Scaffold protein GhMORG1 enhances the resistance of cotton to Fusarium oxysporum by facilitating the MKK6‐MPK4 cascade

In eukaryotes, MAPK scaffold proteins are crucial for regulating the function of MAPK cascades. However, only a few MAPK scaffold proteins have been reported in plants, and the molecular mechanism through which scaffold proteins regulate the function of the MAPK cascade remains poorly understood. Here, we identified GhMORG1, a GhMKK6‐GhMPK4 cascade scaffold protein that positively regulates the resistance of cotton to Fusarium oxysporum. GhMORG1 interacted with GhMKK6 and GhMPK4, and the overexpression of GhMORG1 in cotton protoplasts dramatically increased the activity of the GhMKK6‐GhMPK4 cascade. Quantitative phosphoproteomics was used to clarify the mechanism of GhMORG1 in regulating disease resistance, and thirty‐two proteins were considered as the putative substrates of the GhMORG1‐dependent GhMKK6‐GhMPK4 cascade. These putative substrates were involved in multiple disease resistance processes, such as cellular amino acid metabolic processes, calcium ion binding and RNA binding. The kinase assays verified that most of the putative substrates were phosphorylated by the GhMKK6‐GhMPK4 cascade. For functional analysis, nine putative substrates were silenced in cotton, respectively. The resistance of cotton to F. oxysporum was decreased in the substrate‐silenced cottons. These results suggest that GhMORG1 regulates several different disease resistance processes by facilitating the phosphorylation of GhMKK6‐GhMPK4 cascade substrates. Taken together, these findings reveal a new plant MAPK scaffold protein and provide insights into the mechanism of plant resistance to pathogens.     

Bacillus subtilis BY‐kinase PtkA controls enzyme activity and localization of its protein substrates

Bacillus subtilis BY‐kinase PtkA was previously shown to phosphorylate, and thereby regulate the activity of two classes of protein substrates: UDP‐glucose dehydrogenases and single‐stranded DNA‐binding proteins. Our recent phosphoproteome study identified nine new tyrosine‐phosphorylated proteins in B. subtilis. We found that the majority of these proteins could be phosphorylated by PtkA in vitro. Among these new substrates, single‐stranded DNA exonuclease YorK, and aspartate semialdehyde dehydrogenase Asd were activated by PtkA‐dependent phosphorylation. Because enzyme activity was not affected in other cases, we used fluorescent protein tags to study the impact of PtkA on localization of these proteins in vivo. For several substrates colocalization with PtkA was observed, and more importantly, the localization pattern of the proteins enolase, YjoA, YnfE, YvyG, Ugd and SsbA was dramatically altered in ΔptkA background. Our results confirm that PtkA can control enzyme activity of its substrates in some cases, but also reveal a new mode of action for PtkA, namely ensuring correct cellular localization of its targets.     

p38 MAPK activation, JNK inhibition, neoplastic growth inhibition, and increased gap junction communication in human lung carcinoma and Ras-transformed cells by 4-phenyl-3-butenoic acid

Human lung neoplasms frequently express mutations that down‐regulate expression of various tumor suppressor molecules, including mitogen‐activated protein kinases such as p38 MAPK. Conversely, activation of p38 MAPK in tumor cells results in cancer cell cycle inhibition or apoptosis initiated by chemotherapeutic agents such as retinoids or cisplatin, and is therefore an attractive approach for experimental anti‐tumor therapies. We now report that 4‐phenyl‐3‐butenoic acid (PBA), an experimental compound that reverses the transformed phenotype at non‐cytotoxic concentrations, activates p38 MAPK in tumorigenic cells at concentrations and treatment times that correlate with decreased cell growth and increased cell‐cell communication. H2009 human lung carcinoma cells and ras‐transformed rat liver epithelial cells treated with PBA showed increased activation of p38 MAPK and its downstream effectors which occurred after 4 h and lasted beyond 48 h. Untransformed plasmid control cells showed low activation of p38 MAPK compared to ras‐transformed and H2009 carcinoma cells, which correlates with the reduced effect of PBA on untransformed cell growth. The p38 MAPK inhibitor, SB203580, negated PBA's activation of p38 MAPK downstream effectors. PBA also increased cell–cell communication and connexin 43 phosphorylation in ras‐transformed cells, which were prevented by SB203580. In addition, PBA decreased activation of JNK, which is upregulated in many cancers. Taken together, these results suggest that PBA exerts its growth regulatory effect in tumorigenic cells by concomitant up‐regulation of p38 MAPK activity, altered connexin 43 expression, and down‐regulation of JNK activity. PBA may therefore be an effective therapeutic agent in human cancers that exhibit down‐regulated p38 MAPK activity and/or activated JNK and altered cell–cell communication. J. Cell. Biochem. 113: 269–281, 2012. © 2011 Wiley Periodicals, Inc.     

GRK2-Dependent Desensitization Downstream of G Proteins

G protein-coupled receptor kinases (GRKs) are serine/threonine kinases first discovered by its role in receptor desensitization. Phosphorylation of the C-terminal tail of GPCRs by GRKs triggers the docking of β-arrestins and the functional uncoupling of G proteins and receptors. In addition, we and others have uncovered new direct ways by which GRKs could impinge into intracellular signalling pathways independently of receptor phosphorylation. In particular, we have characterized that elevated GRK2 levels can reduce CCR2-mediated activation of the ERK MAPK route in a manner that is independent of kinase activity and also of G proteins. This inhibition of ERK occurred in the absence of any reduction on MEK phosphorylation, what implicates that GRK2 is acting at the level of MEK or at the MEK-ERK interface to achieve a downregulation of ERK phosphorylation. In fact, we describe here that a direct association between GRK2 and MEK proteins can be detected in vitro. p38 MAPK pathway also appears to be regulated directly by GRK2 in a receptor-independent manner. p38 can be phosphorylated by GRK2 in threonine 123, a residue sitting at the entrance of a docking groove by which this MAPK associates to substrates and upstream activators. The T123phospho-mimetic mutant of p38 shows a reduced ability to bind to MKK6, concomitant with an impaired p38 activation, and a decreased phosphorylation of downstream substrates such as MEF2, MK2 and ATF2. Elevated levels of GRK2 downregulate p38-dependent cellular responses, such as differentiation of preadipocytic cells, while LPS-induced cytokine release is enhanced in macrophages from GRK2 (+/?) mice. In sum, we describe in this article different ways by which GRK2 directly regulates MAPK-mediated cellular events. This regulation of the MAPK modules by GRK2 could be relevant in pathological situations where the levels of this kinase are altered, such as during inflammatory diseases or cardiovascular pathologies.     

A Förster resonance energy transfer sensor for live‐cell imaging of mitogen‐activated protein kinase activity in Arabidopsis

The catalytic activity of mitogen‐activated protein kinases (MAPKs) is dynamically modified in plants. Since MAPKs have been shown to play important roles in a wide range of signaling pathways, the ability to monitor MAPK activity in living plant cells would be valuable. Here, we report the development of a genetically encoded MAPK activity sensor for use in Arabidopsis thaliana. The sensor is composed of yellow and blue fluorescent proteins, a phosphopeptide binding domain, a MAPK substrate domain and a flexible linker. Using in vitro testing, we demonstrated that phosphorylation causes an increase in the Förster resonance energy transfer (FRET) efficiency of the sensor. The FRET efficiency can therefore serve as a readout of kinase activity. We also produced transgenic Arabidopsis lines expressing this sensor of MAPK activity (SOMA) and performed live‐cell imaging experiments using detached cotyledons. Treatment with NaCl, the synthetic flagellin peptide flg22 and chitin all led to rapid gains in FRET efficiency. Control lines expressing a version of SOMA in which the phosphosite was mutated to an alanine did not show any substantial changes in FRET. We also expressed the sensor in a conditional loss‐of‐function double‐mutant line for the Arabidopsis MAPK genes MPK3 and MPK6. These experiments demonstrated that MPK3/6 are necessary for the NaCl‐induced FRET gain of the sensor, while other MAPKs are probably contributing to the chitin and flg22‐induced increases in FRET. Taken together, our results suggest that SOMA is able to dynamically report MAPK activity in living plant cells.     

TLR2 and TLR4 activation induces p38 MAPK‐dependent phosphorylation of S6 kinase 1 in C2C12 myotubes

Toll‐like receptors 2 (TLR2) and 4 (TLR4) are present in the plasma membrane of skeletal muscle cells where their functions remain incompletely resolved. They can bind various extracellular ligands, such as FSL‐1, lipopolysaccharide (LPS) and/or palmitic acid (PA). We have investigated the link between PA, TLR2/4 and ribosomal S6 kinase 1 (S6K1) in C2C12 myotubes. Incubation with agonists of either TLR2 or TLR4, and with a high concentration of PA, increased S6K1 phosphorylation. Canonical upstream kinases of S6K1, protein kinase B (PKB) and mammalian target of rapamycin complex 1 (mTORC1), were regulated in the opposite way by PA, indicating that these kinases were probably not involved. By using the SB202190 inhibitor, p38 MAPK (mitogen‐activated protein kinase) was found to be a key mediator of PA‐induced phosphorylation of S6K1. Downregulation of either tlr2 or tlr4 gene expression by small interfering RNAs prevented the activation of both p38 MAPK and S6K1 by FSL‐1, LPS or PA. Thus TLR2 and TLR4 agonists can increase the level of S6K1 phosphorylation in a p38 MAPK‐dependent way in C2C12 myotubes. As PA induced the same intracellular signalling, a novel atypical pathway for PA is induced at the cellular membrane level and results in a higher phosphorylation state of S6K1.     

The transmembrane protein Sho1 cooperates with the mucin Msb2 to regulate invasive growth and plant infection in Fusarium oxysporum

In the vascular wilt pathogen Fusarium oxysporum, the mitogen‐activated protein kinase (MAPK) Fmk1 is essential for plant infection. The mucin‐like membrane protein Msb2 regulates a subset of Fmk1‐dependent functions. Here, we examined the role of the tetraspan transmembrane protein Sho1 as an additional regulator of the Fmk1 pathway and determined its genetic interaction with Msb2. Targeted Δsho1 mutants were generated in wild‐type and Δmsb2 backgrounds to test possible interactions between the two genes. The mutants were examined for hyphal growth under different stress conditions, phosphorylation of the MAPK Fmk1 and an array of Fmk1‐dependent virulence functions. Similar to Msb2, Sho1 was required for the activation of Fmk1 phosphorylation, as well as Fmk1‐dependent gene expression and invasive growth functions, including extracellular pectinolytic activity, cellophane penetration, plant tissue colonization and virulence on tomato plants. Δsho1 mutants were hypersensitive to the cell wall‐perturbing compound Calcofluor White, and this phenotype was exacerbated in the Δmsb2 Δsho1 double mutant. These results highlight that Sho1 and Msb2 have partially overlapping functions upstream of the Fmk1 MAPK cascade, to promote invasive growth and plant infection, as well as cell wall integrity, in F. oxysporum.     

Relations between MAPK phosphorylation and ABA level in <Emphasis Type="Italic">Malus sieversii</Emphasis> under water stress

A correlation between protein kinase phosphorylation and ABA level was studied in Malus sieversii (Ledeb.) Roem. seedlings under water stress. The seedlings were treated with PEG 6000 for imitation of water stress, and the MAPK activity and ABA content in each treatment were then determined. We demonstrated that the increase in the activities of the total protein kinase (TPK) and mitogen-activated protein kinase (MAPK) after treatment with 20% PEG 6000 appeared to result in a high level of ABA. MAPK activity accounted for 76.8% of TPK activity. The activity peaks of TPK and MAPK preceded the highest level of ABA accumulation. It is interesting that the ABA level in roots and leaves of seedlings pretreated with 2 × 10⁻² mM exogenous ABA for 20 min following treatment by 20% PEG 6000 was much higher than that of seedlings treated with exogenous ABA only. We analyzed the influence of MAPK inhibitor ITU (5-iodotubercidin) on ABA accumulation in the seedlings of M. sieversii under water stress and showed that 1 μM ITU significantly decreased the ABA level induced by a water loss. However, the phosphoesterase inhibitor PAO (phenylarisine oxide) enhanced ABA accumulation, indicating that the phosphorylated MAPK was correlated to ABA synthesis. Together, these results suggest that MAPK phosphorylation played an important role in ABA accumulation under water stress, and MAPK might mediate the signal transduction of ABA synthesis.     

The phosphorylation status of T522 modulates tissue‐specific functions of SIRT1 in energy metabolism in mice

SIRT1, the most conserved mammalian NAD⁺‐dependent protein deacetylase, is an important metabolic regulator. However, the mechanisms by which SIRT1 is regulated in vivo remain unclear. Here, we report that phosphorylation modification of T522 on SIRT1 is crucial for tissue‐specific regulation of SIRT1 activity in mice. Dephosphorylation of T522 is critical for repression of its activity during adipogenesis. The phospho‐T522 level is reduced during adipogenesis. Knocking‐in a constitutive T522 phosphorylation mimic activates the β‐catenin/GATA3 pathway, repressing PPARγ signaling, impairing differentiation of white adipocytes, and ameliorating high‐fat diet‐induced dyslipidemia in mice. In contrast, phosphorylation of T522 is crucial for activation of hepatic SIRT1 in response to over‐nutrition. Hepatic SIRT1 is hyperphosphorylated at T522 upon high‐fat diet feeding. Knocking‐in a SIRT1 mutant defective in T522 phosphorylation disrupts hepatic fatty acid oxidation, resulting in hepatic steatosis after high‐fat diet feeding. In addition, the T522 dephosphorylation mimic impairs systemic energy metabolism. Our findings unveil an important link between environmental cues, SIRT1 phosphorylation, and energy homeostasis and demonstrate that the phosphorylation of T522 is a critical element in tissue‐specific regulation of SIRT1 activity in vivo.     

Negative feedback and ultrasensitivity can bring about oscillations in the mitogen-activated protein kinase cascades.

Functional organization of signal transduction into protein phosphorylation cascades, such as the mitogen-activated protein kinase (MAPK) cascades, greatly enhances the sensitivity of cellular targets to external stimuli. The sensitivity increases multiplicatively with the number of cascade levels, so that a tiny change in a stimulus results in a large change in the response, the phenomenon referred to as ultrasensitivity. In a variety of cell types, the MAPK cascades are imbedded in long feedback loops, positive or negative, depending on whether the terminal kinase stimulates or inhibits the activation of the initial level. Here we demonstrate that a negative feedback loop combined with intrinsic ultrasensitivity of the MAPK cascade can bring about sustained oscillations in MAPK phosphorylation. Based on recent kinetic data on the MAPK cascades, we predict that the period of oscillations can range from minutes to hours. The phosphorylation level can vary between the base level and almost 100% of the total protein. The oscillations of the phosphorylation cascades and slow protein diffusion in the cytoplasm can lead to intracellular waves of phospho-proteins.     

The Arabidopsis CERK1‐associated kinase PBL27 connects chitin perception to MAPK activation

Perception of microbe‐associated molecular patterns by host cell surface pattern recognition receptors (PRRs) triggers the intracellular activation of mitogen‐activated protein kinase (MAPK) cascades. However, it is not known how PRRs transmit immune signals to MAPK cascades in plants. Here, we identify a complete phospho‐signaling transduction pathway from PRR‐mediated pathogen recognition to MAPK activation in plants. We found that the receptor‐like cytoplasmic kinase PBL27 connects the chitin receptor complex CERK1‐LYK5 and a MAPK cascade. PBL27 interacts with both CERK1 and the MAPK kinase kinase MAPKKK5 at the plasma membrane. Knockout mutants of MAPKKK5 compromise chitin‐induced MAPK activation and disease resistance to Alternaria brassicicola. PBL27 phosphorylates MAPKKK5 in vitro, which is enhanced by phosphorylation of PBL27 by CERK1. The chitin perception induces disassociation between PBL27 and MAPKKK5 in vivo. Furthermore, genetic evidence suggests that phosphorylation of MAPKKK5 by PBL27 is essential for chitin‐induced MAPK activation in plants. These data indicate that PBL27 is the MAPKKK kinase that provides the missing link between the cell surface chitin receptor and the intracellular MAPK cascade in plants.     

Deletion of dop in Mycobacterium smegmatis abolishes pupylation of protein substrates in vivo

Proteasome‐bearing bacteria make use of a ubiquitin‐like modification pathway to target proteins for proteasomal turnover. In a process termed pupylation, proteasomal substrates are covalently modified with the small protein Pup that serves as a degradation signal. Pup is attached to substrate proteins by action of PafA. Prior to its attachment, Pup needs to undergo deamidation at its C‐terminal residue, converting glutamine to glutamate. This step is catalysed in vitro by Dop. In order to characterize Dop activity in vivo, we generated a dop deletion mutant in Mycobacterium smegmatis. In the Δdop strain, pupylation is severely impaired and the steady‐state levels of two known proteasomal substrates are drastically increased. Pupylation can be re‐established by complementing the mutant with either DopWt or a Pup variant carrying a glutamate at its ultimate C‐terminal position (PupGGE). Our data show that Pup is deamidated by Dop in vivo and that likely Dop alone is responsible for this activity. Furthermore, we demonstrate that a putative N‐terminal ATP‐binding motif is crucial for catalysis, as a single point mutation (E10A) in this motif abolishes Dop activity both in vivo and in vitro.     

Serotonin activation of the ERK pathway in Hermissenda: contribution of calcium-dependent protein kinase C

The mitogen‐activated protein kinase (MAPK) cascade is an important contributor to synaptic plasticity and learning in both vertebrates and invertebrates. In the nudibranch mollusk Hermissenda, phosphorylation and activation of the extracellular signal‐regulated protein kinase (ERK), a key member of a MAPK cascade, is produced by one‐trial and multitrial Pavlovian conditioning. Several signal transduction pathways that are activated by 5‐hydroxytryptamine (5‐HT) and may contribute to conditioning have been identified in type B photoreceptors. However, the regulation of ERK activity by ‘upstream’ signaling molecules has not been previously investigated in Hermissenda. In the present study we examined the role of protein kinase C (PKC) in the serotonin (5‐HT) activation of the ERK pathway. The phorbol ester TPA produced an increase in ERK phosphorylation that was blocked by the PKC inhibitors GF109203X or Gö6976. TPA‐dependent ERK phosphorylation was also blocked by the MEK1 inhibitors PD098059 or U0126. The increased phosphorylation of ERK by 5‐HT was reduced but not blocked by pretreatment with the calcium chelator BAPTA‐AM or pretreatment with Gö6976 or GF109203X. These results indicate that Ca²⁺‐dependent PKC activation contributes to ERK phosphorylation, although a PKC‐independent pathway is also involved in 5‐HT‐dependent ERK phosphorylation and activation.     

Neuronal p38α mediates age‐associated neural stem cell exhaustion and cognitive decline

Neuronal activity regulates cognition and neural stem cell (NSC) function. The molecular pathways limiting neuronal activity during aging remain largely unknown. In this work, we show that p38MAPK activity increases in neurons with age. By using mice expressing p38α‐lox and CamkII‐Cre alleles (p38α?‐N), we demonstrate that genetic deletion of p38α in neurons suffices to reduce age‐associated elevation of p38MAPK activity, neuronal loss and cognitive decline. Moreover, aged p38α?‐N mice present elevated numbers of NSCs in the hippocampus and the subventricular zone. These results reveal novel roles for neuronal p38MAPK in age‐associated NSC exhaustion and cognitive decline.     

ATR and MKP1 play distinct roles in response to UV‐B stress in Arabidopsis

Ultraviolet‐B (UV‐B) stress activates MAP kinases (MAPKs) MPK3 and MPK6 in Arabidopsis. MAPK activity must be tightly controlled in order to ensure an appropriate cellular outcome. MAPK phosphatases (MKPs) effectively control MAPKs by dephosphorylation of phosphothreonine and phosphotyrosine in their activation loops. Arabidopsis MKP1 is an important regulator of MPK3 and MPK6, and mkp1 knockout mutants are hypersensitive to UV‐B stress, which is associated with reduced inactivation of MPK3 and MPK6. Here, we demonstrate that MPK3 and MPK6 are hyperactivated in response to UV‐B in plants that are deficient in photorepair, suggesting that UV‐damaged DNA is a trigger of MAPK signaling. This is not due to a block in replication, as, in contrast to atr, the mkp1 mutant is not hypersensitive to the replication‐inhibiting drug hydroxyurea, hydroxyurea does not activate MPK3 and MPK6, and atr is not impaired in MPK3 and MPK6 activation in response to UV‐B. We further show that mkp1 leaves and roots are UV‐B hypersensitive, whereas atr is mainly affected at the root level. Tolerance to UV‐B stress has been previously associated with stem cell removal and CYCB1;1 accumulation. Although UV‐B‐induced stem cell death and CYCB1;1 expression are not altered in mkp1 roots, CYCB1;1 expression is reduced in mkp1 leaves. We conclude that the MKP1 and ATR pathways operate in parallel, with primary roles for ATR in roots and MKP1 in leaves.     

Phosphoenolpyruvate phosphotransferase system regulates detection and processing of the quorum sensing signal autoinducer-2

Autoinducer‐2 (AI‐2) a signal produced by a range of phylogenetically distant microorganisms, enables inter‐species cell–cell communication and regulates many bacterial phenotypes. Certain bacteria can interfere with AI‐2‐regulated behaviours of neighbouring species by internalizing AI‐2 using the Lsr transport system (encoded by the lsr operon). AI‐2 imported by the Lsr is phosphorylated by the LsrK kinase and AI‐2‐phosphate is the inducer of the lsr operon. Here we show that in Escherichia coli the phosphoenolpyruvate phosphotransferase system (PTS) is required for Lsr activation and is essential for AI‐2 internalization. Although the phosphorylation state of Enzyme I of PTS is important for this regulation, LsrK is necessary for the phosphorylation of AI‐2, indicating that AI‐2 is not phosphorylated by PTS. Our results suggest that AI‐2 internalization is initiated by a PTS‐dependent mechanism, which provides sufficient intracellular AI‐2 to relieve repression of the lsr operon and, thus induce depletion of AI‐2 from the extracellular environment. The fact that AI‐2 internalization is not only controlled by the community‐dependent accumulation of AI‐2, but also depends on the phosphorylation state of PTS suggests that E. coli can integrate information on the availability of substrates with external communal information to control quorum sensing and its interference.