Novel tau/neurofilament protein kinases

Abstract

Novel TAU/neurofilament protein kinases, PK40 and PK36, are essentially purified and characterized. Novel immunoassays relating to the kinases and inhibitors for the kinases also are provided. Finally, DNA sequences encoding the kinases and cell lines relating to the kinases are provided.

1 . A preparation containing an isolated, essentially pure, nonskeletal-associated kinase, the kinase capable of phosphorylating dephosphorylated NF-M to an extent sufficient to cause a shift on SDS-PAGE of the apparent M r  of dephosphorylated NF-M toward that of native NF-M.  
     
     
         2 . A preparation as claimed in  claim 1  wherein the kinase is capable of phosphorylating both KSP sites of TAU and is capable of abolishing the TAU epitope on TAU.  
     
     
         3 . A preparation as claimed in  claim 1  wherein the kinase is capable of phosphorylating and reconstituting phospho-epitopes on completely dephosphorylated NF-triplet or purified dephosphorylated NF-M.  
     
     
         4 . A preparation as claimed in  claim 1  wherein the kinase is capable of being inhibited by excess ATP.  
     
     
         5 . A preparation as claimed in  claim 1  wherein the kinase has a K m  of about 93 for ATP.  
     
     
         6 . A preparation as claimed in  claim 1  wherein the kinase is capable of phosphorylating completely dephosphorylated NF-M to an extent sufficient to cause a complete shift and wherein the kinase is capable of phosphorylating completely dephosphorylated NF-H to an extent sufficient to cause a partial shift on SDS-PAGE of the apparent M r  of completely dephosphorylated NF-H toward that of native NF-H.  
     
     
         7 . A preparation as claimed in  claim 1  wherein the kinase is capable of phosphorylating completely dephosphorylated TAU to an extent sufficient to cause a complete shift on SDS-PAGE of the apparent M r  of completely dephosphorylated TAU to that of native TAU.  
     
     
         8 . A prepartion as claimed in  claim 1  wherein the kinase is capable of phosphorylating TAU to an extent sufficient to result in an SDS-PAGE pattern characteristic of human TAU proteins extracted from PHF.  
     
     
         9 . A preparation as claimed in claim 1 wherein the kinase has a sequence characterized by sequence numbers 1 and 2.  
     
     
         10 . A preparation as claimed in any one of claims  1 - 9  wherein the kinase has an apparent molecular weight of 40 kD.  
     
     
         11 . A preparation as claimed in  claim 1  wherein the kinase is capable of phosphorylating completely dephosphorylated NF-M to an extent sufficient to cause at least a partial shift on SDS-PAGE of the apparent M r  of completely dephosphorylated NF-M toward that of native NF-M.  
     
     
         12 . A preparation as claimed in  claim 11  wherein the kinase has a K m  of about 50 for ATP.  
     
     
         13 . A preparation as claimed in claims  1  and 11-13 wherein the kinase has an apparent M r  of 36 kD.  
     
     
         14 . A method for detecting a mammalian kinase comprising, 
 preparing a fraction of biological material derived from a mammal, it being unknown whether the fraction contains the kinase, the fraction being substantially free of epitopes that are characteristic of a protein in a particular state of phosphorylation and that are reactive with a test antibody,    contacting the fraction with a protein free of the epitope under conditions so as to permit the phosphorylation of the neuroprotein if the mammalian kinase is present, and    testing for the presence of the epitope using the test antibody.    
     
     
         15 . A method for detecting a mammalian kinase comprising: 
 preparing a fraction of biological material derived from a mammal, it being unknown whether the fraction contains the kinase, the fraction being substantially free of epitopes that are characteristic of a protein in a particular state of phosphorylation and that are reactive with a test antibody,    contacting the fraction with a protein that includes the epitope under conditions so as to permit the phosphorylation of the protein if the mammalian kinase is present, and    testing for the presence of the epitope using the test antibody.    
     
     
         16 . A method as claimed in  claim 15  further characterized by contacting the fraction with a neuroprotein.  
     
     
         17 . A method as claimed in claims  14  or  15  further characterized by contacting the fraction with a completely dephosphorylated neuroprotein.  
     
     
         18 . A method as claimed in claims  14  or  15  further characterized by contacting the fraction with a completely dephosphorylated NF-H.  
     
     
         19 . A method as-claimed in claims  14  or  15  further characterized by contacting the fraction with a completely dephosphorylated TAU.  
     
     
         20 . A method as claimed in claims  14 - 15  wherein the test antibody is a test antibody selected from the group consisting of SMI-31, SMI 33 and SMI-34.  
     
     
         21 . An immunoassay comprising: 
 an antibody that is reactive with an epitope characteristic of a particular state of phosphorylation of a.-protein, and    the protein in a state of phosphorylation that does not bind to the antibody.    
     
     
         22 . An immunoassay as claimed in  claim 21  wherein the protein is a neuroprotein.  
     
     
         23 . An immunoassay as claimed in  claim 21  wherein the protein is dephosphorylated relative to the native protein.  
     
     
         24 . An immunoassay as claimed in  claim 21  wherein the protein is TAU.  
     
     
         25 . An immunoassay as claimed in  claim 23  wherein the protein is TAU.  
     
     
         26 . An immunoassay employing dephosphorylated NF.  
     
     
         27 . An immunoassay as claimed in  claim 26  wherein the assay employs completely dephosphorylated NF-triplet.  
     
     
         28 . An immunoassay as claimed in  claim 26  wherein the assay employs completely dephosphorylated NF-M.  
     
     
         29 . An immunoassay as claimed in  claim 26  wherein the assay employs completely dephosphorylated NF-H.  
     
     
         30 . A monoclonal antibody selectively specific for PK40.  
     
     
         31 . A monoclonal antibody capable of binding to and inhibiting the kinase activity of PK40.  
     
     
         32 . An immunoassay for detecting PK40 employing the monoclonal antibody of  claim 30 .  
     
     
         33 . A polyclonal antibody selectively specific for PK40.  
     
     
         34 . A polyclonal antibody as claimed in  claim 33  wherein the antibody is capable of inhibiting the kinase activity of PK40.  
     
     
         35 . An immunoassay for detecting PK40 employing the polyclonal antibody of  claim 33 .  
     
     
         36 . A monoclonal antibody selectively specific for PK36.  
     
     
         37 . A monoclonal antibody capable of binding to and inhibiting the kinase activity of PK36.  
     
     
         38 . An immunoassay for detecting PK36 employing the monoclonal antibody of  claim 36 .  
     
     
         39 . A polyclonal antibody selectively specific for PK36.  
     
     
         40 . A polyclonal antibody as claimed in  claim 39  wherein the antibody is capable of inhibiting the kinase activity of PK36.  
     
     
         41 . An immunoassay for detecting PK36 employing the polyclonal antibody of  claim 39 .  
     
     
         42 . A method for inhibiting neuroprotein phosphorylation activity in a cell comprising, 
 introducing into a cell an inhibitor of PK40 or PK36 in an amount sufficient to inhibit phosphorylating activity of the PK40 or PK36.    
     
     
         43 . A method as claimed in  claim 42  wherein a fragment of a substrate of PK40 or PK36 is introduced into the cell.  
     
     
         44 . A method as claimed in  claim 42  wherein an antibody capable of binding to PK40 is introduced into the cell.  
     
     
         45 . A method as claimed in  claim 42  wherein an antibody capable of binding to PK36 is introduced into the cell.  
     
     
         46 . A method as claimed in  claim 42  wherein ATP or an analog thereof is introduced into the cell.  
     
     
         47 . A method as claimed in  claim 42  wherein the inhibitor is administered in a sufficient amount to prevent the formation of neurofilament tangles.  
     
     
         48 . A vector containing an oligonucleotide encoding PK40 or a unique fragment thereof.  
     
     
         49 . A vector as claimed in  claim 48  wherein the oligonucleotide is a CDNA.  
     
     
         50 . A vector as claimed in  claim 48  wherein the oligonucleotide corresponds to human PK40-or a unique fragment thereof.  
     
     
         51 . A vector containing an oligonucleotide encoding PK36 or a unique fragment thereof.  
     
     
         52 . A vector as claimed in  claim 51  wherein the oligonucleotide is cDNA.  
     
     
         53 . A vector as claimed in  claim 51  wherein the oligonucleotide corresponds to human PK36 or a unique fragment thereof.  
     
     
         54 . A cell line transformed or transfected with an oligonucleotide encoding PK40 or a unique fragment thereof.  
     
     
         55 . A cell line transformed or transfected with an oligonucleotide encoding PK36 or a unique fragment thereof.  
     
     
         56 . A kinase produced by the cell line of either of claims  54  or  55 .