Assays for protein kinases using fluorescent

• US 5,912,137 A
• Filed: 07/16/1996
• Issued: 06/15/1999
• Est. Priority Date: 07/16/1996
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Tsien, Roger Y., Cubitt, Andrew B.
• Primary Examiner(s): Prouty, Rebecca E.
• Application Number:
  US08/679,865
• Time in Patent Office: 1,064 Days
• Field of Search: 530/350, 530/352, 536/23.4, 435/15
• US Class Current: 435/15
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: G01N 2333/91205 Phosphotransferases in general
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

This invention provides assays for protein kinase activity using fluorescent proteins engineered to include sequences that can be phosphorylated by protein kinases. The proteins exhibit different fluorescent properties in the non-phosphorylated and phosphorylated states.

Claims

1. A fluorescent protein substrate for a protein kinase, comprising:

an Aequorea-related fluorescent protein, wherein said Aequorea-related fluorescent protein is modified within ten amino acids of its amino-terminus by insertion of a phosphorylation site for a protein kinase and said Aequorea-related fluorescent protein exhibits a different fluorescent property in the phosphorylated state than in the un-phosphorylated state.

2. The fluorescent protein substrate of claim 1,

wherein said phosphorylation site is for a protein kinase A, a cGMP-dependent protein kinase, a protein kinase C, a Ca²⁺ /calmodulin-dependent protein kinase I, a Ca²⁺ /calmodulin-dependent protein kinase II or a MAP kinase activated protein kinase type 1.

3. The fluorescent protein substrate of claim 2,

wherein said phosphorylation site is RRXSZ (SEQ ID NO:

3);

RRXTZ (SEQ ID NO;

4), wherein X is any amino acid and Z is a hydrophobic amino acid;

BKISASEFDR PLR (SEQ ID NO;

5), where B represents either lysine (K) or arginine (R), and the first S is the site of phosphorylation;

XRXXSXRX (SEQ ID NO;

7), wherein X is any amino acid;

KKKKRFSFK (SEQ ID NO;

8);

LRRLSDSNF (SEQ ID NO;

9);

KKLNRTLTVA (SEQ ID NO;

10);

or KKANRTLSVA (SEQ ID NO;

11).

4. The fluorescent protein substrate of claim 1,

wherein said Aequorea-related fluorescent protein comprises a substitution at S65 or Y66.

5. The fluorescent protein substrate of claim 1,

wherein said Aeguorea-related fluorescent protein is P4, P4-3, W7, W2, P4-1, or comprises a substitution selected from the group consisting of S65T, S65A, S65L, Y66F or Y66W.

6. The fluorescent protein substrate of claim 1,

wherein said Aequorea-related fluorescent protein comprises a folding mutation.

7. The fluorescent protein substrate of claim 1,

wherein said phosphorylation site is contained within the sequence MRRRRSIITG (SEQ ID NO:

12) or MRRRRSII IIFTG (SEQ ID NO;

13).

8. The fluorescent protein substrate of claim 1,

wherein said Aequorea-related fluorescent protein comprises a substitution at S65A, N149K, V163A or 167T.

9. The fluorescent protein substrate of claim 2,

wherein said phosphorylation site is for a protein kinase A.

10. The fluorescent protein substrate of claim 2,

wherein said phosphorylation site is for a cGMP-dependent protein kinase.

11. The fluorescent protein substrate of claim 2,

wherein said phosphorylation site is for a protein kinase C.

12. The fluorescent protein substrate of claim 2,

wherein said phosphorylation site is for a Ca²⁺ /calmodulin-dependent protein kinase I.

13. The fluorescent protein substrate of claim 2,

wherein said phosphorylation site is for a Ca²⁺ /calmodulin-dependent protein kinase II.

14. The fluorescent protein substrate of claim 2,

wherein said phosphorylation site is for a MAP kinase activated protein kinase type 1.

15. A fluorescent proteins comprising:

an Aequorea-related fluorescent protein with at least a contiguous sequence of 200 amino acids having at least 95% identity with the amino acid sequence of SEQ ID NO;

2,

wherein said Aequorea-related fluorescent protein is modified within ten amino acids of its amino-terminus by insertion of a phosphorylation site for a protein kinase and the fluorescent protein exhibits a different fluorescent property in said phosphorylated state than in the un-phosphorylated state.

16. The fluorescent protein of claim 15,

wherein said phosphorylation site is for a protein kinase A, a cGMP-dependent protein kinase, a protein kinase C, a Ca²⁺ /calmodulin-dependent protein kinase I, a Ca²⁺ /calmodulin-dependent protein kinase II or a MAP kinase activated protein kinase type 1.

17. The fluorescent protein of claim 15,

wherein said Aequorea-related fluorescent protein comprises a substitution at S65 or Y66.

18. The fluorescent protein of claim 15,

wherein said Aequorea-related fluorescent protein comprises a folding mutation.

19. The fluorescent protein of claim 15,

wherein said Aequorea-related fluorescent protein comprises a substitution at N149K.

20. The fluorescent protein of claim 15,

wherein said Aequorea-related fluorescent protein comprises a substitution at V163A.

21. The fluorescent protein of claim 15,

wherein said Aequorea-related fluorescent protein comprises a substitution at I167T.

22. A kit, comprising:

a fluorescent protein comprising an Aequorea-related fluorescent protein with at least a contiguous sequence of 200 amino acids having at least 95% identity with the amino acid sequence of SEQ ID NO;

2, wherein said Aequorea-related fluorescent protein is modified within ten amino acids of its amino-terminus by insertion of a phosphorylation site for a protein kinase and said Aequorea-related fluorescent protein exhibits a different fluorescent property in the phosphorylated state than in the un-phosphorylated state, and

a phosphate donor.

23. A composition, comprising:

a fluorescent protein substrate for a protein kinase comprising an Aequorea-related fluorescent protein,

wherein said Aequorea-related fluorescent protein is modified within ten amino acids of its amino-terminus by insertion of a phosphorylation site for a protein kinase and said Aequorea-related fluorescent protein exhibits a different fluorescent property in the phosphorylated state than in the un-phosphorylated state, and

a test compound.

24. A composition, comprising:

a fluorescent protein comprising an Aequorea-related fluorescent protein with at least a contiguous sequence of 200 amino acids having at least 95% identity with the amino acid sequence of SEQ ID NO;

2,

wherein said Aequorea-related fluorescent protein is modified within ten amino acids of its amino-terminus by insertion of a phosphorylation site for a protein kinase and said Aequorea-related fluorescent protein exhibits a different fluorescent property in said phosphorylated state than in the un-phosphorylated state, and

a test compound.