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.
Highlights
An adaptor protein, CrkII, which is involved in a variety of signaling cascades such as cell growth, migration, and apoptosis, becomes phosphorylated on Tyr221 upon stimulation
Plasmids—pPicchu was derived from the pCAGGS eukaryotic expression vector [24] and encoded a chimeric protein, phosphorylation indicator of CrkII chimeric unit (Picchu), which consisted of a yellow-emitting mutant of green fluorescent protein (YFP) [25], CrkII, and a cyan-emitting mutant of green fluorescent protein (CFP) from the amino terminus
Phosphorylation Indicator of CrkII—We designed a protein that consisted of CrkII and a pair of YFP and CFP, so that intramolecular binding of the SH2 domain of CrkII to phosphorylated Tyr221 brought CFP close to YFP and increased fluorescent resonance energy transfer (FRET) from CFP to YFP (Fig. 1A)
Summary
Plasmids—pPicchu was derived from the pCAGGS eukaryotic expression vector [24] and encoded a chimeric protein, Picchu, which consisted of a yellow-emitting mutant of green fluorescent protein (YFP) [25], CrkII, and a cyan-emitting mutant of green fluorescent protein (CFP) from the amino terminus (see Fig. 1A). The carboxyl terminus of CrkII was truncated to the amino acids indicated as suffixes to the plasmid names. We amplified each of the domains by polymerase chain reaction with primers con-
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