Fluorescence (Förster) resonance energy transfer imaging of oncogene activity in living cells

Abstract

A hallmark of cancer cells is their uncontrolled activation of growth signal transduction cascades comprised of oncogene products. Overexpression and activating mutations of the growth factor receptors Ras and Raf are frequently observed in human cancer cells. Several research groups, including our own, have been developing probes based on the principle of fluorescence (Förster) resonance energy transfer (FRET) to visualize how signaling molecules, including oncogene products, are regulated in normal and cancerous cells in the living state. In this review, we will briefly introduce the principle of FRET-based probes, present an overview of the probes reported to date, and discuss the perspectives of these probes and fluorescent imaging systems in cancer biology.

Figure 1

Schematic view of a signal transduction event. Upon growth factor stimuli, receptors on the plasma membrane become tyrosine‐phosphorylated and recruit adaptor proteins. The adaptor proteins directly or indirectly bring their binding partner, a guanine nucleotide exchange factor (GEF), which promotes guanine nucleotide exchange of GDP for GTP and activates G proteins. The GTP‐bound active G proteins bind to effectors and transduce signals for biological outputs, such as cytoskeletal rearrangement or proliferation. GTPase‐activating proteins (GAP) negatively regulate the G protein activity.

Figure 2

Structures of the Raichu/Picchu FRET probes. Two conformations, namely off (upper) and on (lower), are shown schematically for each probe. (A) Cameleon: Binding of calcium (green ball) to calmodulin (CaM) induces a conformational change in Cameleon, thereby bringing the donor (BFP) into close proximity with the acceptor (GFP) and evoking sensitized FRET. (B) Raichu‐Ras: GTP‐bound Ras in the probe binds intramolecularly with the Ras‐binding domain of Raf, which results in increased FRET from CFP and YFP. (C) Picchu: CrkII, which is composed of Src homology (SH) 2 and SH3 domains, is sandwiched between YFP and CFP. Upon activation of Abl kinase or epidermal growth factor receptor (EGFR), tyrosine 221 (denoted as Y221) is phosphorylated (indicated as P, and circled in red). Intramolecular binding of the SH2 domain to Y221 increases the level of FRET. (D) Src monitor: The SH2 domain of Src and a substrate peptide sequence (subpep) are sandwiched between YFP and CFP. Upon activation of Src, the tyrosine in the substrate peptide is phosphorylated (indicated as P, and circled in red) and then recognized by the SH2 domain of the probe. (E) Prin‐c‐Raf: Raf kinase is sandwiched between YFP and CFP. Raf adopts two conformations, namely the closed inactive and open active forms. FRET is high and low in the former and latter conformations, respectively. Upon activation, the Ras‐binding domain (RBD) and kinase region of the probe bind to Ras and MEK, respectively, concomitant with a decrease in FRET. (F) AKAR and ART: In AKAR, the phosphoserine‐binding domain of 14–3‐3τ and a substrate peptide sequence (subpep) are sandwiched between YFP and CFP. Protein kinase A (PKA) phosphorylates the serine residue in the substrate region (indicated as P, and circled in red), which is then recognized by the phosphoserine‐binding domain of 14–3‐3τ. In ART, red‐ and blue‐shifted GFP (RGFP and BGFP) are fused to the substrate‐peptide sequence of Kemptide. The conformational change of Kemptide induced by serine phosphorylation (indicated by the circled P) is monitored by FRET. (G) CKAR: The FHA2 phosphothreonine‐binding domain from Rad51 (denoted as FHA2) and a substrate peptide sequence (subpep) are sandwiched between CFP and YFP. A conformational change of the probe is induced by protein kinase C (PKC)‐dependent phosphorylation (indicated by the circled P).

Figure 3

Examples of Ras and Rap imaging. (A) COS‐7 cells expressing Raichu‐Ras were stimulated with 50 ng/mL of epidermal growth factor. FRET and differential interference contrast images were recorded at the indicated time points. (B) HeLa cells expressing Raichu‐Rap1 and Epac were stimulated with 50 µM and 100 µM of forskolin and the phosphodiesterase inhibitor IBMX, respectively, to elevate intracellular cAMP. FRET images were recorded at the indicated time points.

Figure 4

Rho is activated both at the trailing and leading edges of migrating cells. HeLa cells expressing Raichu‐RhoA were replated onto glass‐bottom dishes coated with collagen to observe their stochastic migration. FRET and differential interference contrast (DIC) images were recorded at the indicated time points. The white scale bars represent 10 µm.