Patent ID: 8323919
Filing Date: 2012-12-04
Classification: C12N,G01N

Abstract:
1. An in vitro method of identifying a glycogen synthase kinase 3 (GSK3) modulator, comprising contacting a GSK3 and a 4E-BP1-derived GSK3 substrate under GSK3 kinase activity reaction conditions, in the presence and absence of a test substance; wherein the GSK3 comprises a protein selected from the group consisting of: (i) the GSK3 of SEQ ID No: 1 or 3, (ii) a protein consisting of a sequence at least 95% identical to SEQ ID NO: 1 or 3 and functional to phosphorylate SEQ ID NO: 5, on residues Thr (iii) a GSK3 encoded by the complement of a nucleic acid which hybridizes to SEQ ID No: 2 or 4 under highly stringent hybridization conditions comprising hybridization in a solution containing 6×SSC, 5× Denhardt's solution, 30% formamide, and 100 micrograms/ml denatured salmon sperm at 37° C. overnight, followed by washing in a solution of 0.1×SSC and 0.1% SDS at 65° C. for 15 minutes and functional to phosphorylate SEQ ID NO: 5, on residues Thr wherein the 4E-BP1-derived GSK3 substrate comprises a non-primed protein selected from the group consisting of: (a) the polypeptide of SEQ ID No. 5, (b) a polypeptide consisting of a sequence at least 90% identical to SEQ ID NO: 5 and having at least one Thr-X-X-X-Thr motif, where each X is an independently selected amino acid, and each of said at least one motif can be phosphorylated by the protein of SEQ ID NO: 1 or 3, (c) the polypeptide of SEQ ID No. 7, (d) a polypeptide consisting of a sequence at least 90% identical to SEQ ID NO: 7 and having at least one Thr-X-X-X-Thr motif, where each X is an independently selected amino acid, and each of said at least one motif can be phosphorylated by the protein of SEQ ID NO: 1 or 3, (e) the polypeptide of SEQ ID No. 8, (f) a polypeptide consisting of a sequence at least 90% identical to SEQ ID NO: 8 and having at least one Thr-X-X-X-Thr motif, where each X is an independently selected amino acid, and each of said at least one motif can be phosphorylated by the protein of SEQ ID NO: 1 or 3, (g) a polypeptide consisting of a sequence encoded by the complement of a nucleic acid which hybridizes to SEQ ID No: 6 under highly stringent hybridization conditions comprising hybridization in a solution containing 6×SSC, 5× Denhardt's solution, 30% formamide, and 100 micrograms/ml denatured salmon sperm at 37° C. overnight, followed by washing in a solution of 0.1×SSC and 0.1% SDS at 65° C. for 15 minutes, said polypeptide having at least one motif, Thr-X-X-X-Thr, where each X is an independently selected amino acid, and each of said at least one motif can be phosphorylated by the protein of SEQ ID NO: 1 or 3, and (h) a fragment of a polypeptide consisting of a sequence at least 90% identical to SEQ ID NO: 5 and having at least one Thr-X-X-X-Thr motif, where each X is an independently selected amino acid, and each of said at least one motif can be phosphorylated by the protein of SEQ ID NO: 1 or 3; wherein the method detects phosphorylation of the 4E-BP1-derived GSK3 substrate by the GSK3 polypeptide and; wherein changes in the phosphorylation of the 4E-BP1-derived GSK3 substrate by the GSK3 polypeptide in the presence, compared to the absence, of the test substance identifies the test substance as a GSK3 modulator.