Figure Q9–4 Fluorescent reporter protein designed to detect tyrosine phosphorylation (problem 9–9). (a) Domain structure of reporter protein. Four domains are indicated: CFp, YFp, tyrosine kinase substrate peptide, and a phosphotyrosine-binding domain. (B) Fret assay. YFp/CFp is normalized to 1.0 at time zero. the reporter was incubated in the presence (or absence) of abl and atp for the indicated times. Arrow indicates time of addition of a tyrosine phosphatase. (From a.Y. ting, k.h. kain, r.L. klemke and r.Y. tsien, Proc. Natl Acad. Sci. USA 98:15003–15008, 2001. With permission from national academy of sciences.) separated. When the CFP and YFP domains are brought close together, however, fluorescence resonance energy transfer (FRET) allows excitation of CFP to stimulate emission by YFP. FRET shows up experimentally as an increase in the ratio of emission at 526 nm versus 476 nm (YFP/ CFP) when CFP is excited by 434 nm light.
Incubation of the reporter protein with Abl protein tyrosine kinase in the presence of ATP gave an increase in YFP/CFP emission (Figure Q9–4B). In the absence of ATP or the Abl protein, no FRET occurred. FRET was also eliminated by addition of a tyrosine phosphatase (Figure Q9–4B). Describe as best you can how the reporter protein detects active Abl protein tyrosine kinase.
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