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1.
The ubiquitous Ca2+-sensing protein calmodulin (CaM) fulfills its numerous signaling functions through a wide range of modular binding and activation mechanisms. By activating adenylyl cyclases (ACs) 1 and 8, Ca2+ acting via calmodulin impacts on the signaling of the other major cellular second messenger cAMP. In possessing two CaM-binding domains, a 1-5-8-14 motif at the N terminus and an IQ-like motif (IQlm) at the C terminus, AC8 offers particularly sophisticated regulatory possibilities. The IQlm has remained unexplored beyond the suggestion that it bound CaM, and the larger C2b region of which it is part was involved in the relief of autoinhibition of AC8. Here we attempt to distinguish the function of individual residues of the IQlm. From a complementary approach of in vitro and cell population AC activity assays, as well as CaM binding, we propose that the IQlm alone, and not the majority of the C2b, imparts CaM binding and autoinhibitory functions. Moreover, this duality of function is spatially separated and depends on amino acid side-chain character. Accordingly, residues critical for CaM binding are positively charged and clustered toward the C terminus, and those essential for the maintenance of autoinhibition are hydrophobic and more N-terminal. Secondary structure prediction of the IQlm supports this separation, with an ideally placed break in the α-helical nature of the sequence. We additionally find that the N and C termini of AC8 interact, which is an association specifically abrogated by fully Ca2+-bound, but not Ca2+-free, CaM. These data support a sophisticated activation mechanism of AC8 by CaM, in which the duality of the IQlm function is critical.The divalent calcium ion, Ca2+, plays a key role in modulating cellular processes as diverse as fertilization and apoptosis (1, 2). Ca2+ concentrations inside the cell are held ∼100 nm, despite a perpetually higher level of 1–2 mm in the extracellular medium. This steep gradient across the plasma membrane allows for a large influx when Ca2+ channels open, with subsequent signaling events that often rely on transduction via Ca2+-binding proteins (3). The archetypal Ca2+ sensor is calmodulin (CaM),2 a small, acidic protein so strictly conserved that all vertebrate CaM genes encode an identical 148-residue sequence (4). Multifunctional in its downstream effects, CaM can bind to at least 300 target proteins with novel partners continuing to be discovered (5). The list of effectors includes two isoforms of the adenylyl cyclase (AC) superfamily, AC1 and AC8, which comprise the Ca2+-stimulable subset of the nine particulate ACs (6). In intact cells, AC8 exhibits a predilection for store-operated, or capacitative, Ca2+ entry (CCE) (7, 8). This mode of Ca2+ entry is triggered by the emptying of endo/sarcoplasmic reticular stores by physiological or pharmacological stimuli (9, 10). Although the mechanism of the regulation of AC8 in nonexcitable cells by CCE (or voltage-gated Ca2+ entry in neurons) can be viewed to rely on facets of cellular compartmentalization (1113), the detailed molecular mechanism whereby Ca2+ stimulates the enzyme is unclear. Consequently, the present investigation addresses the molecular mechanism whereby Ca2+, acting via calmodulin, stimulates AC8.The broad features of CaM that hold the key to its multiple regulatory strategies are understood. CaM is organized into two homologous globular domains (or lobes) united by a short linker segment (14). Both the N-terminal (N-lobe) and the C-terminal lobe (C-lobe) include two EF-hands (specialized Ca2+-coordinating helix-loop-helix motifs (15)), endowing CaM with four Ca2+-binding sites. Ca2+ binding to either lobe of CaM induces a structural reconfiguration determined by the two helices of each EF-hand separating from near-antiparallel to perpendicular arrays (14, 16). This exposes hydrophobic trenches in the C- and N-lobes sequentially (because the former has the highest affinity for Ca2+), which are notably lined with a disproportionate number of flexible methionine side chains, ready to accommodate a remarkable array of unrelated sequences. Initially, the mode of Ca2+-loaded CaM association with targets was considered to be uniform; the N- and C-lobes collapsed around the target peptides of e.g. smooth muscle myosin light chain kinase (17), skeletal muscle myosin light chain kinase (18), and CaMKIIα (19) with the N-lobe favoring the C-terminal target sequence and vice versa. However, CaM has since proven to be more versatile and unpredictable in how it associates with and regulates effectors, with reports of N-lobe-N-terminal/C-lobe-C-terminal interaction (20), target dimerization promoted by CaM (21), CaM binding to fatty acyl modifications (22), and other deviations from the early model.Nevertheless, three main forms of CaM regulation have been proposed as follows: relief of autoinhibition; active site remodeling; and dimerization of target domains (23). Whether the precise mechanism of CaM binding and subsequent regulation of AC8 falls into these categories of CaM regulation is not resolved. A previous study (24) established that AC8 possesses two calmodulin-binding domains (CaMBDs). CaM recognition sequences generally show little homology, although classifications based on relative positions of key hydrophobic residues have been usefully applied (4). The N-terminal CaMBD of AC8 conforms to a “1-5-8-14 motif” having large hydrophobic side chains from Trp, Val, or Ile residues at these spatially conserved sites. The C-terminal CaMBD contains an IQ-like motif (IQlm), in accordance with a signature (IVL)QXXXR(K) arrangement, to which CaM binds directly (25). By truncating one terminus or both termini, Gu and Cooper (24) asserted that the N terminus contributed little to direct CaM regulation of AC8, whereas the C terminus was critical for the maintenance of an auto-inhibited state, which was relieved upon binding of CaM. Thus, functional elements of both autoinhibition and pre-association, imparted by noncontiguous CaMBDs, have been suggested, thereby excluding AC8 from a simple model of CaM binding to an autoinhibitory domain leading to activation, as is sometimes observed (23).Recently, the proposal that the two CaMBDs play separate roles in AC8 activation was reinforced (26). This study suggested that CaM tethering by the N-terminal 1-5-8-14 motif provides the catalytically relevant C-terminal CaMBD with privileged access to CaM, thereby circumventing the need for AC8 to compete for CaM, whose free concentration in the cell is far exceeded by that of its targets (27, 28). 1–14 motifs are more generally employed in relieving autoinhibitory influences (23), so the use by AC8 of a 1-5-8-14 motif as a CaM-tethering site is unusual. The proposal that CaM pre-associates here was based on limited mutagenesis of residues within the 1-5-8-14 motif of AC8 and the use of CaM mutants whose Ca2+ binding capability was abolished completely or limited to discrete lobes. In contrast to the 1-5-8-14 motif at the N terminus, very little is known of the IQlm at the C terminus, in terms of the roles of the individual residues in CaM binding or autoinhibition, or even on the interplay between CaMBDs at the N and C termini of AC8.Against this background, the present study sought to assess the contribution of IQlm residues to the function of AC8, focusing on consequences of key mutations on CaM-binding efficiency, regulation by Ca2+/CaM, and maintenance of the autoinhibited state. Through this series of experiments, the level of coordination between the IQ-like and 1-5-8-14 motifs became evident. The provision of a pre-associated CaM molecule was found to allow for potentially deleterious mutations of the IQlm to be tolerated. Within this latter motif, Leu1196, Val1197, and Leu1200 are residues essential to autoinhibition, whereas the main responsibility of binding CaM directly at the C terminus lies with Arg1202 and Arg1204. In this regard, the AC8 IQlm spatially separates the two functions of CaM binding and maintenance of autoinhibition, a separation that is supported by a predicted break in the helicity of the IQlm region. Thus, a new variation is revealed in the manner by which a target exploits the CaM device into a sophisticated activation mechanism.  相似文献   

2.
Carbon dioxide is fundamental to the physiology of all organisms. There is considerable interest in the precise molecular mechanisms that organisms use to directly sense CO2. Here we demonstrate that a mammalian recombinant G-protein-activated adenylyl cyclase and the related Rv1625c adenylyl cyclase of Mycobacterium tuberculosis are specifically stimulated by CO2. Stimulation occurred at physiological concentrations of CO2 through increased kcat. CO2 increased the affinity of enzyme for metal co-factor, but contact with metal was not necessary as CO2 interacted directly with apoenzyme. CO2 stimulated the activity of both G-protein-regulated adenylyl cyclases and Rv1625c in vivo. Activation of G-protein regulated adenylyl cyclases by CO2 gave a corresponding increase in cAMP-response element-binding protein (CREB) phosphorylation. Comparison of the responses of the G-protein regulated adenylyl cyclases and the molecularly, and biochemically distinct mammalian soluble adenylyl cyclase revealed that whereas G-protein-regulated enzymes are responsive to CO2, the soluble adenylyl cyclase is responsive to both CO2 and bicarbonate ion. We have, thus, identified a signaling enzyme by which eukaryotes can directly detect and respond to fluctuating CO2.Inorganic carbon (Ci)3 is central to prokaryotic and eukaryotic physiology. The predominant biologically active forms of Ci are CO2 and and their relative contributions to the total Ci pool are pH-dependent. Biological roles for CO2 and include photosynthetic carbon fixation (1), pH homeostasis (2), carbon metabolism (3), activation of virulence in pathogenic organisms (4), sperm maturation (5), and as an alarmone in Drosophila (6, 7).Given its importance in biology, the identification of CO2 responsive signaling pathways is key to understanding how organisms cope with fluctuating CO2. Two seven transmembrane receptors, Gr21a and Gr63a, have been shown to confer CO2 responsiveness in Drosophila neurons (6, 7). Guanylyl cyclase D expressing olfactory neurons also mediate sensitivity to CO2 in mice (8). A role for cGMP-activated channels in CO2 sensing has been observed in CO2 avoidance behavior in Caenorhabditis (9, 10). Despite these impressive advances, no eukaryotic signaling enzymes unequivocally demonstrated to respond to CO2 have been identified.The mammalian soluble adenylyl cyclase (sAC) synthesizes the second messenger 3′,5′-cAMP and is directly stimulated by (1113). Stimulation of sAC by has an unequivocal role in sperm maturation (5, 1416). sAC is a member of the Class III family of adenylyl cyclases (ACs), a family that also includes the G-protein-regulated ACs and many examples from prokaryotic genomes (17, 18). The Class III ACs can be divided into four subclasses (a–d) based upon polymorphisms within the active site (19). sAC is a member of Class IIIb, a subclass characterized partly by replacement of a substrate binding Asp with Thr. The Class IIIa ACs include the mammalian G-protein-stimulated ACs and numerous prokaryotic examples. These have been previously assumed to be non-responsive to Ci (12).All prokaryotic Class IIIb ACs examined to date respond to Ci including enzymes from organisms as diverse as Anabaena PCC 7120, Mycobacterium tuberculosis, Stigmatella aurantiaca, and Chloroflexus aurantiacus (20, 21). Two Class IIIb ACs, Slr1991 of Synechocystis PCC 6803 and CyaB1 of Anabaena PCC 7120, have been proven to respond to CO2 and not , giving rise to the idea of AC as a true gas-sensing molecule (22, 23). The finding that Class IIIb ACs respond to CO2 and not necessitates an examination of the assumption that G-protein-regulated ACs and related prokaryotic enzymes do not respond to Ci.Here we demonstrate, contrary to previous work, that a recombinant G-protein-regulated AC and the Class IIIa Rv1625c AC of M. tuberculosis H37Rv show a pH-dependent response to Ci due to specific stimulation by CO2 at physiologically relevant concentrations. CO2 interacted directly with the apoprotein and modulated the activity of both the prokaryotic enzyme and G-protein-regulated AC in vivo. Finally, we contrasted the responses of sAC- and G-protein-regulated ACs to different species of Ci and propose that the mammalian cAMP signaling pathway is able to discriminate between CO2 and in vivo.  相似文献   

3.
Cyclic di-GMP (c-di-GMP) is a broadly conserved, intracellular second-messenger molecule that regulates biofilm formation by many bacteria. The synthesis of c-di-GMP is catalyzed by diguanylate cyclases (DGCs) containing the GGDEF domain, while its degradation is achieved through the phosphodiesterase activities of EAL and HD-GYP domains. c-di-GMP controls biofilm formation by Pseudomonas fluorescens Pf0-1 by promoting the cell surface localization of a large adhesive protein, LapA. LapA localization is regulated posttranslationally by a c-di-GMP effector system consisting of LapD and LapG, which senses cytoplasmic c-di-GMP and modifies the LapA protein in the outer membrane. Despite the apparent requirement for c-di-GMP for biofilm formation by P. fluorescens Pf0-1, no DGCs from this strain have been characterized to date. In this study, we undertook a systematic mutagenesis of 30 predicted DGCs and found that mutations in just 4 cause reductions in biofilm formation by P. fluorescens Pf0-1 under the conditions tested. These DGCs were characterized genetically and biochemically to corroborate the hypothesis that they function to produce c-di-GMP in vivo. The effects of DGC gene mutations on phenotypes associated with biofilm formation were analyzed. One DGC preferentially affects LapA localization, another DGC mainly controls swimming motility, while a third DGC affects both LapA and motility. Our data support the conclusion that different c-di-GMP-regulated outputs can be specifically controlled by distinct DGCs.  相似文献   

4.
The mammalian Na+/Ca2+ exchanger, NCX1.1, serves as the main mechanism for Ca2+ efflux across the sarcolemma following cardiac contraction. In addition to transporting Ca2+, NCX1.1 activity is also strongly regulated by Ca2+ binding to two intracellular regulatory domains, CBD1 and CBD2. The structures of both of these domains have been solved by NMR spectroscopy and x-ray crystallography, greatly enhancing our understanding of Ca2+ regulation. Nevertheless, the mechanisms by which Ca2+ regulates the exchanger remain incompletely understood. The initial NMR study showed that the first regulatory domain, CBD1, unfolds in the absence of regulatory Ca2+. It was further demonstrated that a mutation of an acidic residue involved in Ca2+ binding, E454K, prevents this structural unfolding. A contradictory result was recently obtained in a second NMR study in which Ca2+ removal merely triggered local rearrangements of CBD1. To address this issue, we solved the crystal structure of the E454K-CBD1 mutant and performed electrophysiological analyses of the full-length exchanger with mutations at position 454. We show that the lysine substitution replaces the Ca2+ ion at position 1 of the CBD1 Ca2+ binding site and participates in a charge compensation mechanism. Electrophysiological analyses show that mutations of residue Glu-454 have no impact on Ca2+ regulation of NCX1.1. Together, structural and mutational analyses indicate that only two of the four Ca2+ ions that bind to CBD1 are important for regulating exchanger activity.Cardiac contraction/relaxation relies upon Ca2+ fluxes across the plasma membrane (sarcolemma) of cardiomyocytes. Rapid Ca2+ influx (primarily through L-type Ca2+ channels) triggers the release of additional Ca2+ from the sarcoplasmic reticulum (SR),4 resulting in cardiomyocyte contraction. Removal of cytosolic Ca2+ by reuptake into the SR (through the SR Ca2+-ATPase) and expulsion from the cell (primarily through the Na+/Ca2+ exchanger, NCX1.1) results in relaxation (1). Altered Ca2+ cycling is observed in a number of pathophysiological situations including ischemia, hypertrophy, and heart failure (2). Understanding the function and regulation of NCX1.1 is thus of fundamental importance to understand cardiac physiology.NCX1.1 utilizes the electrochemical potential of the Na+ gradient to extrude Ca2+ in a ratio of three Na+ ions to one Ca2+ ion (3). In addition to transporting both Na+ and Ca2+, NCX1.1 is also strongly regulated by these two ions. Intracellular Na+ can induce NCX1.1 to enter an inactivated state, whereas Ca2+ bound to regulatory sites removes Na+-dependent inactivation and also activates Na+/Ca2+ exchange (3). These regulatory sites are located on a large cytoplasmic loop (∼500 residues located between transmembrane helices V and VI) containing two calcium binding domains (CBD1 and CBD2), which sense cytosolic Ca2+ levels. We have previously shown that Ca2+ binding to the primary site in CBD2 is required for full exchange regulation (4); CBD1, however, is a site of higher affinity and appears to dominate the activation of exchange activity by Ca2+.Both CBDs have an immunoglobulin fold formed from two antiparallel β sheets generating a β sandwich with a differing number of Ca2+ ions coordinated at the tip of the domain (4, 5). CBD1 binds four Ca2+ ions, whereas CBD2 binds only two Ca2+ ions. An initial NMR study revealed a local unfolding of the upper portion of CBD1 upon release of Ca2+ (6). In contrast, CBD2 did not display an unfolding response upon Ca2+ removal. A comparative analysis between CBDs revealed a difference in charge at residues in equivalent positions near the Ca2+ coordination site; Glu-454 in CBD1 is replaced by Lys-585 in CBD2. The unstructuring of CBD1 upon Ca2+ removal was alleviated by reversing the charge of the acidic residue (E454K) involved in Ca2+ coordination (6). Previously, we solved the structures of the Ca2+-bound and -free conformations of CBD2 and revealed a charge compensation mechanism involving Lys-585 (4). The positively charged lysine residue assumes the position of one of the Ca2+ ions upon Ca2+ depletion, permitting CBD2 to retain its overall fold (4). A similar phenomenon is predicted to take place in E454K-CBD1 mutant. In addition, Hilge et al. (6) showed that the E454K mutation of CBD1 decreases Ca2+ affinity to a level similar to that of CBD2 and suggested that the E454K mutation would cause the loss of primary regulation of NCX1.1 by CBD1.The significance of some of these observations is unclear as a recent NMR study (7) of CBD1 under more physiologically relevant conditions revealed no significant alteration in tertiary structure in the absence of Ca2+. It was hypothesized that Ca2+ binding induces localized conformational and dynamic changes involving several of the binding site residues. To clarify this issue, we solved the crystal structure of the E454K-CBD1 mutant and examined the functional effects of different CBD1 mutations in the full-length NCX1.1. The results indicate that charge compensation is indeed provided by the residue Lys-454 to replace one Ca2+, whereas the overall E454K-CBD1 structure is only slightly perturbed. The charge compensation, however, has no impact on Ca2+ regulation of NCX1.1.  相似文献   

5.
A hallmark feature of Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) regulation is the generation of Ca2+-independent autonomous activity by Thr-286 autophosphorylation. CaMKII autonomy has been regarded a form of molecular memory and is indeed important in neuronal plasticity and learning/memory. Thr-286-phosphorylated CaMKII is thought to be essentially fully active (∼70–100%), implicating that it is no longer regulated and that its dramatically increased Ca2+/CaM affinity is of minor functional importance. However, this study shows that autonomy greater than 15–25% was the exception, not the rule, and required a special mechanism (T-site binding; by the T-substrates AC2 or NR2B). Autonomous activity toward regular R-substrates (including tyrosine hydroxylase and GluR1) was significantly further stimulated by Ca2+/CaM, both in vitro and within cells. Altered Km and Vmax made autonomy also substrate- (and ATP) concentration-dependent, but only over a narrow range, with remarkable stability at physiological concentrations. Such regulation still allows molecular memory of previous Ca2+ signals, but prevents complete uncoupling from subsequent cellular stimulation.  相似文献   

6.
7.
Altered cytosolic Ca2+ is implicated in the aetiology of many diseases including diabetes but there are few studies on the mechanism(s) of the altered Ca2+ regulation. Using human lymphocytes, we studied cytosolic calcium (Cai) and various Ca2+ transport mechanisms in subjects with Type 2 diabetes mellitus and control subjects. Ca2+-specific fluorescent probes (Fura-2 and Fluo-3) were used to monitor the Ca2+ signals. Thapsigargin, a potent and specific inhibitor of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), was used to study Ca2+- store dependent Ca2+ fluxes. Significant (P < 0.05) elevation of basal Cai levels was observed in lymphocytes from diabetic subjects. Cai levels were positively correlated with fasting, plasma glucose and HbAlc. There was also a significant (P < 0.05) reduction in plasma membrane calcium (PMCA) ATPase activity in diabetic subjects compared to controls. Cells from Type 2 diabetics exhibited an increased Ca2+ influx (as measured both by Fluo-3 fliorescence and C45a assays) as a consequence of of thapsigargin-mediated Ca2+ store depletion. Upon addition of Mn2+ (a surrogate of Ca2+), the fura-2 fluorescence decayed in an exponential fashion and the rate and extent of this decline was steeper and greater in cells from type 2 diabetic patients. There was also a significant (P < 0.05) difference in the Na+/Ca2+ exchange activity in Type 2 diabetic patients, both under resting conditions and after challenging the cells with thapsigargin, when the internal store Ca2+ sequestration was circumvented. Pharmacological activation of protein kinase C (PKC) in cells from patients resulted in only partial inhibition of Ca2+ entry. We conclude that cellular Ca2+ accumulation in cells from Type 2 diabetes results from (a) reduction in PMCA ATPase activity, (b) modulation of Na+/Ca2+ exchange and (3) increased Ca2+ influx across the plasma membrane.  相似文献   

8.
Synaptic transmission is highly plastic and subject to regulation by a wide variety of neuromodulators and neuropeptides. In the present study, we have examined the role of isoforms of the cytochrome b561 homologue called no extended memory (nemy) in regulation of synaptic strength and plasticity at the neuromuscular junction (NMJ) of third instar larvae in Drosophila. Specifically, we generated two independent excisions of nemy that differentially affect the expression of nemy isoforms. We show that the nemy45 excision, which specifically reduces the expression of the longest splice form of nemy, leads to an increase in stimulus evoked transmitter release and altered synaptic plasticity at the NMJ. Conversely, the nemy26.2 excision, which appears to reduce the expression of all splice forms except the longest splice isoform, shows a reduction in stimulus evoked transmitter release, and enhanced synaptic plasticity. We further show that nemy45 mutants have reduced levels of amidated peptides similar to that observed in peptidyl-glycine hydryoxylating mono-oxygenase (PHM) mutants. In contrast, nemy26.2 mutants show no defects in peptide amidation but rather display a decrease in Tyramine β hydroxylase activity (TβH). Taken together, these results show non-redundant roles for the different nemy isoforms and shed light on the complex regulation of neuromodulators.  相似文献   

9.
Arrestin regulates many facets of G-protein coupled receptor signaling. In Drosophila, Arrestin 1 (Arr1) is expressed at a lower level than Arrestin 2 (Arr2), and the role of Arr1 in visual physiology is less understood. Here we generated transgenic flies expressing enhanced green fluorescent protein tagged Arr1 (Arr1-eGFP) and explored its trafficking in live photoreceptors. We show that Arr1-eGFP is localized in the cytoplasm and displays light-dependent translocation to the rhabdomere possibly by interacting with photoactivated rhodopsin 1 (Rh1*). In the adult, translocation of Arr1-eGFP occurs with slower kinetics when compared with that of Arr2-eGFP. This slower kinetic activity may be attributable to a reduced level of phosphorylated Rh1*. Indeed, a reduced level of phosphorylated Rh1* recruits a lower level of Arr1-eGFP to rhabdomeres. To investigate whether Arr1 is required for the deactivation of phosphorylated Rh1*, we show that in flies with reduced Arr1 prolonged depolarizing afterpotential can be triggered with fewer light pulses, indicating that inactivation of phosphorylated Rh1* is compromised when the Arr1 level is reduced. Consistently, Arr1 is no longer required for deactivation of Rh1 in flies expressing phosphorylation-deficient Rh1. Previously it was reported that Arr1 displays light-dependent internalization. Unexpectedly, in adult photoreceptors we failed to observe endocytosis of Arr1-eGFP. In contrast, we show that in pupal photoreceptors Arr1-eGFP becomes internalized and sequestered in vesicles within the cytoplasm. Taken together, we propose that Arr1 plays distinct roles during development and adulthood. Arr1 orchestrates the recycling of phosphorylated Rh1* in pupae whereas it regulates the deactivation in adult.  相似文献   

10.
11.
Ca2+ modulates the visual response in both vertebrates and invertebrates. In Drosophila photoreceptors, an increase of cytoplasmic Ca2+ mimics light adaptation. Little is known regarding the mechanism, however. We explored the role of the sole Drosophila Ca2+/calmodulin-dependent protein kinase II (CaMKII) to mediate light adaptation. CaMKII has been implicated in the phosphorylation of arrestin 2 (Arr2). However, the functional significance of Arr2 phosphorylation remains debatable. We identified retinal CaMKII by anti-CaMKII antibodies and by its Ca2+-dependent autophosphorylation. Moreover, we show that phosphorylation of CaMKII is greatly enhanced by okadaic acid, and indeed, purified PP2A catalyzes the dephosphorylation of CaMKII. Significantly, we demonstrate that anti-CaMKII antibodies co-immunoprecipitate, and CaMKII fusion proteins pull down the catalytic subunit of PP2A from fly extracts, indicating that PP2A interacts with CaMKII to form a protein complex. To investigate the function of CaMKII in photoreceptors, we show that suppression of CaMKII in transgenic flies affects light adaptation and increases prolonged depolarizing afterpotential amplitude, whereas a reduced PP2A activity brings about reduced prolonged depolarizing afterpotential amplitude. Taken together, we conclude that CaMKII is involved in the negative regulation of the visual response affecting light adaptation, possibly by catalyzing phosphorylation of Arr2. Moreover, the CaMKII activity appears tightly regulated by the co-localized PP2A.Visual transduction is the process that converts the signal of light (photons) into a change of membrane potential in photoreceptors (see Ref. 1 for review). Visual signaling is initiated upon the activation of rhodopsins by light: light switches on rhodopsin to generate metarhodopsin, which activates the heterotrimeric Gq in Drosophila (2). Subsequently, the GTP-bound Gαq subunit activates phospholipase Cβ4 encoded by the norpA (no receptor potential A) gene (3). Phospholipase Cβ4 catalyzes the breakdown of phosphoinositol 4,5-bisphosphate to generate diacylglycerol, which or its metabolite has been implicated in gating the transient receptor potential (TRP)2 and TRP-like channels (4, 5). TRP is the major Ca2+ channel that mediates the light-dependent depolarization response leading to an increase of cytosolic Ca2+ in photoreceptors. The rise of intracellular Ca2+ modulates several aspects of the visual response including activation, deactivation, and light adaptation (6). For example, Ca2+ together with diacylglycerol activates a classical protein kinase C, eye-PKC, which is critical for the negative regulation of visual signaling by modulating deactivation and light adaptation (711).Light adaptation is the process by which photoreceptors adjust the visual sensitivity in response to ambient background light by down-regulating rhodopsin-mediated signaling. Light adaptation can be arbitrarily subdivided into long term and short term adaptation and may involve multiple regulations to reduce the efficiency of rhodopsin, G protein, or cation channels. For example, translocation of both Gq (12, 13) and TRP-like channels (14, 15) out of the visual organelle may contribute to long term adaptation in Drosophila. In contrast, short term adaptation may be orchestrated by modulating the activity of signaling proteins by protein kinases. Hardie and co-workers (16) demonstrated that an increase of cytoplasmic [Ca2+] mimicked light adaptation, leading to inhibition of the light-induced current. These authors also showed that light adaptation is independent of eye-PKC. Thus the effect of cytoplasmic Ca2+ to control light adaptation is likely mediated via calmodulin and CaMKII. The contribution of CaMKII to light adaptation has not been explored.CaMKII is a multimeric Ca2+/calmodulin-dependent protein kinase that modulates diverse signaling processes (17). Drosophila contains one CaMKII gene (18) that gives rise to at least four protein isoforms (19). These CaMKII isoforms share over 85% sequence identities with the α isoform of vertebrate CaMKII. For insights into the in vivo physiological role of CaMKII, Griffith et al. (20) generated transgenic flies (ala) expressing an inhibitory domain of the rat CaMKII under the control of a heat shock promoter, hsp70. They demonstrated that, upon heat shock treatment, the overexpression of the inhibitory peptide resulted in a suppression of the endogenous CaMKII activity in the transgenic flies (20). It has been shown that inhibition of CaMKII affects learning and memory (20) and neuronal functions (2124). In photoreceptors, CaMKII has been implicated in the phosphorylation of the major visual arrestin, Arr2 (25, 26). However, how phosphorylation of Arr2 by CaMKII modifies the visual signaling remains to be elucidated.Here we report the biochemical and electrophysiological analyses of CaMKII in Drosophila retina. We demonstrate that suppression of CaMKII in ala1 transgenic flies leads to a phenotype indicative of defective light adaptation. The ala1 flies also display greater visual response, suggesting a defect in Arr2. These results support the notion that CaMKII plays a role in the negative regulation of the visual response. Our biochemical analyses demonstrate that dephosphorylation of CaMKII is mediated by protein phosphatase 2A (PP2A). Importantly, we show that PP2A interacts with CaMKII, indicating that CaMKII forms a stable protein complex with PP2A to ensure a tight regulation of the kinase activity. Thus a partial loss of function in PP2A would elevate the CaMKII activity. Indeed, we show that mts heterozygotes display reduced prolonged depolarizing potential (PDA) amplitude. This PDA phenotype strongly suggests that Arr2 becomes more effective to terminate the visual signaling in mts flies. Together, our findings indicate that the ability of Arr2 to terminate metarhodopsin is increased upon phosphorylation by CaMKII, and the retinal CaMKII activity is regulated by PP2A.  相似文献   

12.
13.
Purified rat brain Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) is stimulated by brain gangliosides to a level of about 30% the activity obtained in the presence of Ca2+/calmodulin (CaM). Of the various gangliosides tested, GT1b was the most potent, giving half-maximal activation at 25 microM. Gangliosides GD1a and GM1 also gave activation, but asialo-GM1 was without effect. Activation was rapid and did not require calcium. The same gangliosides also stimulated the autophosphorylation of CaM-kinase II on serine residues, but did not produce the Ca2+-independent form of the kinase. Ganglioside stimulation of CaM-kinase II was also present in rat brain synaptic membrane fractions. Higher concentrations (125-250 microM) of GT1b, GD1a, and GM1 also inhibited CaM-kinase II activity. This inhibition appears to be substrate-directed, as the extent of inhibition is very dependent on the substrate used. The molecular mechanism of the stimulatory effect of gangliosides was further investigated using a synthetic peptide (CaMK 281-309), which contains the CaM-binding, inhibitory, and autophosphorylation domains of CaM-kinase II. Using purified brain CaM-kinase II in which these regulatory domains were removed by limited proteolysis. CaMK 281-309 strongly inhibited kinase activity (IC50 = 0.2 microM). GT1b completely reversed this inhibition, but did not stimulate phosphorylation of the peptide on threonine-286. These results demonstrate that GT1b can partially mimic the effects of Ca2+/CaM on native CaM-kinase II and on peptide CaMK 281-309.  相似文献   

14.
As a second messenger, Ca2+ plays a major role in cold induced transduction via stimulus-specific increases in [Ca2+]cyt, which is called calcium signature. During this process, CAXs (Ca2+/H+ exchangers) play critical role. For the first time, a putative Ca2+/H+ exchanger GhCAX3 gene from upland cotton (Gossypium hirsutum cv. ‘YZ-1′) was isolated and characterized. It was highly expressed in all tissues of cotton except roots and fibers. This gene may act as a regulator in cotton’s response to abiotic stresses as it could be up-regulated by Ca2+, NaCl, ABA and cold stress. Similar to other CAXs, it was proved that GhCAX3 also had Ca2+ transport activity and the N-terminal regulatory region (NRR) through yeast complementation assay. Over-expression of GhCAX3 in tobacco showed less sensitivity to ABA during seed germination and seedling stages, and the phenotypic difference between wild type (WT) and transgenic plants was more significant when the NRR was truncated. Furthermore, GhCAX3 conferred cold tolerance in yeast as well as in tobacco seedlings based on physiological and molecular studies. However, transgenic plant seeds showed more sensitivity to cold stress compared to WT during seed germination, especially when expressed in N-terminal truncated version. Finally, the extent of sensitivity in transgenic lines was more severe than that in WT line under sodium tungstate treatment (an ABA repressor), indicating that ABA could alleviate cold sensitivity of GhCAX3 seeds, especially in short of its NRR. Meanwhile, we also found that overexpression of GhCAX3 could enhance some cold and ABA responsive marker genes. Taken together, these results suggested that GhCAX3 plays important roles in the cross-talk of ABA and cold signal transduction, and compared to full-length of GhCAX3, the absence of NRR could enhance the tolerance or sensitivity to cold stress, depending on seedling’s developmental stages.  相似文献   

15.
16.

Background

Trichomonas vaginalis has an unusually large genome (∼160 Mb) encoding ∼60,000 proteins. With the goal of beginning to understand why some Trichomonas genes are present in so many copies, we characterized here a family of ∼123 Trichomonas genes that encode transmembrane adenylyl cyclases (TMACs).

Methodology/Principal Findings

The large family of TMACs genes is the result of recent duplications of a small set of ancestral genes that appear to be unique to trichomonads. Duplicated TMAC genes are not closely associated with repetitive elements, and duplications of flanking sequences are rare. However, there is evidence for TMAC gene replacements by homologous recombination. A high percentage of TMAC genes (∼46%) are pseudogenes, as they contain stop codons and/or frame shifts, or the genes are truncated. Numerous stop codons present in the genome project G3 strain are not present in orthologous genes of two other Trichomonas strains (S1 and B7RC2). Each TMAC is composed of a series of N-terminal transmembrane helices and a single C-terminal cyclase domain that has adenylyl cyclase activity. Multiple TMAC genes are transcribed by Trichomonas cloned by limiting dilution.

Conclusions/Significance

We conclude that one reason for the unusually large genome of Trichomonas is the presence of unstable families of genes such as those encoding TMACs that are undergoing massive gene duplication and concomitant development of pseudogenes.  相似文献   

17.
The Na+/H+ exchanger isoform 1 is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammals by extruding an intracellular H+ in exchange for one extracellular Na+. We characterized structural and functional aspects of the critical transmembrane (TM) segment XI (residues 449-470) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM XI was mutated to cysteine in the background of the cysteine-less protein and the sensitivity to water-soluble sulfhydryl reactive compounds MTSET ((2-(trimethylammonium) ethyl)methanethiosulfonate) and MTSES ((2-sulfonatoethyl) methanethiosulfonate) was determined for those residues with at least moderate activity remaining. Of the residues tested, only proteins with mutations L457C, I461C, and L465C were inhibited by MTSET. The activity of the L465C mutant was almost completely eliminated, whereas that of the L457C and I461C mutants was partially affected. The structure of a peptide representing TM XI (residues Lys447-Lys472) was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. The structure consisted of helical regions between Asp447-Tyr454 and Phe460-Lys471 at the N and C termini of the peptide, respectively, connected by a region with poorly defined, irregular structure consisting of residues Gly455-Gly459. TM XI of NHE1 had a structural similarity to TM XI of the Escherichia coli Na+/H+ exchanger NhaA. The results suggest that TM XI is a discontinuous helix, with residue Leu465 contributing to the pore.The mammalian Na+/H+ exchanger isoform 1 (NHE1)4 is a ubiquitous integral membrane protein that regulates intracellular pH. It mediates removal of a single intracellular proton in exchange for an extracellular sodium ion (1). NHE1 has many functions aside from protection of cells from intracellular acidification (2). It promotes cell growth and differentiation (3), regulates sodium fluxes and cell volume after challenge by osmotic shrinkage (4), and has been demonstrated to be involved in modulating cell motility (5). In addition its activity is important in invasiveness of neoplastic breast cancer cells (6). NHE1 also plays critical roles in heart disease. It has a contributing role in heart hypertrophy and in the damage that occurs during ischemia and reperfusion. Inhibition of NHE1 with Na+/H+ exchanger inhibitors protects the myocardium during various disease states (7-10).NHE1 is composed of two general regions, an N-terminal membrane domain of ∼500 amino acids and a C-terminal regulatory domain of ∼315 amino acids (1, 8). The membrane domain is responsible for ion movement and an analysis of topology by cysteine scanning accessibility suggested it has 3 membrane-associated segments and 12 integral transmembrane segments (11) (Fig. 1A). The mechanism of transport of the membrane domain is of great interest both from a scientific viewpoint and in the design of improved NHE1 inhibitors that may be necessary for clinical use (1). In this regard, we have recently characterized the functionally important residues and the structure of both TM IV and TM VII. Prolines 167 and 168 of TM IV were critical to NHE1 function (12) and cysteine-scanning mutagenesis was used to show that Phe161 is a pore lining residue critical to transport. Analysis of the structure of TM IV showed that TM IV is composed of one region of β-turns, an extended middle region including Pro167-Pro168, and a helical region (13). TM VII was much more typical of a transmembrane helix although it was interrupted with a break in the helix at the functionally critical residues Gly261-Glu262 (14).Open in a separate windowFIGURE 1.Models of the Na+/H+ exchanger. A, simplified topological model of the transmembrane domain of the NHE1 isoform of the Na+/H+ exchanger as described earlier (11). EL, extracellular loop; IL, intracellular loop. B, model of amino acids present in TM XI.Another important TM segment of the Na+/H+ exchanger is TM XI (Fig. 1B). Several different lines of evidence have suggested that it is critical to NHE1 function. A recent study generated chimeras of NHE1 from various species and found that a region including TM XI was important in determining NHE1 inhibitor sensitivity (15). More specifically, mutagenesis of several amino acids of TM XI has shown that it is likely involved in either ion transport or proper targeting to the plasma membrane. Two mutants in TM XI, Y454C and R458C, are retained in the endoplasmic reticulum (16). In addition, mutation of Gly455 and Gly456 in TM XI shift the pHi dependence of the exchanger to the alkaline side, whereas mutation of Arg440 in intracellular loop 5 at the N-terminal end of TM XI shifts the pHi dependence to make it more acidic (17, 18). Also, the structure of the bacterial Na+/H+ exchanger NhaA has been elucidated. Both TM IV and TM XI play a critical role forming an assembly that cross, with each being a helix, an extended polypeptide and a short helix (19). We found that TM IV of NHE1 has a similar structure and function to that of TM IV of NhaA (2, 13), leaving open the possibility that TM XI of NHE1 is also similar in structure and function to TM XI of NhaA.For these reasons, we undertook a systematic examination of the structural and functional aspects of TM XI of the NHE1 isoform of the Na+/H+ exchanger. The sequence of human TM XI of NHE1 is 449QFIIAYGGLRGAIAFSLGYLLD470. In this study we use cysteine scanning mutagenesis and site-specific mutagenesis to identify and characterize critical pore lining residues of the protein. We also use nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of a synthetic peptide representing TM XI in dodecylphosphocholine (DPC) micelles. Evidence has suggested that TM segments of membrane proteins possess all the structural information required to form their higher order structures in their amino acid sequence (20). This has been demonstrated in earlier studies on membrane protein segments such as the cystic fibrosis transmembrane conductance regulator (21), a fungal G-protein-coupled receptor (22), bacteriorhodopsin (23, 24), and rhodopsin (25), where it was shown that isolated TM segments from membrane proteins had structures in good agreement with the segments of the entire protein. Also, the use of DPC micelles has been shown to be an excellent membrane mimetic environment for these studies (26, 27). Our study identifies Leu465 as contributing to the pore of the protein and shows that the structure of TM XI consists of two helices corresponding to Asp447-Tyr454 and Phe460-Lys471 at the N and C termini, respectively, connected by a flexible region at residues 455-459. The structure of TM XI was similar to the x-ray structure of TM XI of NhaA.  相似文献   

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Epigenetic mechanisms regulate the expression of virulence traits in diverse pathogens, including protozoan and fungi. In the human fungal pathogen Candida albicans, virulence traits such as antifungal resistance, white-opaque switching, and adhesion to lung cells are regulated by histone deacetylases (HDACs). However, the role of HDACs in the regulation of the yeast-hyphal morphogenetic transitions, a critical virulence attribute of C. albicans, remains poorly explored. In this study, we wished to determine the relevance of other HDACs on C. albicans morphogenesis. We generated mutants in the HDACs HOS1, HOS2, RPD31, and HDA1 and determined their ability to filament in response to different environmental stimuli. We found that while HOS1 and RPD31 have no or a more limited role in morphogenesis, the HDACs HOS2 and HDA1 have opposite roles in the regulation of hyphal formation. Our results demonstrate an important role for HDACs on the regulation of yeast-hyphal transitions in the human pathogen C. albicans.  相似文献   

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