Inherent Regulation of EAL Domain-catalyzed Hydrolysis of Second Messenger Cyclic di-GMP

The universal second messenger cyclic di-GMP (cdG) is involved in the regulation of a diverse range of cellular processes in bacteria. The intracellular concentration of the dinucleotide is determined by the opposing actions of diguanylate cyclases and cdG-specific phosphodiesterases (PDEs). Whereas most PDEs have accessory domains that are involved in the regulation of their activity, the regulatory mechanism of this class of enzymes has remained unclear. Here, we use biophysical and functional analyses to show that the isolated EAL domain of a PDE from Escherichia coli (YahA) is in a fast thermodynamic monomer-dimer equilibrium, and that the domain is active only in its dimeric state. Furthermore, our data indicate thermodynamic coupling between substrate binding and EAL dimerization with the dimerization affinity being increased about 100-fold upon substrate binding. Crystal structures of the YahA-EAL domain determined under various conditions (apo, Mg²⁺, cdG·Ca²⁺ complex) confirm structural coupling between the dimer interface and the catalytic center. The built-in regulatory properties of the EAL domain probably facilitate its modular, functional combination with the diverse repertoire of accessory domains.     

Activation of Tyrosine Hydroxylase in PC12 Cells by the Cyclic GMP and Cyclic AMP Second Messenger Systems

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is subject to regulation by a variety of agents. Previous workers have found that cyclic AMP-dependent protein kinase and calcium-stimulated protein kinases activate tyrosine hydroxylase. We wanted to determine whether cyclic GMP might also be involved in the regulation of tyrosine hydroxylase activity. We found that treatment of rat PC12 cells with sodium nitroprusside (an activator of guanylate cyclase), 8-bromocyclic GMP, forskolin (an activator of adenylate cyclase), and 8-bromocyclic AMP all produced an increase in tyrosine hydroxylase activity measured in vitro or an increased conversion of [14C]tyrosine to labeled catecholamine in situ. Sodium nitroprusside also increased the relative synthesis of cyclic GMP in these cells. In the presence of MgATP, both cyclic GMP and cyclic AMP increased tyrosine hydroxylase activity in PC12 cell extracts. The heat-stable cyclic AMP-dependent protein kinase inhibitor failed to attenuate the activation produced in the presence of cyclic GMP. It eliminated the activation produced in the presence of cyclic AMP. Sodium nitroprusside also increased tyrosine hydroxylase activity in vitro in rat corpus striatal synaptosomes and bovine adrenal chromaffin cells. In all cases, the cyclic AMP-dependent activation of tyrosine hydroxylase was greater than that of the cyclic GMP-dependent second messenger system. These results indicate that both cyclic GMP and cyclic AMP and their cognate protein kinases activate tyrosine hydroxylase activity in PC12 cells.     

The Second Messenger Cyclic Di-GMP Regulates Clostridium difficile Toxin Production by Controlling Expression of sigD

The Gram-positive obligate anaerobe Clostridium difficile causes potentially fatal intestinal diseases. How this organism regulates virulence gene expression is poorly understood. In many bacterial species, the second messenger cyclic di-GMP (c-di-GMP) negatively regulates flagellar motility and, in some cases, virulence. c-di-GMP was previously shown to repress motility of C. difficile. Recent evidence indicates that flagellar gene expression is tightly linked with expression of the genes encoding the two C. difficile toxins TcdA and TcdB, which are key virulence factors for this pathogen. Here, the effect of c-di-GMP on expression of the toxin genes tcdA and tcdB was determined, and the mechanism connecting flagellar and toxin gene expressions was examined. In C. difficile, increasing c-di-GMP levels reduced the expression levels of tcdA and tcdB, as well as that of tcdR, which encodes an alternative sigma factor that activates tcdA and tcdB expression. We hypothesized that the C. difficile orthologue of the flagellar alternative sigma factor SigD (FliA; σ²⁸) mediates regulation of toxin gene expression in response to c-di-GMP. Indeed, ectopic expression of sigD in C. difficile resulted in increased expression levels of tcdR, tcdA, and tcdB. Furthermore, sigD expression enhanced toxin production and increased the cytopathic effect of C. difficile on cultured fibroblasts. Finally, evidence is provided that SigD directly activates tcdR expression and that SigD cannot activate tcdA or tcdB expression independent of TcdR. Taken together, these data suggest that SigD positively regulates toxin genes in C. difficile and that c-di-GMP can inhibit both motility and toxin production via SigD, making this signaling molecule a key virulence gene regulator in C. difficile.     

The Second Messenger, Cyclic AMP, Is Not Sufficient for Myelin Gene Induction in the Peripheral Nervous System

Abstract: The adenylyl cyclase-cyclic AMP (cAMP) second messenger pathway has been proposed to regulate myelin gene expression; however, a clear correlation between endogenous cAMP levels and myelin-specific mRNA levels has never been demonstrated during the induction or maintenance of differentiation by the myelinating Schwann cell. Endogenous cAMP levels decreased to 8–10% of normal nerve by 3 days after crush or permanent transection injury of adult rat sciatic nerve. Whereas levels remained low after transection injury, cAMP levels reached only 27% of the normal values by 35 days after crush injury. Because P₀ mRNA levels were 60% of normal levels by 14 days and 100% by 21 days after crush injury, cAMP increased only well after P₀ gene induction. cAMP, therefore, does not appear to trigger myelin gene induction but may be involved in myelin assembly or maintenance. Forskolin, an activator of adenylyl cyclase, increased endoneurial cAMP levels only in the normal nerve, and in the crushed nerve beginning at 16 days after injury, but at no time in the transected nerve. Only by treating transected nerve with 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cAMP phosphodiesterases, in combination with forskolin was it possible to increase cAMP levels. No induction of myelin genes, however, was observed with short- or long-term treatment with IBMX and forskolin in the transected nerve. A three-fold increase in phosphodiesterase activity was observed at 35 days after both injuries, and a nonmyelinated nerve was shown to have even higher activity. These experiments, therefore, suggest an important role for phosphodiesterase in the inactivation of this second messenger-dependent stimuli when Schwann cells are non-myelinating, such as after sciatic nerve injury or in the nonmyelinated nerve, which again implies that cAMP may be required for the maintenance of the myelin sheath.     

Two-Step Synthesis and Hydrolysis of Cyclic di-AMP in Mycobacterium tuberculosis

Cyclic di-AMP is a recently discovered signaling molecule which regulates various aspects of bacterial physiology and virulence. Here we report the characterization of c-di-AMP synthesizing and hydrolyzing proteins from Mycobacterium tuberculosis. Recombinant Rv3586 (MtbDisA) can synthesize c-di-AMP from ATP through the diadenylate cyclase activity. Detailed biochemical characterization of the protein revealed that the diadenylate cyclase (DAC) activity is allosterically regulated by ATP. We have identified the intermediates of the DAC reaction and propose a two-step synthesis of c-di-AMP from ATP/ADP. MtbDisA also possesses ATPase activity which is suppressed in the presence of the DAC activity. Investigations by liquid chromatography -electrospray ionization mass spectrometry have detected multimeric forms of c-di-AMP which have implications for the regulation of c-di-AMP cellular concentration and various pathways regulated by the dinucleotide. We have identified Rv2837c (MtbPDE) to have c-di-AMP specific phosphodiesterase activity. It hydrolyzes c-di-AMP to 5′-AMP in two steps. First, it linearizes c-di-AMP into pApA which is further hydrolyzed to 5′-AMP. MtbPDE is novel compared to c-di-AMP specific phosphodiesterase, YybT (or GdpP) in being a soluble protein and hydrolyzing c-di-AMP to 5′-AMP. Our results suggest that the cellular concentration of c-di-AMP can be regulated by ATP concentration as well as the hydrolysis by MtbPDE.     

神经迁移因子在血管系统中的表达与功能

神经迁移因子是近10年来在发育神经生物学中的研究热点,主要由ephrin、neuropilin、Slit和netrin四大家族成员构成,其主要功能是吸引或排斥神经元轴突的迁移,在神经系统中发挥着重要作用。现在,越来越多的实验证据表明：神经迁移因子的作用不仅仅局限在神经系统发育过程中,在血管发生或新生血管形成中同样具有不可替代的功能。     

The Influence of Stress on the Affective Modulation of the Startle Response to Nicotine Cues

Recent research suggesting that nicotine cues are appetitive in nature promotes the affective modulation of the startle reflex (AMSR) paradigm as a potentially valuable psychophysiological tool for elucidating mechanisms involved in nicotine addiction. Despite numerous studies indicating stress as a key factor in nicotine dependence, specific behavioral mechanisms linking stress and smoking have yet to be explicated. The current study aimed to determine the effects of stress, a negative affective state intimately linked with nicotine use, on the psychophysiological responding of nicotine dependent individuals during smoking cues. Twenty-nine nicotine dependent individuals were randomly assigned to the trier social stress test or control condition directly prior to administration of the AMSR paradigm, which examined their physiological responses to appetitive, neutral, aversive, and nicotine cue images. Both groups evinced significantly decreased startle magnitudes in response to nicotine cues as compared to aversive images. However, exposure to stress did not significantly modulate the startle reflex while viewing nicotine cues. Stress induction does not appear to modulate the AMSR paradigm when evaluating responses to nicotine images. These findings suggest that AMSR is robust to effects of acute stress induction in nicotine dependent individuals which may increase its viability as a clinical tool for assessing success in smoking cessation interventions.     

Chemosensory Cues to Conspecific Emotional Stress Activate Amygdala in Humans

Alarm substances are airborne chemical signals, released by an individual into the environment, which communicate emotional stress between conspecifics. Here we tested whether humans, like other mammals, are able to detect emotional stress in others by chemosensory cues. Sweat samples collected from individuals undergoing an acute emotional stressor, with exercise as a control, were pooled and presented to a separate group of participants (blind to condition) during four experiments. In an fMRI experiment and its replication, we showed that scanned participants showed amygdala activation in response to samples obtained from donors undergoing an emotional, but not physical, stressor. An odor-discrimination experiment suggested the effect was primarily due to emotional, and not odor, differences between the two stimuli. A fourth experiment investigated behavioral effects, demonstrating that stress samples sharpened emotion-perception of ambiguous facial stimuli. Together, our findings suggest human chemosensory signaling of emotional stress, with neurobiological and behavioral effects.     

The Calcium Ion Is a Second Messenger in the Nitrate Signaling Pathway of Arabidopsis

Understanding how plants sense and respond to changes in nitrogen availability is the first step toward developing strategies for biotechnological applications, such as improvement of nitrogen use efficiency. However, components involved in nitrogen signaling pathways remain poorly characterized. Calcium is a second messenger in signal transduction pathways in plants, and it has been indirectly implicated in nitrate responses. Using aequorin reporter plants, we show that nitrate treatments transiently increase cytoplasmic Ca²⁺ concentration. We found that nitrate also induces cytoplasmic concentration of inositol 1,4,5-trisphosphate. Increases in inositol 1,4,5-trisphosphate and cytoplasmic Ca²⁺ levels in response to nitrate treatments were blocked by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, a pharmacological inhibitor of phospholipase C, but not by the nonfunctional phospholipase C inhibitor analog {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}}U73343. In addition, increase in cytoplasmic Ca²⁺ levels in response to nitrate treatments was abolished in mutants of the nitrate transceptor NITRATE TRANSPORTER1.1/Arabidopsis (Arabidopsis thaliana) NITRATE TRANSPORTER1 PEPTIDE TRANSPORTER FAMILY6.3. Gene expression of nitrate-responsive genes was severely affected by pretreatments with Ca²⁺ channel blockers or phospholipase C inhibitors. These results indicate that Ca²⁺ acts as a second messenger in the nitrate signaling pathway of Arabidopsis. Our results suggest a model where NRT1.1/AtNPF6.3 and a phospholipase C activity mediate the increase of Ca²⁺ in response to nitrate required for changes in expression of prototypical nitrate-responsive genes.Plants are sessile organisms that evolved sophisticated sensing and response mechanisms to adapt to changing environmental conditions. Calcium, a ubiquitous second messenger in all eukaryotes, has been implicated in plant signaling pathways (Harper et al., 2004; Hetherington and Brownlee, 2004; Reddy and Reddy, 2004; Hepler, 2005). Multiple abiotic and biotic cues elicit specific and distinct spatiotemporal patterns of change in the concentration of cytosolic Ca²⁺ ([Ca2+]_cyt) in plants (Sanders et al., 2002; Hetherington and Brownlee, 2004; Reddy and Reddy, 2004; Hepler, 2005). Abscisic acid and heat shock treatments cause a rapid intracellular Ca²⁺ increase that is preceded by a transient increase in the level of inositol 1,4,5-trisphosphate (IP₃; Sanchez and Chua, 2001; Zheng et al., 2012). Ca²⁺ signatures are detected, decoded, and transmitted to downstream responses by a set of Ca²⁺ binding proteins that functions as Ca²⁺ sensors (White and Broadley, 2003; Dodd et al., 2010).Nitrate is the main source of N in agriculture and a potent signal that regulates the expression of hundreds of genes (Wang et al., 2004; Vidal and Gutiérrez, 2008; Ho and Tsay, 2010). Despite progress in identifying genome-wide responses, only a handful of molecular components involved in nitrate signaling has been identified. Several pieces of evidence indicate that NITRATE TRANSPORTER1.1 (NRT1.1)/Arabidopsis (Arabidopsis thaliana) NITRATE TRANSPORTER1 PEPTIDE TRANSPORTER FAMILY6.3 (AtNPF6.3) is a nitrate sensor in Arabidopsis (Ho et al., 2009; Gojon et al., 2011; Bouguyon et al., 2015). NRT1.1/AtNPF6.3 is required for normal expression of more than 100 genes in response to nitrate in Arabidopsis roots (Wang et al., 2009). Downstream of NRT1.1/AtNPF6.3, CALCINEURIN B-LIKE INTERACTING SER/THR-PROTEINE KINASE8 (CIPK8) is required for normal nitrate-induced expression of primary nitrate response genes, and the CIPK23 kinase is able to control the switch from low to high affinity of NRT1.1/AtNPF6.3 (Ho et al., 2009; Hu et al., 2009; Ho and Tsay, 2010; Castaings et al., 2011). CIPKs act in concert with CALCINEURIN B-LIKE proteins, plant-specific calcium binding proteins that activate CIPKs to phosphorylate downstream targets (Albrecht et al., 2001). Early experiments using maize (Zea mays) and barley (Hordeum vulgare) detached leaves showed that nitrate induction of two nitrate primary response genes was altered by pretreating leaves with the calcium chelator EGTA or the calcium channel blocker LaCl₃ (Sakakibara et al., 1997; Sueyoshi et al., 1999), suggesting an interplay between nitrate response and calcium-related signaling pathways. However, the role of calcium as a second messenger in the nitrate signaling pathway has not been directly addressed.We show that nitrate treatments cause a rapid increase of IP₃ and [Ca2+]_cyt levels and that blocking phospholipase C (PLC) activity inhibits both IP₃ and [Ca2+]_cyt increases after nitrate treatments. We provide evidence that NRT1.1/AtNPF6.3 is required for increasing both IP₃ and [Ca2+]_cyt in response to nitrate treatments. Altering [Ca2+]_cyt or blocking PLC activity hinders regulation of gene expression of nitrate-responsive genes. Our results indicate that Ca²⁺ is a second messenger in the nitrate signaling pathway of Arabidopsis.     

Spatiotemporal Dynamics of Phosphorylation in Lipid Second Messenger Signaling

The plasma membrane serves as a dynamic interface that relays information received at the cell surface into the cell. Lipid second messengers coordinate signaling on this platform by recruiting and activating kinases and phosphatases. Specifically, diacylglycerol and phosphatidylinositol 3,4,5-trisphosphate activate protein kinase C and Akt, respectively, which then phosphorylate target proteins to transduce downstream signaling. This review addresses how the spatiotemporal dynamics of protein kinase C and Akt signaling can be monitored using genetically encoded reporters and provides information on how the coordination of signaling at protein scaffolds or membrane microdomains affords fidelity and specificity in phosphorylation events.The alteration of protein or lipid structure by phosphorylation is one of the most effective ways to transduce extracellular signals into cellular actions. Phosphorylation can alter enzyme activity, regulate protein stability, affect protein interactions or localization, or influence other post-translational modifications. A plethora of cellular processes, including cell growth, differentiation, and migration, are tightly regulated by phosphorylation. Cellular homeostasis is achieved by means of a precisely regulated balance between phosphorylation and dephosphorylation, and disruption of this balance results in pathophysiologies. Kinases and phosphatases are antagonizing effector enzymes that respond to second messengers and mediate phosphorylation/dephosphorylation events.Two prominent lipid second messenger pathways are those mediated by diacylglycerol (DAG)¹ and phosphatidylinositol 3,4,5-trisphosphate (PIP₃) (Fig. 1A). These membrane-embedded second messengers recruit effector kinases containing specific membrane-targeting modules to membranes, thus activating them. Specifically, DAG recruits C1-domain-containing proteins, notably protein kinase C (PKC), whereas PIP₃ recruits pleckstrin homology (PH) domain-containing proteins, such as Akt. Specificity and fidelity in signaling are often achieved via the compartmentalization of signaling on protein scaffolds and membrane microdomains, which can control the access of enzymes to particular substrates. In this review, we provide a brief background of the DAG and PIP₃ pathways and their effector kinases, PKC and Akt, respectively. We discuss sensors that have been developed to measure lipid second messenger levels and kinase activity at various subcellular compartments, the role of scaffolds and membrane microdomains in compartmentalizing signaling, and the consequences of dysregulation of second messenger signaling in disease.Open in a separate windowFig. 1.Diagram of diacylglycerol and PIP₃ signaling pathways. A, agonist stimulation activates cell surface receptors such as receptor tyrosine kinases or G-protein-coupled receptors, which active phospholipase C (PLC) or phosphoinositide-3 kinase (PI3K), both of which act on phosphatidylinositol 4,5-bisphosphate (PIP₂). PLC hydrolyzes PIP₂ to produce diacylglycerol (DAG) and inositol triphosphate (IP₃). IP₃ binds to the IP₃ receptor (IP₃R) at the endoplasmic reticulum (ER), releasing Ca²⁺. DAG and Ca²⁺ recruit conventional protein kinase C (PKC) to the plasma membrane (via its C1 and C2 domains, respectively) and activate it. Ca²⁺ release also leads to the production of DAG at the Golgi. Diacylglycerol kinase (DGK) suppresses DAG-mediated signaling by converting DAG to phosphatidic acid (PA). Alternatively, PI3K phosphorylates PIP₂ into phosphatidylinositol 3,4,5-bisphosphate (PIP₃), which recruits Akt to the plasma membrane via its pleckstrin homology (PH) domain, where it gets activated. PTEN terminates PIP₃ signaling by converting it to PIP₂. B, enzymes involved in DAG and PIP₂ production and removal. PIP₂ is converted to PIP₃ by PI3K, whereas PTEN opposes this reaction by dephosphorylating PIP₃. PIP₂ can also be converted to DAG and IP₃ by PLC. DAG can then be removed by DGKs as it gets converted to PA. Phosphatidic acid phosphatases (PAPs) and sphingomyelin synthases (SMSs) convert PA back to DAG.

Lipid Second Messengers

Lipid second messengers are signaling molecules produced in response to extracellular stimuli. Targeting enzymes and their substrates to the same membrane constrains them to a space of reduced dimensionality, thus increasing the apparent concentration of the signaling complex and the likelihood and amplitude of signaling (1).

DAG Pathway

Upon activation by agonists, receptor tyrosine kinases and G-protein-coupled receptors can activate phospholipase C, which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP₂) found at the plasma membrane into the second messengers DAG and inositol 1,4,5-trisphosphate (IP₃) (2). DAG recruits proteins that contain C1 domains, small globular DAG-binding domains, to membranes (3), whereas IP₃ freely diffuses inside the cell and binds to the IP₃ receptor at the endoplasmic reticulum. This releases another second messenger, Ca²⁺, which induces further production of DAG at the Golgi (4). Two main classes of proteins that bind DAG are the protein kinases PKC and PKD and the Rac-GAPs chimerins; the affinity of their C1 domains for DAG can vary greatly and is discussed below in the context of PKC. DAG can also be generated from phosphatidic acid (PA) by phosphatidic acid phosphatases or sphingomyelin synthases. The removal of DAG, and thus termination of its signaling, is achieved by diacylglycerol kinases (DGKs) that convert it into PA (Fig. 1B).

PIP₃ Pathway

The second messenger PIP₃ is generated upon the stimulation of receptor tyrosine kinases or G-protein-coupled receptors that activate phosphoinositide 3-kinase (PI3K) to phosphorylate PIP₂ (Fig. 1A). PIP₃ binds certain PH domains, recruiting enzymes such as Akt to the membrane (5). PIP₃ signaling is terminated by the tumor suppressor phosphatase and tensin homologue (PTEN), a lipid phosphatase that converts PIP₃ to PIP₂ (Fig. 1B).

Genetically Encoded Second Messenger Sensors

Second messenger levels can be measured in live cells, in real time, using genetically encoded sensors (6–11). The first generation of sensors comprised a fluorescent protein fused to a domain that specifically binds a second messenger (i.e. C1 or PH domain). Recruitment of the tagged domain to membranes containing the second messenger is monitored and used as a proxy for its levels (6, 10, 11). Recently, reporters containing two fluorescent proteins that undergo changes in fluorescence resonance energy transfer (FRET) as they change their distance or relative orientation upon binding of a second messenger have been developed (7–9). These reporters provide more quantitative data, as they rely on ratiometric measurements of two fluorophores as opposed to the translocation of a single fluorophore to membranes, thus minimizing artifacts resulting from cell movements, photobleaching, or variable cell thickness (12). Targeting of these second messenger sensors to particular subcellular compartments provides information on the kinetics and location of second messenger production.

Measuring DAG

One of the first DAG reporters comprised the C1 domain of PKCγ fused to a green fluorescent protein, and its agonist-evoked translocation to membranes served as a readout for DAG production (6). Reporters employing ratiometric measurements were later developed and contain a C1 domain and a FRET pair, such as the DAG reporters DAGR (Fig. 2A), which uses intermolecular FRET (7), and Daglas (Fig. 2B), which uses intramolecular FRET (8). These reporters can be targeted to specific subcellular compartments using a membrane localization sequence, allowing the detection of changes in DAG levels at various intracellular membranes. Such membrane-specific reports have revealed that the Golgi and endoplasmic reticulum have relatively high basal levels of DAG, whereas the plasma membrane lacks measurable basal DAG (9). DAG at the plasma membrane is produced following acute agonist stimulation, and this signaling is swiftly terminated (Fig. 3) as DGK converts DAG to PA (8, 13). In contrast, stimulated DAG levels at the Golgi are relatively sustained, whereas those at mitochondria are not detectably altered by ATP stimulation.Open in a separate windowFig. 2.Genetically encoded reporters for measuring DAG and PIP₃ levels and PKC and Akt activities. A, the diacylglycerol (DAG) reporter, DAGR, comprises a CFP–YFP FRET pair flanking a DAG-binding C1 domain. Translocation of the reporter to membranes upon elevation of DAG leads to intermolecular FRET. B, the DAG reporter Daglas and PIP₃ sensor Fllip are anchored to the membrane via a membrane localization sequence (MLS) and are composed of a FRET pair flanking a lipid-binding domain (LBD) (C1 domain for Daglas and PH domain for Fllip) and a glycine–glycine (GG) hinge. Engagement of the LBD to the membrane induces a conformational change in the reporter, increasing FRET efficiency. The FRET signal is reversed by enzymes that remove the second messengers (i.e. DGK and PTEN). C, Upward and Downward DAG are based on PKCδ and contain a circularly permuted GFP between the pseudosubstrate (PS) and the C1 domain. Binding of the C1 domain to DAG increases (for Upward DAG) or decreases (for Downward DAG) the fluorescence intensity by altering the conformation of the GFP β-barrel. D, InPAkt is a PIP₃ reporter comprising the PH domain of Akt, a pseudoligand that binds the PH domain with a lower affinity than PIP₃, and a FRET pair. Upon PIP₃ production, the PH domain releases the pseudoligand and binds PIP₃, increasing FRET. E, the PKC reporter, CKAR, and the Akt reporter, BKAR, are made up of an FHA2 domain, a PKC- or Akt-specific substrate peptide, and a FRET pair. When phosphorylated, the substrate sequence binds the FHA2 domain, and this conformational change results in a decrease in FRET. F, the nPKC and atypical PKC reporter KCP-1 is based on the PKC substrate pleckstrin. PKC phosphorylation at three sites between the PH and dishevelled–Egl-10–pleckstrin domains of pleckstrin results in a conformational change that increases FRET between enhanced YFP and GFP2. G, Aktus and AktAR measure Akt activity as phosphorylation of an Akt-specific substrate peptide causes it to bind a phosphopeptide-binding domain (PPBD) (14–3-3η for Aktus and FHA1 for AktAR), increasing FRET from CFP to YFP. H, the bioluminescent Akt reporter (BAR) contains a split luciferase, an FHA2 domain, and an Akt substrate peptide. Phosphorylation by Akt causes the split luciferase to dissociate, decreasing bioluminescence. I, Akind is based on Akt''s kinase and PH domains. Upon recruitment of Akt to PIP₃-enriched membranes and its subsequent phosphorylation, Akt undergoes a conformational change that leads to an increase in FRET between the fluorophores.Open in a separate windowFig. 3.Schematic diagram displaying DAG and PIP₃ levels at various intracellular membranes and the PKC and Akt activity they induce at these locations. UTP stimulates rapid but short-lived production of DAG at the plasma membrane. In contrast, stimulated DAG levels at the Golgi are relatively sustained, leading to more sustained PKC activity at the Golgi than at the plasma membrane. Typically cPKCs are recruited to the plasma membrane because their Ca²⁺-regulated C2 domain selectively recognizes PIP₂, which is enriched at the plasma membrane. In contrast, nPKCs signal primarily at the DAG-enriched Golgi (111). The nucleus shows little UTP-induced PKC activity, whereas the cytosol has the greatest, although transient, UTP-stimulated PKC activity. Upon PDGF stimulation, PIP₃ is more rapidly generated at endomembranes such as the Golgi than at the plasma membrane. PDGF-induced Akt activity rapidly increases at the plasma membrane, whereas in the cytosol the kinetics of activation are slower. The nucleus contains high but delayed Akt activity, despite the lack of PIP₃ production at the nuclear membrane, suggesting that active Akt translocates to the nucleus where there is little phosphatase suppression.More recently, a sensor has been engineered to simultaneously measure two second messengers in the same cell. The Green Upward or Downward DAG (Fig. 2C) was paired with a Ca²⁺ sensor (not shown) for the concomitant measurement of DAG and Ca²⁺ (9). Both of these sensors are based on fluorescent proteins that undergo changes in fluorescence intensity upon the binding of second messengers. The DAG sensor (based on PKCδ) and the Ca²⁺ sensor (based on calmodulin and a calmodulin-binding domain (14)) are linked by a peptide that, upon cleavage, produces equal amounts of the two reporters as distinct peptides (15). Considering that DAG and Ca²⁺ are often co-elevated, measuring these second messengers concomitantly gives a more complete view of signal transduction and has the advantage of providing a direct comparison of second messenger production downstream of the activation of various receptors with different agonists. However, because these reporters rely on changes in fluorescence intensity instead of a FRET ratio, care should be taken when using them, as their readout is dependent on the absolute concentration of the sensor, and intensity changes resulting from focus drift or cell movements can occur during imaging.

Measuring PIP₃

Changes in PIP₃ levels can be assessed by monitoring the agonist-dependent relocalization of fluorescently tagged PIP₃-selective PH domains to membranes (10, 12). However, because these methods monitor translocation to all membranes, pools of PIP₃ at specific membranes cannot be readily monitored. Thus, more quantifiable FRET-based PIP₃ sensors that can be targeted to particular subcellular localizations have been developed. For example, an indicator for phosphoinositides based on Akt''s PH domain, InPAkt (Fig. 2D), showed that the plasma membrane contains basal levels of PIP₃ that are maintained through a balance between PI3K and phosphatases, whereas the nucleus has no detectible production of this second messenger (16). Another PIP₃ sensor, Fllip (Fig. 2B), comprising the PH domain of GRP1 and a FRET pair, is anchored to a membrane via a membrane localization sequence (17). This reporter revealed that platelet-derived growth factor (PDGF) stimulates greater production of PIP₃ at the Golgi and endoplasmic reticulum than at the plasma membrane, and that PIP₃ is generated at these endomembranes by endocytosed receptor tyrosine kinases that activate a localized pool of PI3K.

PKC and Akt as Effectors

PKC and Akt, effector kinases of DAG and PIP₃, respectively, phosphorylate myriad downstream targets (reviewed in Refs. 18–20), many of which have been identified through phosphoproteomic screens (21–23).

PKC

The PKC family consists of nine genes that are divided into three categories based on the domain structure of the enzymes they encode, and hence the second messengers they require for activation. Both conventional PKCs (cPKCs) (α, β, γ) and novel PKCs (nPKCs) (δ, ε, θ, η) bind DAG through tandem C1 domains; cPKCs also bind membranes in a Ca²⁺-dependent manner through a C2 domain. Atypical PKCs (ζ and ι/λ) bind neither DAG nor Ca²⁺ and are regulated by protein–protein interactions through a PB1 domain (24). cPKCs and nPKCs are constitutively phosphorylated at three conserved phosphorylation sites termed the activation loop, turn motif, and hydrophobic motif (25). These phosphorylations are necessary for proper PKC folding, and thus for its activation; for PKCα, these modifications occur with a half-time of 5 to 10 min following biosynthesis (26). The activation loop is phosphorylated by the phosphoinositide-dependent kinase PDK-1 (27–29), an event that triggers two tightly coupled phosphorylations at the turn and hydrophobic motifs (25, 30). mTORC2 is required to initiate the phosphorylation cascade, and in cells lacking mTORC2, PKC is not phosphorylated and thus is shunted for degradation (31–33); however, the mechanism of this regulation is unknown. The hydrophobic motif is autophosphorylated by an intramolecular reaction in vitro, but whether this is the mechanism of modification in cells or whether it is the direct target of another kinase such as mTORC2 remains controversial (33, 34). When first translated, PKC is in an open conformation, with the autoinhibitory pseudosubstrate out of the active site (35). Upon phosphorylation, PKC matures into a catalytically competent, but inactive, species that is maintained in an autoinhibited (closed) conformation in which the pseudosubstrate occupies the substrate-binding cavity. Upon intracellular Ca²⁺ release and DAG production, cPKCs are recruited to membranes through their Ca²⁺-sensitive C2 domain. This relocalization reduces the dimensionality in which the C1 domain has to probe for its membrane-embedded ligand, DAG, thus increasing the effectiveness of this search by several orders of magnitude (36). Binding of one of PKC''s C1 domains to DAG provides the energy necessary to expel the pseudosubstrate from the substrate-binding site, allowing the phosphorylation of downstream targets. For some isozymes, such as PKCδ, the second C1 domain (C1B) is the major binder (37). The affinity of C1 domains for DAG is toggled from low to high by a single residue within the DAG binding cavity: a Trp at position 22 confers an affinity for DAG that is 2 orders of magnitude higher than that conferred by a Tyr at that position (38). Consequently, nPKCs, which contain a high-affinity C1B domain, can respond to DAG alone, whereas cPKCs, which have a low-affinity C1B domain, require the elevation of Ca²⁺ concomitantly with DAG production in order to become activated. Reporters such as the Green Upward or Downward DAG (Fig. 2C) paired with a Ca²⁺ sensor (9), described above, would be useful tools for discerning which agonists solely activate cPKCs versus nPKCs.The amplitude of PKC signaling is diligently balanced through its phosphorylation state (controlling its steady-state levels), the presence of its lipid second messenger, DAG (acutely controlling activity), and, for cPKCs, Ca²⁺ levels. Thus PKC activity can be antagonized via direct dephosphorylation by protein phosphatases such as the PH domain leucine-rich repeat protein phosphatase (PHLPP) or by removal of the lipid second messenger through phosphorylation by the lipid kinase DGK (Fig. 1) (39). The peptidyl-prolyl isomerase Pin1 was recently shown to be necessary for cPKC dephosphorylation and degradation following agonist activation, as it isomerizes the phosphorylated turn motif of cPKCs, thus facilitating PKC dephosphorylation (40).

Akt

Akt, also known as PKB because of its homology to PKA and PKC, is a serine/threonine kinase that promotes cell growth and survival (41). The three Akt isozymes (Akt1, Akt2, and Akt3) contain an N-terminal PH domain that mediates PIP₃-dependent membrane recruitment. Akt is maintained in an inactive conformation by an interaction between the PH and kinase domains, and unlike PKC, Akt is directly activated by phosphorylation at its activation loop and hydrophobic motif following agonist-dependent recruitment to membranes (42). Thus, Akt phosphorylation at these sites can be used as a proxy for activity under certain conditions (43). Akt is co-translationally phosphorylated at the turn motif by mTORC2 (contrasting with the post-translational modification of PKC), an event that is not necessary for function but increases the stability of the protein (44). PIP₃ recruits Akt to the plasma membrane via its PH domain, unmasking the kinase domain to permit phosphorylation of the activation loop by PDK-1 (45) and subsequent phosphorylation of the hydrophobic motif. mTORC2 facilitates hydrophobic motif phosphorylation (46, 47), possibly by assisting in unmasking the kinase domain for phosphorylation. Indeed, manipulations that displace the PH domain effectively bypass the requirement for mTORC2 for phosphorylation of the hydrophobic motif (but not the turn motif) (48). Phosphorylated Akt is locked in an active conformation and can disengage from the membrane and relocalize to other intracellular regions, such as the nucleus (49), to phosphorylate diverse substrates (19). Akt signaling is terminated by the hydrolysis of PIP₃ to PIP₂ by PTEN or by direct dephosphorylation of the activation loop by protein phosphatase 2A or of the hydrophobic motif by PHLPP (50–52). PHLPP1 dephosphorylates Akt2 and Akt3, whereas PHLPP2 dephosphorylates Akt1 and Akt3 (52), suggesting that these isozymes are differentially compartmentalized, likely via the unique PDZ (PSD-95, disheveled, and ZO1) ligand of each PHLPP isozyme (53). Interestingly, stoichiometric quantification of Akt phosphorylation at the activation loop and hydrophobic motif sites using LC-MS revealed that in untreated T cells, less than 1% of Akt is phosphorylated at both of these sites, and a low level of Akt phosphorylation is sufficient to contribute to tumorigenesis (54), highlighting the importance of keeping Akt activity low for maintaining cellular homeostasis.

Identifying PKC and Akt Substrates

A number of PKC and Akt substrates have been identified through biochemical methods, and several phosphoproteomic screens have been devised to identify new substrates and novel roles of these kinases. For example, a functional proteomic screen identified enhancer of mRNA decapping 3 as a substrate of Akt that regulates the mRNA decay and translation repression pathways downstream of insulin signaling (21), and another identified a number of chaperone proteins and protein disulfide isomerases as potential Akt substrates in rat mesangial cells, suggesting that Akt might regulate chaperone function (22). An in vivo quantitative phosphoproteomics study employing stable isotope labeling by amino acids in cell culture implicated a highly deregulated kinase network composed of PKC, PAK4, and SRC in squamous cell carcinoma, but not in papilloma (55).Vast efforts combining phosphorylation sites identified in vivo though mass spectrometry, in vitro through protein microarrays, and computationally through prediction algorithms have uncovered more substrates, setting the foundation for the development of human phosphorylation network maps (23, 56). However, despite efforts to enrich for physiologically relevant phosphorylation events by accounting for subcellular localization and scaffolding, these networks still have shortcomings, as the algorithms predict some false positives and miss a large fraction of known phosphorylation sites.

Kinase Activity Reporters

Genetically encoded reporters allow the visualization of the spatiotemporal dynamics of kinase activity in individual cells. These reporters can be targeted to various subcellular localizations and to protein scaffolds to measure localized activity, which can be more physiologically relevant than bulk activity in the cytosol.

PKC

Because cPKCs and nPKCs are constitutively phosphorylated at the C-terminal sites, and because the phosphate at the activation loop does not modulate activity once the C-terminal tail is phosphorylated (25), their activity cannot be measured with phosphorylation-specific antibodies, as is done for most other kinases. However, PKC activity can be monitored using activity reporters such as the C kinase activity reporter (Fig. 2E), which is composed of a CFP–YFP FRET pair flanking a PKC-specific substrate and an FHA2 phosphothreonine-binding domain (7). Upon phosphorylation of this substrate by PKC, the reporter undergoes a conformational change that decreases FRET. As phosphorylation of the reporter is reversible (i.e. phosphatases can dephosphorylate the reporter), it provides a real-time readout of PKC activity.C kinase activity reporter has been targeted to various intracellular locations to enable specific monitoring of PKC activity at these regions. Using these targeted reporters, Gallegos et al. (13) found that the activation of PKC with the agonist UTP leads to rapid and relatively sustained PKC activity at the Golgi, driven by the persistence of DAG at this membrane (Fig. 3). UTP-dependent PKC activity in the cytosol is, however, quickly terminated by phosphatases, and activity in the nucleus is low because of high phosphatase suppression in this compartment. The mitochondria also have little UTP-stimulated activity; however, using a mitochondrially targeted PKCδ-specific activity reporter, Mito-δCKAR, Zheng et al. (57) revealed that PKCδ translocates to, and is active at, the outer membrane of mitochondria upon stimulation with phorbol esters, and that its intrinsic catalytic activity is required for its interaction with the mitochondria.The Schultz lab has developed a reporter for nPKCs and atypical PKCs, KCP-1 (Fig. 2F), that is based on the PKC substrate pleckstrin (58). This reporter does not utilize a phosphopeptide-binding domain; rather, phosphorylation of residues between its PH and dishevelled–Egl-10–pleckstrin domains causes a conformational change in the reporter, resulting in a change in FRET. Thus, interactions between the phosphorylated sites and other endogenous proteins are reduced.

Akt

A number of FRET-based Akt reporters have been developed, most of which measure the phosphorylation of a synthetic substrate of Akt in live cells; these include Aktus, AktAR (Fig. 2G), and BKAR (Fig. 2E) (59–61). Aktus has low sensitivity and requires overexpression of Akt, whereas BKAR and AktAR can sense endogenous Akt activity. Because activated Akt can disengage from the membrane and diffuse to other subcellular locations, targeting these reporters to various subcellular compartments is particularly useful. BKAR targeted to the plasma membrane revealed that phosphatase suppression of Akt is low at this location (60). The phosphatase suppression of Akt activity is greater in the cytosol than it is at the plasma membrane, so Akt is more rapidly inactivated in the cytosol (Fig. 3), most likely by protein phosphatases that dephosphorylate Akt''s substrates as opposed to Akt itself. Conversely, the nucleus has low phosphatase suppression of Akt, so once Akt diffuses to the nucleus its activity is much more sustained (60). AktAR (61) has a greater dynamic range for detecting Akt activity than BKAR and was used to measure Akt activity in plasma membrane microdomains (addressed below).Whereas fluorescent reporters can measure rapid signaling kinetics at subcellular levels, bioluminescent reporters have the advantage of producing their own light and therefore bypass issues with autofluorescence, photobleaching, and tissue damage from the excitation light (62). A bioluminescent Akt reporter, BAR, was engineered using a split luciferase, an Akt specific substrate, and a phosphopeptide-binding domain (Fig. 2H). This reporter has the capability to measure Akt activity in a noninvasive manner in vivo (63). In addition to Akt activity reporters that measure the phosphorylation of a synthetic substrate by Akt, reporters that measure conformational changes of Akt induced by its translocation and phosphorylation (and thus activity state) have also been developed (64–66). The Akt indicator Akind (57), comprising the PH and catalytic domains of Akt and a FRET pair (Fig. 2I), was used to visualize Akt translocation to and activity at lamellipodial protrusions. Conformational changes attendant to the phosphorylation of Akt result in an increase in FRET. The use of another reporter of Akt action, ReAktion (not shown) (58), led to the proposal that activation loop phosphorylation of Akt decreases its membrane binding affinity, thus allowing disengagement from the membrane and relocalization to other cellular compartments. One advantage of these reporters is that both the activity and the translocation of Akt can be measured concurrently. However, the reporters themselves can phosphorylate endogenous substrates of Akt and could theoretically displace Akt from its scaffolds, thus potentially perturbing the system more than the introduction of an exogenous Akt substrate.

Localized Signaling and the Importance of Scaffolding

Protein scaffolds coordinate and allow specificity and fidelity by compartmentalizing kinases and their downstream substrates, as well as the phosphatases that can rapidly terminate the signal (67). Even though lipid second messengers acutely regulate the activity of PKC and Akt, scaffolds can mediate their access to particular substrates.

PKC Scaffolds

Although multiple PKC isozymes respond to the same second messengers, there is some specificity in their function and signaling, mediated in part by their cell-specific pattern of expression, their differential affinities for certain lipids, and by protein scaffolds. PKC is anchored to numerous protein scaffolds through interactions mediated by its regulatory domain, its pseudosubstrate, or, in the case of PKCα and the atypical PKCs, a PDZ ligand. The first PKC scaffolds were identified by Mochly-Rosen and colleagues as receptors for activated C kinase, which are proposed to selectively bind active PKC and enhance its activity toward substrates anchored at that location (68, 69). A kinase anchoring proteins (AKAPs), which were first identified as PKA scaffolds (70), also anchor PKC in proximity to its targets, but in its inactive state, thus enabling rapid downstream signaling upon PKC activation. For example, AKAP-Lbc coordinates PKCη and PKA to phosphorylate PKD and release it from the scaffold (71), whereas AKAP79 functions as a scaffold for PKC, PKA, and protein phosphatase 2B at the postsynaptic density in neurons (72). One critical aspect of scaffolding is that it can alter the pharmacological profile of tethered kinases, which could have clinical implications for drug design. For example, through the use of targeted PKC activity reporters, Scott and colleagues found that PKC bound to the AKAP79 scaffold is refractory to active site inhibitors, but not allosteric ones. Similarly, the processing phosphorylation of cPKCs by PDK-1 is refractory to active site inhibitors of the co-scaffolded PDK-1 (73).PKCα, -ζ, and -ι also bind PDZ-domain-containing scaffolds through their distinct PDZ ligands. In the case of PKCα, scaffolding by its PDZ ligand is required for cerebellar long-term depression (74). One likely PDZ-domain-binding partner involved in this is protein interacting with Cα kinase (PICK1), which interacts specifically with the PDZ ligand of PKCα (75, 76). Interestingly, PICK1 can have opposing effects on PKCα function in neurons, where it can act as either a mediator or an inhibitor of the phosphorylation of downstream targets. PICK1 targets activated PKCα to synapses to phosphorylate the glutamate receptor subunit GluR2, leading to its endocytosis (77), but it can also act as a barrier to phosphorylation of the metabotropic glutamate receptor mGluR7a by PKCα (78). More recently, a family of Discs large homolog scaffolds that interact with the PDZ ligand of PKCα to facilitate cellular migration was also identified (79).

Akt Scaffolds

Akt translocation to different intracellular regions is contingent on which upstream pathway is activated, partially because of the scaffolding proteins that direct its signaling. For example, the stimulation of endothelial cells with insulin leads to the activation of Akt and its translocation to both the Golgi and mitochondria, whereas stimulation with 17β-estrodiol leads to translocation of Akt to the Golgi but not mitochondria (59). Only a few Akt scaffolds have been identified thus far, but it is becoming more apparent that different receptors use different complexes to direct Akt activity (80). For example, Akt kinase-interacting protein 1 was identified as a scaffold for PI3K/PDK-1/Akt that associates with activated EGF receptors (81). This scaffold is necessary for Akt phosphorylation by PDK-1 downstream of EGF signaling. Conversely, Akt scaffolds can also attenuate Akt signaling by scaffolding it in proximity to its phosphatases. β-arrestin 2 scaffolds Akt and its activation loop phosphatase, PP2A, in response to G-protein-coupled receptor stimulation in dopaminergic neurotransmission (82), and β-arrestin 1 scaffolds Akt1 and its hydrophobic motif phosphatase, PHLPP2, downstream of receptor tyrosine kinases (83). Considering that the three Akt isozymes have some overlapping expression, are activated downstream of a multitude of receptors, have numerous cellular functions, and show specificity in their dephosphorylation by PHLPP, an abundance of Akt scaffolds potentially await identification.

Membrane Microdomains

Lipid rafts, which are cholesterol- and sphingolipid-rich microdomains, not only compartmentalize signaling complexes, but also increase signal transduction by aggregating particular signaling complexes in a small area (1).

PKC

DAG and its effector kinase PKC are often enriched in specialized lipid rafts that form membrane invaginations called caveolae (84, 85). For example, activation of the adenosine A1 receptor in cardiomyocytes causes PKCε and PKCδ to translocate to caveolae. PKCα is also enriched within caveolae through interactions with caveolin, which inhibits its activity, or with the serum deprivation response protein (86, 87).

Akt

Akt signaling not only differs at various subcellular localizations, but also varies within microdomains of a particular membrane. The Akt reporter AktAR (Fig. 2G) was preferentially targeted to different plasma membrane microdomains such as lipid rafts (using the N-terminal region of Lyn kinase) and non-raft regions (using the Kras CAAX motif) to analyze the spatiotemporal dynamics of Akt activity (61). These reporters demonstrate that Akt residing in lipid rafts is activated more potently and with faster kinetics than non-raft Akt. PDK-1 is enriched in membrane microdomains, partially accounting for the increased Akt activity in this compartment (88, 89).Membrane microdomains provide sheltered environments in which signaling is protected from immediate termination. For example, PI3K gets activated and produces PIP₃ in lipid rafts where Akt is recruited, enabling Akt to be rapidly and specifically activated by PDK-1 (90). However, PTEN, which terminates PIP₃ signaling, is primarily found in non-raft microdomains, allowing PIP₃ levels to be temporarily maintained in lipid rafts, underscoring the importance of spatially separating kinases from phosphatases. Disturbing this spatial segregation of negative and positive regulators of a pathway can lead to disease states.

Dysregulation of Lipid Signaling in Disease

Dysregulation of the PIP₃ and DAG signaling pathways leads to numerous pathophysiologies, such as inflammation, cardiovascular disease, diabetes, neurodegeneration, and cancer (91). Imbalances in these pathways are caused by mutations, gene amplifications or deletions, chromosomal translocations, or epigenetic changes of genes in these pathways. In general, enzymes that promote signaling such as PKCι, Akt, and the catalytic subunit of PI3K have been identified as oncogenes (92–94), whereas those that terminate signaling—PTEN, PHLPP, and DGK—have been implicated as tumor suppressors (53, 95–100).Although mutations in Akt are uncommon, Akt signaling is often elevated in cancer. Indeed, the PI3K pathway is one of the most frequently up-regulated pathways in cancer (101–103). Mutations that inactivate PTEN or hyperactivate the catalytic subunit of PI3K are very common and induce constitutive Akt signaling. Interestingly, a functional proteomics study using a reverse-phase protein lysate array revealed that in breast cancers, Akt activation loop and hydrophobic motif phosphorylation strongly inversely correlates with PTEN levels, underscoring the importance of PTEN regulation of Akt activity (104).Mutations in receptors upstream of DAG and PIP₃ can also lead to enhanced signaling. For example, certain EGF receptor mutants constitutively associate with the PI3K/PDK-1/Akt scaffold Akt kinase-interacting protein 1, leading to increased Akt signaling in lung cancer (105). The involvement of some of these genes in cancer is further substantiated by their frequent amplification or deletion in tumors. For example, genes encoding PKCι, Akt1, Akt2, and the catalytic subunit of PI3K are amplified in various cancers (92, 94, 106–109), whereas those encoding PTEN and PHLPP are commonly deleted (110).     

Coding Information in Plant Cells: the Multiple Roles of Ca2+ as a Second Messenger

Abstract: "Kalzium macht alles". With this sentence, the physiologist L. V. Heilbrunn described several decades ago what is still believed by many. The enormous attention this subject has received in the past 15 years has generated a vast amount of information which is helping us to understand how cells perceive and transduce a signal. This review focuses on some recent aspects of Ca²⁺ research and the perspectives that they open for future studies. However, Ca²⁺ is not the only element involved in signal transduction and its action depends on a complex network of signalling molecules, the role of which is discussed. Particular attention is given to the parallels emerging between plant and animal signalling and how we should explore them.     

脯氨酸在植物非生物胁迫耐性形成中的作用

植物作为固着生活的有机体,经常暴露在多变且对其生长发育不利的环境条件中,这些生物或非生物的胁迫因子严重影响着植物的生长、发育、生存和分布。脯氨酸在植物抵抗逆境胁迫过程中起着重要的作用。根据国内外的最新研究进展,结合我们的研究成果,对植物体内脯氨酸的代谢途径、渗透调节、抗氧化、分子伴侣、生长发育信号和毒性等方面进行了综述,并对该研究领域作了展望。     

MicroRNA在植物营养胁迫中的作用

microRNA是具有重要功能的一类非编码小RNA,不仅在植物生长发育过程中具有重要的调控作用,而且在植物营养胁迫中发挥作用。本文综述了microRNA在3种大量元素——氮、磷、硫胁迫中的作用。     

microRNA在植物逆境胁迫应答中的作用

逆境胁迫严重影响着全世界范围内的作物产量。为减少逆境胁迫损伤,植物在长期的进化过程中形成了多级别（转录、转录后和翻译、翻译后）的基因表达调控应答机制。最近研究发现,内源microRNA（miRNA）在植物逆境胁迫应答中具有重要的调节作用。在逆境胁迫发生时,一些miRNA会表达上调,而另一些miRNA会表达下调;miRNA正是通过下调胁迫应答过程的负调节子靶基因和上调胁迫应答过程中的正调节子靶基因,来执行生理调控功能。通过综述miRNA在植物逆境应答中的作用,以期全面的了解逆境胁迫调控网络。