Functional analysis of Arabidopsis immune-related MAPKs uncovers a role for MPK3 as negative regulator of inducible defences

Background : Mitogen-activated protein kinases (MAPKs) are key regulators of immune responses in animals and plants. In Arabidopsis, perception of microbe-associated molecular patterns (MAMPs) activates the MAPKs MPK3, MPK4 and MPK6. Increasing information depicts the molecular events activated by MAMPs in plants, but the specific and cooperative contributions of the MAPKs in these signalling events are largely unclear.[br/] Results: In this work, we analyse the behaviour of MPK3, MPK4 and MPK6 mutants in early and late immune responses triggered by the MAMP flg22 from bacterial flagellin. A genome-wide transcriptome analysis reveals that 36% of the flg22-upregulated genes and 68% of the flg22-downregulated genes are affected in at least one MAPK mutant. So far MPK4 was considered as a negative regulator of immunity, whereas MPK3 and MPK6 were believed to play partially redundant positive functions in defence.[br/] Our work reveals that MPK4 is required for the regulation of approximately 50% of flg22-induced genes and we identify a negative role for MPK3 in regulating defence gene expression, flg22-induced salicylic acid accumulation and disease resistance to Pseudomonas syringae. Among the MAPK-dependent genes, 27% of flg22-upregulated genes and 76% of flg22-downregulated genes require two or three MAPKs for their regulation. The flg22-induced MAPK activities are differentially regulated in MPK3 and MPK6 mutants, both in amplitude and duration, revealing a highly interdependent network.[br/] Conclusions : These data reveal a new set of distinct functions for MPK3, MPK4 and MPK6 and indicate that the plant immune signalling network is choreographed through the interplay of these three interwoven MAPK pathways

Nf1 RasGAP inhibition of LIMK2 mediates a new cross-talk between Ras and Rho pathways.

BACKGROUND: Ras GTPases mediate numerous biological processes through their ability to cycle between an inactive GDP-bound form and an active GTP-bound form. Guanine nucleotide exchange factors (GEFs) favor the formation of the active Ras-GTP, whereas GTPase activating proteins (GAPs) promote the formation of inactive Ras-GDP. Numerous studies have established complex signaling cross-talks between Ras GTPases and other members of the superfamily of small GTPases. GEFs were thought to play a major role in these cross-talks. However, recently GAPs were also shown to play crucial roles in these processes. Among RasGAPs, Nf1 is of special interest. Nf1 is responsible for the genetic disease Neurofibromatosis type I, and recent data strongly suggest that this RasGAP connects different signaling pathways. METHODOLOGY/PRINCIPAL FINDINGS: In order to know if the RasGAP Nf1 might play a role in connecting Ras GTPases to other small GTPase pathways, we systematically looked for new partners of Nf1, by performing a yeast two-hybrid screening on its SecPH domain. LIMK2, a major kinase of the Rho/ROCK/LIMK2/cofilin pathway, was identified in this screening. We confirmed this interaction by co-immunoprecipitation experiments, and further characterized it. We also demonstrated its specificity: the close related homolog of LIMK2, LIMK1, does not interact with the SecPH domain of Nf1. We then showed that SecPH partially inhibits the kinase activity of LIMK2 on cofilin. Our results furthermore suggest a precise mechanism for this inhibition: in fact, SecPH would specifically prevent LIMK2 activation by ROCK, its upstream regulator. CONCLUSIONS/SIGNIFICANCE: Although previous data had already connected Nf1 to actin cytoskeleton dynamics, our study provides for the first time possible detailed molecular requirements of this involvement. Nf1/LIMK2 interaction and inhibition allows to directly connect neurofibromatosis type I to actin cytoskeleton remodeling, and provides evidence that the RasGAP Nf1 mediates a new cross-talk between Ras and Rho signaling pathways within the superfamily of small GTPases

LINGO-1, a protein involved in various neurodevelopmental processes, interacts with neurofibromin (Nf1), the protein responsible for neurofibromatosis type I: molecular studies and functional implications

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Physical interaction between neurofibromin and serotonin 5-HT6receptor promotes receptor constitutive activity

International audienceActive G protein-coupled receptor (GPCR) conformations not only are promoted by agonists but also occur in their absence, leading to constitutive activity. Association of GPCRs with intracellular protein partners might be one of the mechanisms underlying GPCR constitutive activity. Here, we show that serotonin 5 hydroxytryptamine 6 (5-HT6) receptor constitutively activates the Gs/adenylyl cyclase pathway in various cell types, including neurons. Constitutive activity is strongly reduced by silencing expression of the Ras-GTPase activating protein (Ras-GAP) neurofibromin, a 5-HT6 receptor partner. Neurofibromin is a multidomain protein encoded by the NF1 gene, the mutation of which causes Neurofibromatosis type 1 (NF1), a genetic disorder characterized by multiple benign and malignant nervous system tumors and cognitive deficits. Disrupting association of 5-HT6 receptor with neurofibromin Pleckstrin Homology (PH) domain also inhibits receptor constitutive activity, and PH domain expression rescues 5-HT6 receptor-operated cAMP signaling in neurofibromin-deficient cells. Furthermore, PH domains carrying mutations identified in NF1 patients that prevent interaction with the 5-HT6 receptor fail to rescue receptor constitutive activity in neurofibromin-depleted cells. Further supporting a role of neurofibromin in agonist-independent Gs signaling elicited by native receptors, the phosphorylation of cAMP-responsive element-binding protein (CREB) is strongly decreased in prefrontal cortex of Nf1+/− mice compared with WT mice. Moreover, systemic administration of a 5-HT6 receptor inverse agonist reduces CREB phosphorylation in prefrontal cortex of WT mice but not Nf1+/− mice. Collectively, these findings suggest that disrupting 5-HT6 receptor–neurofibromin interaction prevents agonist-independent 5-HT6 receptor-operated cAMP signaling in prefrontal cortex, an effect that might underlie neuronal abnormalities in NF1 patients

New in-capillary electrophoretic kinase assays to evaluate inhibitors of the PI3k/Akt/mTOR signaling pathway

International audienceHuman kinases are one of the most promising targets for cancer therapy. Methods able to measure the effects of drugs on these cell agents remain crucial for biologists and medicinal chemists. The current work therefore sought to develop an in-capillary enzymatic assay based on capillary electrophoresis (CE) to evaluate the inhibition of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt), and the mammalian target of rapamycin (mTOR). These kinases belong to the same signaling pathway PI3K/Akt/mTOR. For this proposal, the capillary was used as a nanoreactor in which a few nanoliters of the kinase, its substrate, adenosine triphosphate (ATP), and the potent inhibitor were separately injected. A transverse diffusion of laminar flow profiles (TDLFP) approach was employed to mix the reactants. Adenosine diphosphate (ADP ) was detected online at 254 nm. The CE assay was first developed on the α isoform of PI3K. It was compared to five commercial kits frequently used to assess kinase inhibition, based on time-resolved fluorescence resonance energy transfer (TR-FRET) and bioluminescence. Each assay was evaluated in terms of sensitivity (S/B), reproducibility (Z'), and variability (r (2)). This CE method was easily extended to assay the inhibition of the β, γ, and δ isoforms of PI3K, and of the other kinases of the pathway, Akt1 and mTOR, since it is based on in-capillary mixing by TDLFP and on ADP quantification by simple UV absorption. This work shows for the first time the evaluation of inhibitors of the kinases of the PI3K/Akt/mTOR pathway using a common in-capillary CE assay. Several inhibitors with a wide range of affinity toward these enzymes were tested

SecPH partially inhibits cofilin phosphorylation by LIMK2, but not by LIMK1.

<p><i>A</i>. <i>Actin cytoskeleton organisation</i>. HeLa cells were cotransfected with pcDNA3, LIMK2 and SecPH or its parental empty plasmid, p3XFlag. Cells were fixed and stained with phalloidin, or anti-HA or anti-flag antibodies. <i>B. Inhibition of LIMK2 cofilin phosphorylation by SecPH.</i> Cells were cotransfected with LIMK2 and SecPH or Galectin-3 (a non-specific control protein). Immunoprecipitated HA-LIMK2 and GST-cofilin were used in the kinase assay. The kinase activity on cofilin of immunoprecipitated HA-LIMK2 from cells cotransfected with Galectin-3 was taken as 1.0. Each value represents the mean ± SE (standard error) of four independent experiments. Statistical significance was determined relative to control using one-way ANOVA (* p<0.05). The HA-immunoprecipitates were also submitted to HA-immunoblotting and to coomassie blue staining. Lysates were submitted to flag-immunoblotting. <i>C. Dose dependent inhibition of LIMK2 cofilin phosphorylation by SecPH.</i> Cells were transfected with LIMK2 and either SecPH or Galectin-3. SecPH and Galectin-3 cell lysates were immunoprecipitated with anti-flag beads, beads were then eluted flag peptide. Immunoprecipitated HA-LIMK2 and GST-cofilin were used for kinase assay and were incubated with increasing amount of immunoprecipitated SecPH or Galectin-3 (0, 6, 12, 18 ul respectively). The kinase activity on cofilin of immunoprecipitated HA-LIMK2 with no addition of immunoprecipitated SecPH or Galectin-3 was taken as 1.0. Each value represents the mean ± SE of four independent experiments. Statistical significance was determined relative to control using one-way ANOVA (*** p<0.0001). Immunoprecipitates were also subjected to immunoblotting and to coomassie blue staining. <i>D. SecPH does not inhibit cofilin phosphorylation by LIMK1.</i> Cells were transfected either with SecPH or with LIMK1. SecPH cell lysates were immunoprecipitated with anti-flag beads, beads were then eluted with flag peptide. Immunoprecipitated HA-LIMK1 was used for kinase assay and was incubated with increasing amount of immunoprecipitated SecPH (0, 6, 12, 18 ul respectively). The kinase activity on cofilin of immunoprecipitated HA-LIMK1 with no addition of immunoprecipitated SecPH was taken as 1.0. Each value represents the mean ± SE of two independent experiments. Immunoprecipitates were also subjected to immunoblotting and to coomassie blue staining.</p

Physical interaction between neurofibromin and serotonin 5-HT 6

International audienceActive G protein-coupled receptor (GPCR) conformations not only are promoted by agonists but also occur in their absence, leading to constitutive activity. Association of GPCRs with intracellular protein partners might be one of the mechanisms underlying GPCR constitutive activity. Here, we show that serotonin 5 hydroxytryptamine 6 (5-HT6) receptor constitutively activates the Gs/adenylyl cyclase pathway in various cell types, including neurons. Constitutive activity is strongly reduced by silencing expression of the Ras-GTPase activating protein (Ras-GAP) neurofibromin, a 5-HT6 receptor partner. Neurofibromin is a multidomain protein encoded by the NF1 gene, the mutation of which causes Neurofibromatosis type 1 (NF1), a genetic disorder characterized by multiple benign and malignant nervous system tumors and cognitive deficits. Disrupting association of 5-HT6 receptor with neurofibromin Pleckstrin Homology (PH) domain also inhibits receptor constitutive activity, and PH domain expression rescues 5-HT6 receptor-operated cAMP signaling in neurofibromin-deficient cells. Furthermore, PH domains carrying mutations identified in NF1 patients that prevent interaction with the 5-HT6 receptor fail to rescue receptor constitutive activity in neurofibromin-depleted cells. Further supporting a role of neurofibromin in agonist-independent Gs signaling elicited by native receptors, the phosphorylation of cAMP-responsive element-binding protein (CREB) is strongly decreased in prefrontal cortex of Nf1+/− mice compared with WT mice. Moreover, systemic administration of a 5-HT6 receptor inverse agonist reduces CREB phosphorylation in prefrontal cortex of WT mice but not Nf1+/− mice. Collectively, these findings suggest that disrupting 5-HT6 receptor–neurofibromin interaction prevents agonist-independent 5-HT6 receptor-operated cAMP signaling in prefrontal cortex, an effect that might underlie neuronal abnormalities in NF1 patients

SecPH affects ROCK kinase activity specifically with respect to LIMK2.

<p><i>A. MLC phosphorylation is not affected by SecPH.</i> Same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047283#pone-0047283-g002" target="_blank">Figure 2B</a>, except 0.5 µg of MLC were added in the kinase reaction mixture. Each value represents the mean ± SE (standard error) of two independent experiments. B. <i>MLC phosphorylation by ROCK-1 is not affected by SecPH.</i> Cells were cotransfected with ROCK1 and SecPH or Galectin-3. Immunoprecipitated c-Myc-ROCK1 was used for the kinase assay in the presence of recombinant LIMK2 or MLC (0.5 µg each). The kinase activity on LIMK2/MLC of immunoprecipitated c-Myc-ROCK from cells cotransfected with Galectin-3 was taken as 1.0. Each value represents the mean ± SE (standard error) of four independent experiments. Immunoprecipitates were also submitted to c-Myc-immunoblotting and lysates to flag-immunoblotting.</p

Interaction between LIMK2 and the SecPH domain of Nf1.

<p><i>A. Diagram of Nf1.</i> GRD (GAP related domain) responsible for the main known function of Nf1 is depicted as well as SecPH, the region used for the two-hybrid screening. <i>B. Interaction revealed by the two-hybrid screening.</i> L40 cells transformed with pBTM116-TBD were mated with Y187 cells transformed with the empty plasmid pACT2 (as a negative control) or pACT2-TFS1 (as a positive control, as demonstrated by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047283#pone.0047283-Chautard1" target="_blank">[32]</a>). L40 cells transformed with pBTM116-SecPH were mated with Y187 cells transformed with pACT2-LIMK1 or pACT2-LIMK2. After mating on YPD, the resultant diploids were selected on a SD-LW medium. The interaction between the LexA fusion proteins encoded by the pACT2 plasmids and the Gal4 fusion proteins encoded by pBTM116 plasmids was tested by checking the growth of diploids on a SD-LWH media containing 3AT (1 mM) and their ability to cleave X-gal (1 mM) thereby attesting the production of β-galactosidase. <i>C. Interaction in HEK-293 transfected cells.</i> HEK-293 cells were cotransfected with either HA-LIMK2 or HA-LIMK1 and flag-SecPH or its parental empty plasmid (p3XFlag). Cell lysates and anti-flag immunoprecipitation eluates were analyzed by immunobloting. <i>D. Immunoprecipitated LIMK2 interacts with recombinant 6His-SecPH.</i> HEK-293 cells were transfected with HA-LIMK2 or its parental empty plasmid, pcDNA3. The corresponding cell lysates were immunoprecipitated with anti-HA beads. Beads were then incubated with 6His-SecPH in lysis buffer. Anti-HA immunoprecipitates were analyzed by immunobloting. <i>E. Transfected LIMK2 interacts with endogenous Nf1.</i> Cells were transfected with HA-LIMK2 or its parental empty plasmid, pcDNA3. Lysates and anti-HA immunoprecipitates were analyzed by immunobloting. <i>F. Endogenous LIMK2 interacts with endogenous Nf1</i> Anti-Nf1 immunoprecipitates from HEK-293 were analyzed by immunobloting. <i>G. Domains of LIMK2 involved in its interaction with SecPH</i>. Top. Schematic diagram of LIMK2 and its various fragments designed for this study. Bottom. Cells were cotransfected with SecPH and one of the domains of LIMK2. Lysates and anti-flag immunoprecipitates were analyzed by immunobloting.</p

Mechanism of SecPH inhibition of LIMK2 kinase activity.

<p><i>A. SecPH does not prevent LIMK2 from interacting with ROCK1</i>. Cells were cotransfected with HA-LIMK2, ROCK1 and SecPH or its empty parental plasmid. Lysates and anti-HA immunoprecipitates were subjected to immunoblotting. <i>B. SecPH affects LIMK2 phosphorylation.</i> Same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047283#pone-0047283-g002" target="_blank">Figure 2B</a>. Statistical significance was determined relative to control using one-way ANOVA (*** p<0.0001). <i>C. Inhibition of LIMK2 cofilin phosphorylation by SecPH in the presence of ROCK1.</i> Cells were cotransfected with ROCK1, LIMK2 and SecPH or Galectin-3 (a non-specific control protein). Immunoprecipitated HA-LIMK2 and GST-cofilin were used for the kinase assay. The kinase activity on cofilin of immunoprecipitated HA-LIMK2 from cells cotransfected with Galectin-3 was taken as 1.0. Each value represents the mean ± SE (standard error) of four independent experiments. The HA-immunoprecipitates were also submitted to immunoblotting and to coomassie blue staining. Lysates were also submitted to flag-immunoblotting. <i>D. SecPH affects LIMK2 T505 phosphorylation by ROCK1.</i> Cells were cotransfected with either LIMK2-TA or LIMK2-WT and SecPH or Galectin-3 (a non-specific control protein). Lysates were subjected to immunoblotting.</p