Detection reagents for tyrosine kinase activity and methods of use thereof
View Patent ↗Dual colored fluorescent indicators of specific tyrosine kinase activity and methods of use thereof are disclosed.
1. A genetic construct encoding a dual colored, specific, tyrosine kinase enzyme indicator molecule for assessing tyrosine kinase activity in a cell, said construct comprising the following sequences in operable linkage in the recited order:
a) a promoter sequence effective to drive expression of said construct in a target cell;
b) a sequence encoding a first fluorescent reporter molecule that is insensitive to changes in phosphorylation;
c) sequences encoding at least two distinct consensus phosphorylation sites for tyrosine phosphorylation wherein the consensus phosphorylation sites are selected from the group consisting of SEQ ID NOS: 43-86;
d) a sequence encoding a circularly permuted second fluorescent reporter molecule, where
said first and second fluorescent reporter molecules fluoresce different colors; and
e) an SH2 interacting domain from a tyrosine kinase selected from the group consisting of
fyn, src, lck, Csk, p85, Ab1, RasGAP, Crk, Nck, She, FRS2, Grb2, SH2B, rAPS, PLC-g, Gab1, Grb7, Grb14, and yes, wherein phosphorylation of tyrosine in one or more of said consensus phosphorylation sites in c) causes said SH2 interacting domain to bind to said consensus phosphorylation site thereby causing the fluorescence signal of the second fluorescent reporter molecule to increase.
2. The genetic construct of claim 1 where the SH2 interacting domain is selected from the group consisting of fyn, src, RasGAP, Crk, and Nck.
3. The genetic construct of claim 2 where the SH2 interacting domain is fyn.
4. The genetic construct of claim 2 where the consensus phosphorylation sites are selected from the group consisting of SEQ ID NOS: 51-53.
5. An expression vector containing the genetic construct of claim 1 .
6. An isolated host cell containing the expression vector of claim 5 .