Figure 2
EAS proteins are targets for ERK2 and exhibit decreased FRETefficiency upon phosphorylation. (A) Emission spectra indicate the effective FRET change for each EAS protein sensor. Time course fluorimetry with EAS-Neg shows that pERK2 induces no FRET change 30 minutes after ATP addition. All other EAS proteins show varying gains in ECFP emission (475 nm) and losses in EYFP emission (525 nm) 40 minutes after ATP addition. (B) Absolute change in the ratio of EYFP/ECFP emission for EAS-Neg is zero over a 30 minute time course, whereas a decrease in ratio for EAS-3, EAS-4, and EAS-5 is readily detectable at 2 minutes, and continues to decay exponentially over the time course. EAS-2 has a detectable change in emission ratio, but the change is minimal as compared to other EAS proteins. Error analysis was determined from three independent experiments. (C) EAS proteins (EAS-2, -3, -4, and -5) are phosphorylated by pERK2 in the presence of γ-[32P]ATP. This confirms that EAS proteins are targets of pERK2 as compared with MBP, a known ERK2 substrate. As expected, EAS-Neg is not phosphorylated by pERK2.