A Pair of Fluorescent Resonance Energy Transfer-based Probes for Tyrosine Phosphorylation of the CrkII Adaptor Protein in Vivo
Year: 2001
Type: article
Abstract: An adaptor protein, CrkII, which is involved in a variety of signaling cascades such as cell growth, migration, and apoptosis, becomes phosphorylated on Tyr(221) upon stimulation. Here, we report on a fluorescent resonance energy 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 within the cell. To overcome this problem, we fused the CAAX box of Ki-Ras to the carboxyl terminus of this probe and restricted its localization mostly to the plasma membrane. With this modified probe, we found that epidermal growth factor-induced phosphorylation of CrkII was initiated at the peripheral plasma membrane, moving toward the center of the cell. Moreover, this CAAX box-fused probe showed improvement in sensitivity and time resolution of the monitoring of CrkII phosphorylation. Thus, this pair of CrkII probes visualizes dynamic changes in the total and local levels of the tyrosine phosphorylation of CrkII in a living cell.
Source: Journal of Biological Chemistry
Institutions: The University of Osaka, RIKEN Center for Brain Science, RIKEN
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W2084850247