Editors' ChoiceSignaling Dynamics

The Lights on Ras Avenue

Science Signaling  10 Dec 2013:
Vol. 6, Issue 305, pp. ec298
DOI: 10.1126/scisignal.2004982

Signal transduction often involves inputs that converge on a common protein or module of proteins. How such convergence produces divergent outputs remains a mystery. One hypothesis is that receiver proteins decode temporal input and convey this information to downstream effectors. The receptor tyrosine kinase family of receptors for ligands, such as platelet-derived growth factor (PDGF) and nerve growth factor (NGF), converge on the guanosine triphosphatase Ras to produce a range of distinct cellular outputs, including cell proliferation, differentiation, and migration. Toettcher et al. used an optogenetic approach to directly activate Ras and found that different temporal profiles of activation resulted in the phosphorylation of different sets of proteins. The optogenetic tool (opto-SOS) comprises two fusion proteins, one in which the catalytic domain of the Ras guanine nucleotide exchange factor Son of Sevenless is fused to the plant protein PIF and another in which the plant protein PhyB is tethered to the plasma membrane with a CAAX-box–type prenylation domain. When exposed to red light (in the presence of the chromophore phycocyanobilin), PIF and PhyB interact and recruit opto-SOS to the membrane, where it activates Ras. Similar to cells treated with PDGF or NGF, opto-SOS–expressing cells exposed to red light showed increased phosphorylation of endogenous ERK (extracellular signal–regulated kinase) and nuclear localization of ERK fused to a blue fluorescent protein (BFP-ERK), as well as enhanced proliferation and morphological changes. ERK activation in opto-SOS–expressing cells exhibited a graded steady-state response to different ratios of wavelengths of stimulatory and inhibitory light, and the response to light stimulation was fast (minutes), persistent, and reversible. The Ras-ERK module functioned as a high-bandwidth, low-pass filter. Light flashes with a period ranging from 4 minutes to 2 hours produced a similar gain (ratio of output to input amplitude), whereas high-frequency stimulation (periods less than 4 minutes) produced minimal output. The delay between BFP-ERK nuclear localization and light stimulation was 3 minutes, indicating an inherent phase shift in the input to output signal transmission. Antibody array–based proteomics to detect phosphorylation changes downstream of Ras-ERK activation revealed three classes of responses: (i) those that responded only to PDGF but not light, (ii) those that responded to PDGF and either transient or sustained light, and (iii) those that responded to PDGF and sustained but not transient light. The last category included phosphorylation of signal transducer and activator of transcription 3 (STAT3). Immunofluorescence analysis of cocultured parental and opto-SOS–expressing cells showed that sustained but not transient light stimulation led to the secretion of leukemia inhibitory factor that stimulated paracrine STAT3 phosphorylation. Thus, the optogenetic approach has revealed that, as in other systems such as neurotransmission, both the strength and timing of signal activation in the Ras-ERK pathway can affect the nature of the cellular response. The optogenetic approach will undoubtedly open the door to investigations into the dynamics of signaling modules that were not formerly possible.

J. E. Toettcher, O. D. Weiner, W. A. Lim, Using optogenetics to interrogate the dynamic control of signal transmission by the Ras/Erk module. Cell 155, 1422–1434 (2013). [Online Journal]