A pair of fluorescent resonance energy transfer-based probes for tyrosine phosphorylation of the CrkII adaptor protein in vivo

An adaptor protein, CrkII, which is involved in a variety of signaling casc ades such as cell growth, migration, and apoptosis, becomes phosphorylated on Tyr.. upon stimulation. Here, we report on a fluorescent resonance energ y transfer-based sensor, which consists of CrkII sandwiched with cyan- and yellow-emitting variants of green fluorescent protein. This protein enabled us to monitor rapid and transient phosphorylation of CrkII upon epidermal growth factor stimulation in a living cell. However, rapid diffusion of the probes prevented us from specifying where the phosphorylation started with in the cell. To overcome this problem, we fused the CAAX box of Ki-Ras to t he carboxyl terminus of this probe and restricted its localization mostly t o the plasma membrane. With this modified probe, we found that epidermal gr owth factor-induced phosphorylation of CrkII was initiated at the periphera l plasma membrane, moving toward the center of the cell. Moreover, this CAA X box-fused probe showed improvement in sensitivity and time resolution of the monitoring of CrkII phosphorylation. Thus, this pair of CrkII probes vi sualizes dynamic changes in the total and local levels of the tyrosine phos phorylation of CrkII in a living cell.