Claims
- 1. A method for identifying an ATM kinase substrate recognition sequence in a protein, which comprises contacting an ATM kinase with a fusion polypeptide and detecting whether binding has occurred between the ATM kinase and the fusion polypeptide, wherein the fusion polypeptide contains a structural portion and a candidate ATM kinase substrate recognition sequence portion.
- 2. The method according to claim 1, wherein the structural portion lacks an ATM kinase recognition sequence.
- 3. The method according to claim 1, wherein the candidate sequence has an ATM kinase recognition sequence motif, including a serine, whereby the polypeptide is phosphorylatable by the ATM kinase on the serine.
- 4. The method according to claim 1, wherein the candidate sequence has a modified ATM kinase recognition sequence motif lacking an amino acid residue phosphorylatable by ATM kinase, whereby the polypeptide is not phosphorylatable by the ATM kinase.
- 5. The method according to claim 1, wherein the structural portion is GST.
- 6. The method according to claim 1, wherein the candidate peptide sequence is selected from the group consisting of:
- 7. A method for identifying a putative ATM target protein, which method comprises analyzing the sequence of the protein to determine whether it contains a sequence corresponding to an ATM substrate recognition consensus sequence motif B1-X-B2-S-Q-X-X (SEQ ID NO:1), wherein B1 is a hydrophobic amino acid, B2 is a hydrophobic amino acid or aspartic acid, X is any amino acid, Q is glutamine, and S is serine.
- 8. The method according to claim 7, wherein the ATM substrate recognition sequence motif is selected from the group consisting of P-P-D-S-Q-E-X (SEQ ID NO:2) and L-P-[L or A]-S-Q-[D or P]-X (SEQ ID NO:3), wherein P is proline, D is aspartic acid, E is glutamic acid, L is leucine, and A is alanine.
- 9. The method according to claim 7, wherein the target protein is involved in a cellular process selected from the group consisting of double stranded DNA break repair, telomere synthesis or repair, the aging process, tumor suppression, insulin signaling, insulin-like growth factor-I signaling, cell cycle control, affecting cell survival after HTLV infection and autophosphorylation.
- 10. The method according to claim 7, further comprising determining whether the sequence corresponding to an ATM substrate recognition consensus sequence motif is phosphorylated by ATM.
- 11. The method according to claim 10, wherein determining whether the sequence corresponding to an ATM substrate recognition consensus sequence motif is phosphorylated by ATM comprises contacting ATM kinase with a fusion polypeptide and detecting whether the fusion polypeptide is phosphorylated, wherein the fusion polypeptide contains a structural portion and the sequence corresponding to an ATM substrate recognition consensus sequence motif.
- 12. The method according to claim 10, wherein determining whether the sequence corresponding to an ATM substrate recognition consensus sequence motif is phosphorylated by ATM comprises contacting ATM kinase with the target protein and detecting whether the target protein is phosphorylated.
- 13. A method for identifying an ATM-regulated process comprising:
a) identifying a target protein comprising an ATM substrate recognition phosphorylation sequence according to the method of claim 7;b) modulating ATM-mediated phosphorylation of the target protein; and c) determining whether modulation of ATM-mediated phosphorylation of the target protein affects a pathway, which indicates that the process is an ATM-regulated process.
- 14. The method according to claim 13, wherein the ATM-regulated pathway is selected from the group consisting of double stranded DNA break repair, telomere synthesis or repair, the aging process, tumor suppression, insulin signaling, insulin-like growth factor-I signaling, cell cycle control, affecting cell survival after HTLV infection and autophosphorylation.
- 15. The method according to claim 13, wherein the target protein is selected from the group consisting of NBS/p95, MRE11, PHASI, CHK1, Werner, PST1, CUT1, ATM, BRCA1, and RAD17.
- 16. The method according to claim 13, wherein modulation of ATM-mediated phosphorylation comprises inhibiting ATM-mediated phosphorylation.
- 17. The method according to claim 13, wherein modulation of ATM-mediated phosphorylation comprises increasing ATM-mediated phosphorylation.
- 18. A method for modulating an ATM-regulated process comprising modulating ATM-mediated phosphorylation of a target protein comprising an ATM kinase substrate recognition sequence in a cell.
- 19. The method according to claim 18, wherein the modulation of ATM-mediated phosphorylation comprises inhibiting ATM-mediated phosphorylation.
- 20. The method according to claim 19, wherein inhibiting ATM-mediated phosphorylation comprises expressing kinase dead ATM mutant in the cell.
- 21. The method according to claim 18, wherein modulation of ATM-mediated phosphorylation comprises increasing ATM-mediated phosphorylation.
- 22. The method according to claim 21, wherein the ATM-mediated phosphorylation is increased by increasing the level of expression of ATM in a cell.
- 23. The method according to claim 18, wherein the ATM-regulated process is selected from the group consisting of double stranded DNA break repair, telomere synthesis or repair, the aging process, tumor suppression, insulin signaling, insulin-like growth factor-I signaling, cell cycle control, affecting cell survival after HTLV infection and autophosphorylation.
- 24. The method according to claim 18, wherein the target protein is selected from the group consisting of NBS/p95, MRE11, PHASI, CHK1, Werner, PST1, CUT1, ATM, BRCA1, and RAD17.
- 25. A nucleic acid encoding a kinase dead ATM mutant.
- 26. A recombinant vector which codes for expression of a defective ATM polypeptide.
- 27. The recombinant vector of claim 26 wherein the defective ATM polypeptide is a kinase dead ATM mutant.
- 28. A recombinant cell line containing the vector of claim 26.
- 29. A recombinant vector which codes on expression for a fusion polypeptide, wherein the fusion polypeptide contains a structural portion and a candidate ATM kinase recognition sequence portion.
- 30. The vector according to claim 29, wherein the structural portion lacks an ATM kinase recognition sequence.
- 31. The vector according to claim 29, wherein the fusion peptide is a GST fusion peptide.
- 32. The vector according to claim 29, wherein the candidate peptide comprises a sequence selected from the group consisting of:
- 33. A method for screening for a compound that modulates ATM-mediated phosphorylation, comprising detecting whether there is a change in the level of ATM-mediated phosphorylation of a polypeptide comprising an ATM substrate recognition sequence in the presence of a candidate compound, wherein an increase in the level of phosphorylation indicates that the compound agonizes ATM-mediated phosphorylation, and a decrease in the level of phosphorylation indicates that the compound antagonizes ATM-mediated phosphorylation.
- 34. The method according to claim 33, wherein the compound selectively modulates ATM-mediated phosphorylation.
- 35. The method according to claim 33, further comprising detecting inhibition of a cellular process mediated by ATM phosphorylation of a target protein, wherein inhibition of the activity is indicative of inhibition of ATM.
- 36. The method according to claim 35, wherein the change of a cellular process is selected from the group consisting of loss of S-phase checkpoint, a defect in the G2/M checkpoint, an increase in radiosensitivity, and increase in sensitivity to chemotherapeutic agents.
- 37. A method for screening for a compound that induces an ATM-regulated pathway in a cell, comprising contacting the cell with a candidate compound, and detecting whether the ATM-mediated process is induced in the cell, wherein the cell is defective for expression of ATM, with the proviso that the pathway is not tumor suppression or cell cycle control.
- 38. The method according to claim 33, wherein screening for a compound that induces an ATM-regulated process in a cell line comprising contacting a cell line that empresses an ATM kinase dead mutant with a candidate compound, and detecting whether the ATM-mediated pathway is induced in the cell line.
- 39. A composition comprising ATM and a polypeptide, wherein the polypeptide comprises an ATM kinase substrate recognition sequence.
- 40. The composition of claim 39, wherein the ATM kinase substrate recognition sequence is selected from the group consisting of
- 41. A peptide comprising a sequence corresponding to an ATM substrate recognition consensus sequence motif B1-X-B2-S-Q-X-X (SEQ ID NO: 1), wherein B1 is a hydrophobic amino acid, B2 is a hydrophobic amino acid or aspartic acid, X is any amino acid, Q is glutamine, and S is serine.
- 42. The peptide of claim 41, wherein the ATM substrate recognition sequence motif is selected from the group consisting of P-P-D-S-Q-E-X (SEQ ID NO:2) and L-P-[L or A]-S-Q-[D or P]-X (SEQ ID NO:3), wherein P is proline, D is aspartic acid, E is glutamic acid, L is leucine, and A is alanine.
- 43. The peptide of claim 41, wherein the ATM kinase substrate recognition sequence is selected from the group consisting of
- 44. A method for identifying a modulator of ATM-mediated activity comprising determining the extent of HTLV integration in the presence of a potential modulator of ATM-mediated activity, wherein the absence of HTLV integration indicates that said potential modulator is a modulator of ATM-mediated activity.
- 45. A method for inhibiting HTLV integration comprising contacting a cell with an ATM inhibitor.
- 46. The method according to claim 18, wherein the ATM-regulated process is affecting cell survival after HTLV infection.
- 47. The method according to claim 18, wherein the ATM-regulated process is insulin-like growth factor-I signaling.
Parent Case Info
[0001] This is a division of application Ser. No. 09/400,653, filed Sep. 21, 1999, which is a continuation-in-part of and claims the priority of U.S. application Ser. No. 09/248,061 filed Feb. 10, 1999. Each of these prior applications is hereby incorporated herein by reference, in its entirety.
Government Interests
[0002] The research leading to the present invention was supported, in part, by National Institute of Health grants CA71387 and ES05777. Accordingly, the Government has certain rights in the invention.
Divisions (1)
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Number |
Date |
Country |
Parent |
09400653 |
Sep 1999 |
US |
Child |
10024123 |
Dec 2001 |
US |
Continuation in Parts (1)
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Number |
Date |
Country |
Parent |
09248061 |
Feb 1999 |
US |
Child |
09400653 |
Sep 1999 |
US |