Universal kinase substrates and methods of use thereof

1. A peptide that is 9 to 50 amino acids in length and comprises a first amino acid sequence having at least 85% sequence identity to an amino acid sequence of formula Ia:

wherein:

R 1a is selected from the group consisting of: E, D;

R 2a is selected from the group consisting of: D, E;

R 3a is P, A;

R 4a is selected from the group consisting of: I;

R 5a is Y;

R 6a is selected from the group consisting of: V, A;

R 7a is T;

R 8a is selected from the group consisting of: L, V; and

R 9 a is selected form the group consisting of: E, A;

or a salt thereof.

2. The peptide of claim 1 , wherein first the amino acid sequence comprises an amino acid sequence of formula (Ia).

3. The peptide of claim 1 , wherein the first amino acid sequence has at least 85% sequence identity to EDPIYVTLE (SEQ ID NO:2) or DEAIYATVA (SEQ ID NO:5).

4. The peptide of claim 1 , wherein the first amino acid sequence is EDPIYVTLE (SEQ ID NO:2) or DEAIYATVA (SEQ ID NO:5).

5. A peptide comprising a first amino acid sequence having at least 85% sequence identity to EDDVYDSVP (SEQ ID NO:7).

6. The peptide of claim 1 , further comprising a second amino acid sequence linked to the N- or C-terminus of the first amino acid sequence through a peptide bond.

7. The peptide of claim 6 , wherein the second amino acid sequence comprises one or more lysine residues.

8. The peptide of claim 7 , wherein the second amino acid sequence comprises GGKGG (SEQ ID NO:21) or GGKKK (SEQ ID NO:22).

9. The peptide of claim 8 , which is EDPIYVTLEGGKKK (SEQ ID NO:19) or DEAIYATVAGGKKK (SEQ ID NO:20).

10. The peptide of claim 6 , wherein the second amino acid sequence is a peptide tag sequence.

11. The peptide of claim 6 , wherein the second amino acid sequence is a terbium binding peptide sequence.

12. The peptide of claim 1 , which is 9 to 50 amino acids in length and consists of the first amino acid sequence having at least 85% sequence identity to the amino acid sequence of formula Ia.

13. The peptide of claim 1 , operably linked to an affinity capture agent or a detectable agent.

14. The peptide of claim 1 , which is a substrate for five or more of protein tyrosine kinases (PTKs).

15. The peptide of claim 1 , which is a substrate for two or more PTKs selected from the group consisting of AATK, ABL, ABL2, ACK, ALK, ARG, AXL, BLK, BMX, BRK, BTK, CSF1R, CSK, DDR1, DDR2, EGFR, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB6, ERBB2, ERBB3, ERBB4, FER, FES, FGFR1, FGFR2, FGFR3, FGFR4, FGR, FLT1, FLT3, FLT4, FMS, FRK, FYN, GSG2, HCK, IGF1R, ILK, INSR, INSRR, IR, IRAK4, ITK, JAK1, JAK2, JAK3, KDR, KIT, KSR1, LCK, LMTK2, LMTK3, LTK, LYN, MATK, c-MER, MERTK, MET, MLTK, MST1R, MUSK, NPR1, NTRK1, NTRK2, NTRK3, PDGFRA, PDGFRB, PDK1/PDHK1, PLK4, PTK2, PTK2B, PTK6, PTK7, PYK2, RET, ROR1, ROR2, ROS1, RYK, SGK493, SRC, SRMS, STYK1, SYK, TEC, TEK, TEX14, TIE1, TNK1, TNK2, TNNI3K, TRKA, TRKB, TRPM7/CHAK1, TXK, TYK2, TYRO3, YES1 and ZAP70.

16. The peptide of claim 1 , which is a substrate for ABL, BTK, LYN, FGR, FYN, SRC, CSK, FES, ACK, EGFR, JAK3, ALK, FLT3, PYK2 and SYK.

17. A composition comprising a peptide as described in claim 1 , a kinase and optionally, radio-labeled ATP.

18. A method for detecting phosphorylation activity of a kinase, comprising:

1) contacting the kinase with a peptide as described in claim 1 to provide a resulting mixture;

2) measuring a signal from a detectable agent; and

3) detecting phosphorylation activity of the kinase when changes in the signal are detected as compared to a control.

19. A method for identifying a substrate of a kinase, comprising:

1) contacting the kinase with a test peptide to provide a first resulting mixture;

2) measuring a signal from a detectable agent in the first resulting mixture;

3) contacting the kinase with a peptide as described in claim 1 to provide a second resulting mixture;

4) measuring a signal from a detectable agent in the second resulting mixture; and

5) standardizing the signal from the first resulting mixture using the signal measurements from the second resulting mixture; and

6) identifying the test peptide as a substrate of the kinase when the standardized signal from the first resulting mixture is greater than a reference value.

20. A method to identity an inhibitor of a kinase comprising:

1) contacting a peptide as described in claim 1 , the kinase and a test compound to provide a resulting mixture;

2) measuring a signal from a detectable agent in the resulting mixture; and

3) identifying the test compound as an inhibitor of the kinase when changes in the signal are detected as compared to a control.

21. A method to design a peptide substrate which detects the activity of a plurality of protein tyrosine kinases (PTKs) comprising:

1) generating a candidate set of peptide substrates comprising the peptides as described in claim 1 ;

2) excluding one or more peptides from the candidate set, which are likely to not be phosphorylated by a plurality of kinases; and

3) screening the remaining peptides from step 2) for the ability to act as a substrate for a plurality of kinases.

22. The peptide of claim 5 , wherein the first amino acid sequence is EDDVYDSVP (SEQ ID NO:7).

23. The peptide of claim 5 , which is EDDVYDSVP (SEQ ID NO:7).

24. The peptide of claim 1 , which is EDPIYVTLE (SEQ ID NO:2).

25. The peptide of claim 1 , which is DEAIYATVA (SEQ ID NO:5).