The activity of the ERK has complex spatial and temporal dynamics that are important for the specificity of downstream effects. However, current biochemical techniques do not allow for the measurement of ERK signaling with fine spatiotemporal resolution. We developed a genetically encoded, FRET-based sensor of ERK activity (the extracellular signal-regulated kinase activity reporter, EKAR), optimized for signal-to-noise ratio and fluorescence lifetime imaging. EKAR selectively and reversibly reported ERK activation in HEK293 cells after epidermal growth factor stimulation. EKAR signals were correlated with ERK phosphorylation, required ERK activity, and did not report the activities of JNK or p38. EKAR reported ERK activation in the dendrites and nucleus of hippocampal pyramidal neurons in brain slices after theta-burst stimuli or trains of back-propagating action potentials. EKAR therefore permits the measurement of spatiotemporal ERK signaling dynamics in living cells, including in neuronal compartments in intact tissues.fluorescence lifetime imaging microscopy ͉ FRET ͉ MAPK T he MAPK family is a class of serine/threonine kinases that includes the ERK, p38, and JNK subfamilies. Members of the ERK subfamily are essential for numerous, diverse physiological functions, including cellular differentiation, proliferation and neuronal plasticity, and their activities are up-regulated in many cancers (1). ERK signaling spans multiple subcellular compartments (1, 2). For example, in neurons ERK is activated at synapses and regulates gene transcription in the nucleus, hundreds of micrometers away (1). The spatial and temporal dynamics of ERK activity are likely critical in establishing the specificity of downstream signals. In PC12 cells, for example, epidermal growth factor (EGF) induces transient ERK activity only in the cytoplasm, leading to cellular proliferation; whereas, neural growth factor (NGF) triggers long-lasting ERK activity in both the cytoplasm and nucleus, resulting in cellular differentiation (2).Traditional methods to measure ERK signaling, by Western blotting or immunostaining for phosphorylated, active ERK, have provided valuable insight into ERK function. However, these methods present a static snapshot of cellular events; they do not allow for the dynamic examination of ERK activity with fine spatial resolution. Recently developed imaging approaches that use fluorescent sensors of signaling activities can overcome these shortcomings (3). FRET-based reporters have been used in living cells to monitor the spatiotemporal patterns of Ca 2ϩ signaling and enzymatic activities (4). We therefore created a genetically encoded FRET-based sensor of ERK activity that selectively reports ERK signaling in living cells. Results Design and Function of EKAR.To create a genetically encoded fluorescent sensor of ERK activity, we customized a generic design for FRET-based kinase activity reporters (5-8). Our ERK activity sensor, named EKAR (extracellular signalregulated kinase activity reporter), includes a fluorescen...

Summary Variable, glutamine-encoding, CAA interruptions indicate that a property of the uninterrupted HTT CAG repeat sequence, distinct from the length of huntingtin’s polyglutamine segment, dictates the rate at which Huntington’s disease (HD) develops. The timing of onset shows no significant association with HTT cis -eQTLs but is influenced, sometimes in a sex-specific manner, by polymorphic variation at multiple DNA maintenance genes, suggesting that the special onset-determining property of the uninterrupted CAG repeat is a propensity for length instability that leads to its somatic expansion. Additional naturally occurring genetic modifier loci, defined by GWAS, may influence HD pathogenesis through other mechanisms. These findings have profound implications for the pathogenesis of HD and other repeat diseases and question the fundamental premise that polyglutamine length determines the rate of pathogenesis in the “polyglutamine disorders.”

The cJun NH(2)-terminal kinase (JNK) signal transduction pathway is established to be an important mechanism of regulation of the cJun transcription factor. Studies of Jnk1(-/-) and Jnk2(-/-) mice suggest that the JNK1 and JNK2 isoforms have opposite effects on cJun expression and proliferation. Here, we demonstrate, using a chemical genetic approach, that both JNK1 and JNK2 are positive regulators of these processes. We show that competition between JNK1 and JNK2 contributes to the opposite phenotypes caused by JNK1 and JNK2 deficiency. Our analysis illustrates the power of a chemical genetics approach for the analysis of signal transduction pathways and also highlights the limitations of single gene knockout strategies for the analysis of signaling pathways that are formed by a network of interacting proteins.