Methods of evaluating phosphatase inhibitors
View Patent ↗The present invention identified OVARC1000473 (SEQ ID NO: 1) and NT2RM1000377 (SEQ ID NO: 3) as clones showing suppression of CREB activation by forskolin, and provides evaluation methods using these genes, and/or proteins encoded by these genes. Furthermore, these proteins were found to enhance cell damage. Compounds that can be screened based on the evaluation methods of this invention are useful as agents for inhibiting the CREB dephosphorylation reaction, agents for suppressing enhancement of cell damage, and preventive and therapeutic agents for memory disorders and/or neurodegenerative disorders.
1. A method for detecting a compound which inhibits dephosphorylation of a cAMP responsive element binding protein (CREB), wherein the method comprises
contacting a protein with (A) CREB and a protein kinase that phosphorylates CREB to phosphorylate CREB, or (B) a phosphorylated CREB in the presence or absence of a test compound, measuring the phosphorylation level of CREB;
wherein a higher level of phosphorylation in the presence of the test compound compared to the absence of the compound is indicative that the compound inhibits dephosphorylation of CREB, and:
wherein (1) the protein comprises the amino acid sequence of SEQ ID NO: 4, (2) the protein is encoded by a polynucleotide that hybridizes under stringent conditions with the full length complement of SEQ ID NO: 3, wherein the stringent conditions comprise a washing step in 0.1×SCC and 0.1% SDS at 65° C., and wherein the protein has CREB dephosphorylation activity; or (3) the protein comprises an amino acid sequence with at least 95% homology to the amino acid sequence of SEQ ID NO: 4, wherein the protein has CREB dephosphorylation activity.
2. The method of claim 1 , wherein the CREB phosphorylation level is measured, using as an index, the transcriptional activity of a CREB-regulated gene.
3. The method of claim 1 , wherein the protein comprises the amino acid sequence of SEQ ID NO:4.
4. The method of claim 1 , wherein, the protein is encoded by a polynucleotide that hybridizes under stringent conditions with the full length complement of SEQ ID NO: 3, wherein the stringent conditions comprise a washing step in 0.1×SCC and 0.1% SDS at 65° C. and wherein the protein has CREB dephosphorylation activity.
5. The method of claim 1 , wherein the protein comprises an amino acid sequence with at least 95% homology to the amino acid sequence of SEQ ID NO: 4, wherein the protein has CREB dephosphorylation activity.
6. The method of claim 1 , wherein the method comprises contacting the protein in the presence of CREB and a kinase to phosphorylate CREB in the presence or absence of the test compond.
7. The method of claim 6 , wherein the CREB phosphorylated site is a serine at position 133 of CREB.
8. The method of claim 1 , wherein the method comprises contacting a test compound with a protein in the presence of a phosphorylated CREB.
9. The method of claim 1 , wherein the CREB phosphorylated site is a serine at position 133 of CREB.
10. The method of claim 1 , wherein the contacting occurs in a cell.
11. The method of claim 10 , wherein the cell comprises a polynucleotide encoding CREB.
12. The method of claim 10 , wherein the cell comprises a polynucleotide encoding the protein.
13. The method of claim 11 , wherein the cell comprises a polynucleotide encoding the protein.