Disruption of the LOV-Jα helix interaction activates phototropin kinase activity
ABSTRACT Light plays a crucial role in activating phototropins, a class of plant photoreceptors that are sensitive to blue and UV-A wavelengths. Previous studies indicated that phototropin uses a bound flavin mononucleotide (FMN) within its light-oxygen-voltage (LOV) domain to generate a protein-flavin covalent bond under illumination. In the C-terminal LOV2 domain of Avena sativa phototropin 1, formation of this bond triggers a conformational change that results in unfolding of a helix external to this domain called Jalpha [Harper, S. M., et al. (2003) Science 301, 1541-1545]. Though the structural effects of illumination were characterized, it was unknown how these changes are coupled to kinase activation. To examine this, we made a series of point mutations along the Jalpha helix to disrupt its interaction with the LOV domain in a manner analogous to light activation. Using NMR spectroscopy and limited proteolysis, we demonstrate that several of these mutations displace the Jalpha helix from the LOV domain independently of illumination. When placed into the full-length phototropin protein, these point mutations display constitutive kinase activation, without illumination of the sample. These results indicate that unfolding of the Jalpha helix is the critical event in regulation of kinase signaling for the phototropin proteins.
- SourceAvailable from: PubMed Central
[Show abstract] [Hide abstract]
- "ght - oxygen - voltage domains undergo versatile light dependent interactions . In the best - studied LOV domain , LOV2 from Avena sativa phototropin , light - induced thioeither bond formation between a cysteine residue and the FMN chromophore leads to partial unfolding of the C - terminal α - helix ( named Jα ) from the rest of the LOV2 domain ( Harper et al . , 2004 ) . This conformation change has been widely used to construct light - controllable proteins in allosteric or steric manners ( Lee et al . , 2008 ; Strickland et al . , 2008 ; Moglich et al . , 2009 ; Wu et al . , 2009 ; Ohlendorf et al . , 2012 ) . Wu et al . ( 2009 ) constructed photoactivatable small GTPase Rac1 ( PA - Rac1 ; Figure "
ABSTRACT: In the nervous system, protein activities are highly regulated in space and time. This regulation allows for fine modulation of neuronal structure and function during development and adaptive responses. For example, neurite extension and synaptogenesis both involve localized and transient activation of cytoskeletal and signaling proteins, allowing changes in microarchitecture to occur rapidly and in a localized manner. To investigate the role of specific protein regulation events in these processes, methods to optically control the activity of specific proteins have been developed. In this review, we focus on how photosensory domains enable optical control over protein activity and have been used in neuroscience applications. These tools have demonstrated versatility in controlling various proteins and thereby cellular functions, and possess enormous potential for future applications in nervous systems. Just as optogenetic control of neuronal firing using opsins has changed how we investigate the function of cellular circuits in vivo, optical control may yet yield another revolution in how we study the circuitry of intracellular signaling in the brain.Frontiers in Molecular Neuroscience 01/2015; 8:37. DOI:10.3389/fnmol.2015.00037 · 4.08 Impact Factor
[Show abstract] [Hide abstract]
- "Since the A9a helix also interacts with the Ja helix (Fig. 4D, top, white circles), the modification at His-495 may alter the interaction of the A9a helix with the Ja helix. This altered interaction could lead to partial unfolding and displacement of the Ja helix away from the LOV2 core in the mutant (Fig. 4D, bottom right, green arrows), as has been observed in the mutated oat phot1 (Harper et al., 2004). Moreover, this displacement could cause the observed instability of the mutant phot1. "
ABSTRACT: In higher plants, blue light (BL) phototropism is primarily controlled by the phototropins, which are also involved in stomatal movements and chloroplast relocation. These photoresponses are mediated by two phototropins, phot1 and phot2. Phot1 mediates responses with higher sensitivity than phot2, and phot2 specifically mediates chloroplast avoidance and dark positioning response. Here, we report the isolation and characterization of a Non-phototropic seedling (Nps1) mutant of tomato (Solanum lycopersicum). The mutant is impaired in low-fluence BL responses including chloroplast accumulation and stomatal opening. Genetic analyses show that the mutant locus is dominant negative in nature. In dark grown seedlings of Nps1mutant phototropin 1 protein accumulates at highly reduced level relative to wild type and lacks BL-induced autophosphorylation. The mutant harbors a single G1484 to A transition in the Hinge1 region of phototropin 1 homologue resulting in an arginine to histidine substitution (R495H) in a highly conserved A'α helix proximal to the LOV2 domain of the translated gene product. Significantly, the R495H substitution occurring in the Hinge1 region of PHOT1 abolishes its regulatory activity in Nps1 seedlings, thereby highlighting the functional significance of A'α helix region in phototropic signaling of tomato.Plant physiology 02/2014; 164(4). DOI:10.1104/pp.113.232306 · 7.39 Impact Factor
[Show abstract] [Hide abstract]
- "While the FMN molecule is noncovalently associated with the LOV domain in darkness, upon absorption of BL, a reversible photocycle is initiated such that the activated FMN forms a covalent adduct with a nearby Cys residue in the LOV domain (Christie et al., 1999, 2002; Salomon et al., 2000). Although their photocycles are similar, the LOV1 domain is thought primarily to regulate receptor di/multimerization (Salomon et al., 2004; Nakasako et al., 2008; Nakasone et al., 2013), whereas LOV2 appears to regulate the C-terminal PKD of phots through a novel BL-induced derepression mechanism (Christie et al., 2002; Harper et al., 2003, 2004; Jones et al., 2007; Jones and Christie, 2008; Nakasako et al., 2008; Tokutomi et al., 2008). In the absence of light, the LOV2 domain is folded in a way that causes steric inhibition of the PKD (Figure 2A). "
ABSTRACT: Phototropism, or the differential cell elongation exhibited by a plant organ in response to directional blue light, provides the plant with a means to optimize photosynthetic light capture in the aerial portion and water and nutrient acquisition in the roots. Tremendous advances have been made in our understanding of the molecular, biochemical, and cellular bases of phototropism in recent years. Six photoreceptors and their associated signaling pathways have been linked to phototropic responses under various conditions. Primary detection of directional light occurs at the plasma membrane, whereas secondary modulatory photoreception occurs in the cytoplasm and nucleus. Intracellular responses to light cues are processed to regulate cell-to-cell movement of auxin to allow establishment of a trans-organ gradient of the hormone. Photosignaling also impinges on the transcriptional regulation response established as a result of changes in local auxin concentrations. Three additional phytohormone signaling pathways have also been shown to influence phototropic responsiveness, and these pathways are influenced by the photoreceptor signaling as well. Here, we will discuss this complex dance of intra- and intercellular responses that are regulated by these many systems to give rise to a rapid and robust adaptation response observed as organ bending.The Plant Cell 01/2014; 26(1). DOI:10.1105/tpc.113.119727 · 9.58 Impact Factor