ⁇ ource ⁇ .
  • Hybridization conditions are as de ⁇ cribed by Cate et al. (1986), except that the final wa ⁇ h in tetramethyl ammonium chloride i ⁇ omitted.
  • DNA in ⁇ erts from po ⁇ itive plaque ⁇ are ⁇ ubcloned directly into the pla ⁇ mid vector pBlue- ⁇ cript SKM13+ (Stratagene, Inc. San Diego, CA) .
  • Po ⁇ itive pla ⁇ mid ⁇ ubclone ⁇ are identified by colony hybridization, with the u ⁇ e of the same oligonucleotide hybridization probe.
  • Minipreparation ⁇ of plasmid DNA are prepared from positive colonies.
  • Clone ⁇ are obtained who ⁇ e codon order matche ⁇ the amino acid ⁇ equence of each of the kinases.
  • a full-length cDNA sequence is a ⁇ embled from the overlapping partial clone ⁇ for each of the kinases.
  • Lamprey neurofilament ⁇ combine in one ⁇ ubunit the features of each mammalian NF triplet protein but are highly phosphorylated only in large axons. J Neurosci 9:698-709. -58-

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Abstract

Purification et identification de nouvelles protéines kinases à neurofilament et TAU, PK40 et PK36. De nouveaux dosages immunologiques concernant lesdites kinases et des inhibiteurs desdites kinases sont décrits, ainsi que des séquences d'ADN qui codent ces kinases et des lignées cellulaires relatives auxdites kinases.
PCT/US1991/005698 1991-08-09 1991-08-09 Nouvelles proteines kinases a neurofilament et tau WO1993003177A1 (fr)

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WO1996030766A1 (fr) * 1995-03-27 1996-10-03 F. Hoffmann-La Roche Ag Inhibition de l'association tau-tau
WO1996035126A1 (fr) * 1995-05-03 1996-11-07 President And Fellows Of Harvard College Evaluation du role de la calcineurine en matiere d'immunosuppression et de neurotoxicite
EP1026237A3 (fr) * 1991-08-09 2001-03-28 Massachusetts Institute Of Technology Proteines-kinases de neurofilament/tau PK40 et PK36
US7335505B2 (en) 2001-01-15 2008-02-26 Wista Laboratories Ltd. Materials and methods relating to protein aggregation in neurodegenerative disease
US7737138B2 (en) 2004-09-23 2010-06-15 Wista Laboratories Ltd. Methods of treatment of a tauopathy condition comprising the use of thioninium compounds
US7888350B2 (en) 2006-03-29 2011-02-15 Wista Laboratories Ltd. 3,7-diamino-10H-phenothiazine salts and their use
US8710051B2 (en) 2006-03-29 2014-04-29 Wis Ta Laboratories Ltd. 3,7-diamino-10H-phenothiazine salts and their use
US10864216B2 (en) 2011-02-11 2020-12-15 Wista Laboratories, Ltd. Phenothiazine diaminium salts and their use

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Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1026237A3 (fr) * 1991-08-09 2001-03-28 Massachusetts Institute Of Technology Proteines-kinases de neurofilament/tau PK40 et PK36
US8278298B2 (en) 1995-03-27 2012-10-02 Wista Laboratories Ltd. Inhibition of tau-tau-association
US6376205B1 (en) 1995-03-27 2002-04-23 University Court Of The University Of Aberdeen Screening methods for agents that modulate or inhibit tau association with tau or map2
EP1067386A3 (fr) * 1995-03-27 2004-11-24 The University Court of The University of Aberdeen Inhibition de l'association Tau-Tau
US6953794B2 (en) 1995-03-27 2005-10-11 The University Court Of The University Of Aberdeen Inhibition of tau-tau association
US7534786B2 (en) 1995-03-27 2009-05-19 Wista Laboratories Ltd. Inhibition of tau-tau association
WO1996030766A1 (fr) * 1995-03-27 1996-10-03 F. Hoffmann-La Roche Ag Inhibition de l'association tau-tau
WO1996035126A1 (fr) * 1995-05-03 1996-11-07 President And Fellows Of Harvard College Evaluation du role de la calcineurine en matiere d'immunosuppression et de neurotoxicite
US6444870B1 (en) 1995-05-03 2002-09-03 President And Fellows Of Harvard College Methods for assessing the role of calcineurin immunosuppression and neurotoxicity
US7335505B2 (en) 2001-01-15 2008-02-26 Wista Laboratories Ltd. Materials and methods relating to protein aggregation in neurodegenerative disease
US7893054B2 (en) 2001-01-15 2011-02-22 Wista Laboratories Ltd. Materials and methods relating to protein aggregation in neurodegenerative disease
US7737138B2 (en) 2004-09-23 2010-06-15 Wista Laboratories Ltd. Methods of treatment of a tauopathy condition comprising the use of thioninium compounds
US7888350B2 (en) 2006-03-29 2011-02-15 Wista Laboratories Ltd. 3,7-diamino-10H-phenothiazine salts and their use
US8710051B2 (en) 2006-03-29 2014-04-29 Wis Ta Laboratories Ltd. 3,7-diamino-10H-phenothiazine salts and their use
US9174954B2 (en) 2006-03-29 2015-11-03 Wista Laboratories Ltd. 3,7-diamino-10H-phenothiazine salts and their use
US11344558B2 (en) 2006-03-29 2022-05-31 Wista Laboratories Ltd. 3, 7-diamino-10H-phenothiazine salts and their use
US11951110B2 (en) 2006-03-29 2024-04-09 Wista Laboratories Ltd. 3, 7-diamino-10H-phenothiazine salts and their use
US10864216B2 (en) 2011-02-11 2020-12-15 Wista Laboratories, Ltd. Phenothiazine diaminium salts and their use
US11180464B2 (en) 2011-02-11 2021-11-23 Wista Laboratories Ltd. Phenothiazine diaminium salts and their use
US12064439B2 (en) 2011-02-11 2024-08-20 Wista Laboratories Ltd. Phenothiazine diaminium salts and their use

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