Stress-induced protein phosphatase 2C is a negative regulator of a mitogen-activated protein kinase

Protein phosphatases of type 2C (PP2Cs) play important roles in eukaryotic signal transduction. In contrast to other eukaryotes, plants such as Arabidopsis have an unusually large group of 69 different PP2C genes. At present, little is known about the functions and substrates of plant PP2Cs. We have previously shown that MP2C, a wound-induced alfalfa PP2C, is a negative regulator of mitogen-activated protein kinase (MAPK) pathways in yeast and plants. In this report, we provide evidence that alfalfa salt stress-inducible MAPK (SIMK) and stress-activated MAPK (SAMK) are activated by wounding and that MP2C is a MAPK phosphatase that directly inactivates SIMK but not the wound-activated MAPK, SAMK. SIMK is inactivated through threonine dephosphorylation of the pTEpY motif, which is essential for MAPK activity. Mutant analysis indicated that inactivation of SIMK depends on the catalytic activity of MP2C. A comparison of MP2C with two other PP2Cs, ABI2 and AtP2CHA, revealed that although all three phosphatases have similar activities toward casein as a substrate, only MP2C is able to dephosphorylate and inactivate SIMK. In agreement with the notion that MP2C interacts directly with SIMK, the MAPK was identified as an interacting partner of MP2C in a yeast two-hybrid screen. MP2C can be immunoprecipitated with SIMK in a complex in vivo and shows direct binding to SIMK in vitro in protein interaction assays. Wound-induced MP2C expression correlates with the time window when SIMK is inactivated, corroborating the notion that MP2C is involved in resetting the SIMK signaling pathway.     

In vivo and in vitro activation of temperature-responsive plant map kinases

Alfalfa cells possess two temperature-responsive Mitogen-Activated Protein Kinases (MAPKs), SAMK (Stress-Activated MAP Kinase) activated at 4 degrees C and HAMK (Heat shock-Activated MAP Kinase) activated at 37 degrees C. Both are inactive at 25 degrees C. We show here that SAMK is activated when cells are transferred from 37 degrees C to 25 degrees C, and HAMK is activated when cells are transferred from 4 degrees C to 25 degrees C. Moreover, we show that heat activation of HAMK also occurs in cell-free extracts. We conclude that (i) SAMK or HAMK activation does not require a particular temperature but a relative temperature shift, and (ii) that either HAMK itself or one or more of its upstream activators can sense temperature change directly.     

Identification and characterization of an ABA‐activated MAP kinase cascade in Arabidopsis thaliana

Abscisic acid (ABA) is a major phytohormone involved in important stress‐related and developmental plant processes. Recent phosphoproteomic analyses revealed a large set of ABA‐triggered phosphoproteins as putative mitogen‐activated protein kinase (MAPK) targets, although the evidence for MAPKs involved in ABA signalling is still scarce. Here, we identified and reconstituted in vivo a complete ABA‐activated MAPK cascade, composed of the MAP3Ks MAP3K17/18, the MAP2K MKK3 and the four C group MAPKs MPK1/2/7/14. In planta, we show that ABA activation of MPK7 is blocked in mkk3‐1 and map3k17mapk3k18 plants. Coherently, both mutants exhibit hypersensitivity to ABA and altered expression of a set of ABA‐dependent genes. A genetic analysis further reveals that this MAPK cascade is activated by the PYR/PYL/RCAR‐SnRK2‐PP2C ABA core signalling module through protein synthesis of the MAP3Ks, unveiling an atypical mechanism for MAPK activation in eukaryotes. Our work provides evidence for a role of an ABA‐induced MAPK pathway in plant stress signalling.     

Phosphodiesterase MoPdeH targets MoMck1 of the conserved mitogen‐activated protein (MAP) kinase signalling pathway to regulate cell wall integrity in rice blast fungus Magnaporthe oryzae

In the rice blast fungus Magnaporthe oryzae, the high‐affinity cyclic adenosine monophosphate (cAMP) phosphodiesterase MoPdeH is important not only for cAMP signalling and pathogenicity, but also for cell wall integrity (CWI) maintenance through an unknown mechanism. By utilizing affinity purification, we found that MoPdeH interacts with MoMck1, one of the components of the mitogen‐activated protein (MAP) kinase cascade that regulates CWI. Overexpression of MoMCK1 suppressed defects in autolysis and pathogenicity of the ΔMopdeH mutant, although partially, suggesting that MoPdeH plays a critical role in CWI maintenance mediated by the MAP kinase pathway. We found that MoMck1 and two other MAP kinase cascade components, MoMkk1 and MoMps1, modulate intracellular cAMP levels by regulating the expression of MoPDEH through a feedback loop. In addition, disruption of MoMKK1 resulted in less aerial hyphal formation, defective asexual development and attenuated pathogenicity. Moreover, MoMkk1 plays a role in the response to osmotic stress via regulation of MoOsm1 phosphorylation levels, whereas endoplasmic reticulum (ER) stress enhances MoMps1 phosphorylation and loss of the MAP kinase cascade component affects the unfolded protein response (UPR) pathway. Taken together, our findings demonstrate that MoPdeH functions upstream of the MoMck1–MoMkk1–MoMps1 MAP kinase pathway to regulate CWI, and that MoPdeH also mediates crosstalk between the cAMP signalling pathway, the osmotic sensing high osmolarity glycerol (HOG) pathway and the dithiothreitol (DTT)‐induced UPR pathway in M. oryzae.     

Dissection of the HOG pathway activated by hydrogen peroxide in Saccharomyces cerevisiae

Cells usually cope with oxidative stress by activating signal transduction pathways. In the budding yeast Sacchromyces cerevisiae, the high osmolarity glycerol (HOG) pathway has long been implicated in transducing the oxidative stress‐induced signal, but the underlying mechanisms are not well defined. Based on phosphorylation of the mitogen‐activated protein kinase (MAPK) Hog1, we reveal that the signal from hydrogen peroxide (H₂O₂) flows through Ssk1, the response regulator of the two‐component system of the HOG pathway. Downstream signal transduction into the HOG MAPK cascade requires the MAP kinase kinase kinase (MAP3K) Ssk2 but not its paralog Ssk22 or another MAP3K Ste11 of the pathway, culminating in Hog1 phosphorylation via the MAP2K Pbs2. When overexpressed, Ssk2 is also activated in an Ssk1‐independent manner. Unlike in mammals, H₂O₂ does not cause endoplasmic reticulum stress, which can activate Hog1 through the conventional unfolded protein response. Hog1 activated by H₂O₂ is retained in the cytoplasm, but is still able to activate the cAMP‐ or stress‐responsive elements by unknown mechanisms.     

Isolation and characterization of a <Emphasis Type="Italic">Paramecium</Emphasis> cDNA clone encoding a putative serine/threonine protein kinase

Mitogen-activated protein (MAP) kinases, a closely related family of protein kinases, are involved in cell cycle regulation and differentiation in yeast and human cells. They have not been documented in ciliates. We used PCR to amplify DNA sequences of a ciliated protozoan—Paramecium caudatum—using primers corresponding to amino acid sequences that are common to MAP kinases. We isolated and sequenced one putative MAP kinase-like serine/threonine kinase cDNA from P. caudatum. This cDNA, called pcstk1 (Paramecium caudatum Serine/Threonine Kinase 1) shared approximately 35% amino acid identity with MAP kinases from yeast. MAP kinases are activated by phosphorylation of specific threonine and tyrosine residues. These two amino acid residues are conserved in the PCSTK1 sequence at positions Thr 159 and Tyr 161. The PSTAIRE motif, which is characteristic of the CDK2 gene family, cannot be found in ORF of PCSTK1. The highest homology score was to human STK9, which contains MAP type kinase domains. Comparisons of expression level have shown that pcstk1 is expressed equally in cells at different stages (sexual and asexual). We discussed the possibility, as in other organisms, that a family of MAP kinase genes exists in P. caudatum.     

Heavy metal stress. Activation of distinct mitogen-activated protein kinase pathways by copper and cadmium

Excessive amounts of heavy metals adversely affect plant growth and development. Whereas some regions naturally contain high levels of heavy metals, anthropogenic release of heavy metals into the environment continuously increases soil contamination. The presence of elevated levels of heavy metal ions triggers a wide range of cellular responses including changes in gene expression and synthesis of metal-detoxifying peptides. To elucidate signal transduction events leading to the cellular response to heavy metal stress we analyzed protein phosphorylation induced by elevated levels of copper and cadmium ions as examples for heavy metals with different physiochemical properties and functions. Exposure of alfalfa (Medicago sativa) seedlings to excess copper or cadmium ions activated four distinct mitogen-activated protein kinases (MAPKs): SIMK, MMK2, MMK3, and SAMK. Comparison of the kinetics of MAPK activation revealed that SIMK, MMK2, MMK3, and SAMK are very rapidly activated by copper ions, while cadmium ions induced delayed MAPK activation. In protoplasts, the MAPK kinase SIMKK specifically mediated activation of SIMK and SAMK but not of MMK2 and MMK3. Moreover, SIMKK only conveyed MAPK activation by CuCl(2) but not by CdCl(2). These results suggest that plants respond to heavy metal stress by induction of several distinct MAPK pathways and that excess amounts of copper and cadmium ions induce different cellular signaling mechanisms in roots.     

MMK2, a novel alfalfa MAP kinase, specifically complements the yeast MPK1 function

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases that are activated in response to a variety of stimuli. Here we report the isolation of an alfalfa cDNA encoding a functional MAP kinase, termedMMK2. The predicted amino acid sequence ofMMK2 shares 65% identity with a previously identified alfalfa MAP kinase, termedMMK1. Both alfalfa cDNA clones encode functional kinases when expressed in bacteria, undergoing autophosphorylation and activation to phosphorylate myelin basic protein in vitro. However, only MMK2 was able to phosphorylate a 39 kDa protein from the detergent-resistant cytoskeleton of carrot cells. The distinctiveness ofMMK2 was further shown by complementation analysis of three different MAP kinase-dependent yeast pathways; this revealed a highly specific replacement of the yeastMPK1 (SLT2) kinase byMMK2, which was found to be dependent on activation by the upstream regulators of the pathway. These results establish the existence of MAP kinases with different characteristics in higher plants, suggesting the possibility that they could mediate different cellular responses.     

Ceramide induces release of mitochondrial proapoptotic proteins in caspase-dependent and -independent manner in HT-29 cells

In this study, the release of mitochondrial proapoptotic intermembrane space proteins induced by ex-ogenous C2-ceramide in human colon carcinoma (HT-29) cell line was investigated. HT-29 cells were treated with 12.5, 25 and 50 μmol/L C2-ceramide in vitro. Flow cytometer was used to detect the mito-chondrial membrane potential (ΔΨm). Subcellular fractions were extracted by Mitochondrial/Cytosol Fractionation Kit after C2-ceramide treatment for 24 h. SDS-PAGE was used to determine the level of cytochrome c (Cyt c), high temperature requirement A2 (HtrA2) and second mitochondrial-derived ac-tivator of caspases (Smac) released from mitochondria, the expression of X-linked inhibitor of apop-tosis protein (XIAP) and caspase-3 for 24 h. The results showed that ΔΨm began to decrease from 6 h after 25 and 50 μmol/L C2-ceramide treatment (P<0.05) and cyclosporin A (CsA) could inhibit the col-lapse of ΔΨm through regulating mitochondrial membrane permeability transition pore. There was no effect of C2-ceramide on the expression of Cyt c, HtrA2 and Smac in the total levels. 12.5, 25 and 50 μmol/L C2-ceramide could induce Cyt c, HtrA2 and Smac to release from mitochondria to cytosol and down-regulate the expression of XIAP (P<0.05). Also there was expression of cleaved caspase-3 with C2-ceramide treatment. After the treatment with caspase inhibitor, C2-ceramide still induced the release of Cyt c and HtrA2, but Smac did not. Therefore, C2-ceramide could induce apoptosis of HT-29 cells through the mitochondria pathway. The release of Cyt c, HtrA2 and Smac from mitochondria did not occur via the same mechanism, the release of Cyt c and HtrA2 was caspase-independent and the re-lease of Smac was caspase-dependent.     

Glycosylation defects activate filamentous growth Kss1 MAPK and inhibit osmoregulatory Hog1 MAPK

The yeast filamentous growth (FG) MAP kinase (MAPK) pathway is activated under poor nutritional conditions. We found that the FG‐specific Kss1 MAPK is activated by a combination of an O‐glycosylation defect caused by disruption of the gene encoding the protein O‐mannosyltransferase Pmt4, and an N‐glycosylation defect induced by tunicamycin. The O‐glycosylated membrane proteins Msb2 and Opy2 are both essential for activating the FG MAPK pathway, but only defective glycosylation of Msb2 activates the FG MAPK pathway. Although the osmoregulatory HOG (high osmolarity glycerol) MAPK pathway and the FG MAPK pathway share almost the entire upstream signalling machinery, osmostress activates only the HOG‐specific Hog1 MAPK. Conversely, we now show that glycosylation defects activate only Kss1, while activated Kss1 and the Ptp2 tyrosine phosphatase inhibit Hog1. In the absence of Kss1 or Ptp2, however, glycosylation defects activate Hog1. When Hog1 is activated by glycosylation defects in ptp2 mutant, Kss1 activation is suppressed by Hog1. Thus, the reciprocal inhibitory loop between Kss1 and Hog1 allows only one or the other of these MAPKs to be stably activated under various stress conditions.     

Endothelial microparticles released by activated protein C protect beta cells through EPCR/PAR1 and annexin A1/FPR2 pathways in islets

Islet transplantation is associated with early ischaemia/reperfusion, localized coagulation and redox‐sensitive endothelial dysfunction. In animal models, islet cytoprotection by activated protein C (aPC) restores islet vascularization and protects graft function, suggesting that aPC triggers various lineages. aPC also prompts the release of endothelial MP that bear EPCR, its specific receptor. Microparticles (MP) are plasma membrane procoagulant vesicles, surrogate markers of stress and cellular effectors. We measured the cytoprotective effects of aPC on endothelial and insulin‐secreting Rin‐m5f β‐cells and its role in autocrine and paracrine MP‐mediated cell crosstalk under conditions of oxidative stress. MP from aPC‐treated primary endothelial (EC) or β‐cells were applied to H₂O₂‐treated Rin‐m5f. aPC activity was measured by enzymatic assay and ROS species by dihydroethidium. The capture of PKH26‐stained MP and the expression of EPCR were probed by fluorescence microscopy and apoptosis by flow cytometry. aPC treatment enhanced both annexin A1 (ANXA1) and PAR‐1 expression in EC and to a lesser extent in β‐cells. MP from aPC‐treated EC (eM_aPC) exhibited high EPCR and annexin A1 content, protected β‐cells, restored insulin secretion and were captured by 80% of β cells in a phosphatidylserine and ANXA1‐dependent mechanism. eMP activated EPCR/PAR‐1 and ANXA1/FPR2‐dependent pathways and up‐regulated the expression of EPCR, and of FPR2/ALX, the ANXA1 receptor. Cytoprotection was confirmed in H₂O₂‐treated rat islets with increased viability (62% versus 48% H₂O₂), reduced apoptosis and preserved insulin secretion in response to glucose elevation (16 versus 5 ng/ml insulin per 10 islets). MP may prove a promising therapeutic tool in the protection of transplanted islets.     

The MAP1B-LC1/UBE2L3 complex catalyzes degradation of cell surface CaV2.2 channels

We reported recently a new mechanism by which the neuronal N-type Ca²⁺ (Ca_V2.2) channel expression may be regulated by ubiquitination. This mechanism involves the interaction between the channel and the light chain (LC1) of the microtubule associated protein B (MAP1B). We also showed that MAP1B-LC1 could interact with the ubiquitin-conjugating E2 enzyme UBE2L3 and that the ubiquitination/degradation mechanism triggered by MAP1B-LC1 could be prevented by inhibiting the ubiquitin-proteasome proteolytic pathway. We now report that MAP1B-LC1 can interact with the 2 main variants of the Ca_V2.2 channels (Ca_V2.2e37a and Ca_V2.2e37b) and that the MAP1B-LC1-mediated regulation most likely involves an internalization of the channels via a dynamin and clathrin-dependent pathway. In addition, here we propose that this novel mechanism of Ca_V channel regulation might be conserved among N-type and P/Q-type channels.     

The MAP1B-LC1/UBE2L3 complex catalyzes degradation of cell surface CaV2.2 channels

We reported recently a new mechanism by which the neuronal N-type Ca²⁺ (Ca_V2.2) channel expression may be regulated by ubiquitination. This mechanism involves the interaction between the channel and the light chain (LC1) of the microtubule associated protein B (MAP1B). We also showed that MAP1B-LC1 could interact with the ubiquitin-conjugating E2 enzyme UBE2L3 and that the ubiquitination/degradation mechanism triggered by MAP1B-LC1 could be prevented by inhibiting the ubiquitin-proteasome proteolytic pathway. We now report that MAP1B-LC1 can interact with the 2 main variants of the Ca_V2.2 channels (Ca_V2.2e37a and Ca_V2.2e37b) and that the MAP1B-LC1-mediated regulation most likely involves an internalization of the channels via a dynamin and clathrin-dependent pathway. In addition, here we propose that this novel mechanism of Ca_V channel regulation might be conserved among N-type and P/Q-type channels.     

C4 photosynthetic modifications in the evolutionary transition from land to water in aquatic grasses

Cladistic analysis supports the conclusion that the Orcuttieae tribe of C₄ grasses reflect evolution from a terrestrial ancestry into seasonal pools. All nine species in the tribe exhibit adaptations to the aquatic environment, evident in the structural characteristics of the juvenile foliage, which persist submerged for 1–3 months prior to metamorphosis to the terrestrial foliage. Aquatic leaves of the least derived or basal genus Neostapfia have few morphological and anatomical characteristics specialized to the aquatic environment and have retained full expression of the C₄ pathway, including Kranz anatomy. Orcuttia species have many derived characteristics and are more specialized to the aquatic environment. These latter species germinate earlier in the season and persist in the submerged stage longer than Neostapfia and evidence from the literature indicates length of submergence is positively correlated with fitness components. Aquatic leaves of Orcuttia species lack Kranz or PCR bundle sheath anatomy, yet ¹⁴C-pulse chase studies indicate >95% malate + aspartate as the initial products of photosynthesis and these products turn over rapidly to phosphorylated sugars, indicating a tight coupling of the C₄ and C₃ cycles. Presence of the C₄ pathway is further supported by enzymological data. Contemporary dogma that Kranz anatomy is a sine qua non for operation of the C₄ pathway is contradicted by the patterns in Orcuttia; however, it is unknown whether the pathway acts as a CO₂ concentrating mechanism in these aquatic plants. Received: 12 June 1997 / Accepted: 10 February 1997     

Salviae Miltiorrhizae BGE Radix Increases Rat Striatal K+-Stimulated Dopamine Release and Activates the Dopamine Release with Protection Against Hydrogen Peroxide-Induced Injury in Rat Pheochromocytoma PC12 Cells

The present study investigated the effect of the medicinal plant Salviae miltiorrhizae radix (SMR) on dopaminergic neurotransmission in comparison with amphetamine. The effect of SM (0.1 g/ml) on K⁺ (20 mM)-stimulated dopamine (DA) release from rat striatal slices was compared with amphetamine (10⁻⁴ M). Amphetamine and SMR significantly increased K⁺-stimulated DA release (P<0.001) from rat striatal slices when compared with K⁺-stimulated alone. On the other hand, to examine whether in vitro SMR treatment induces DA release in PC12 cells, the role of protein kinases has been investigated in the induction of the SMR-mediated events by using inhibitors of protein kinase C (PKC), mitogen activated protein kinase (MAP kinase) or protein kinase A (PKA). PKC inhibitors chelerythrine (50 and 100 nM), Ro31-8220 (100 nM) and the MAP kinase inhibitor, PD98059 (20 μM) inhibited the ability of SMR to elicit the SMR-stimulated DA release. The direct-acting PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA, 100 nM) mimicked the ability of SMR to elicit DA release. On the contrary, a selective PKA inhibitor, 50 μM Rp-8-Br-cAMP, blocked the development of SMR-stimulated DA release. The results demonstrated that SMR may stimulate DA release and that SMR-induced increases in MAP kinase and PKC are important for induction of the enhancement in transporter-mediated DA release and PKA was also required for the enhancement in SMR-stimulated DA release. SMR treatment (0.1–10 μg/ml) to the hydrogen peroxide (H₂O₂)-treated PC12 cells activated the enzyme activities such as catalase, superoxide dismutase and glutathione peroxidase, and decreased the malondialdehyde level, indicating that SMR has also protective effects against free radical-induced cell toxicity. Therefore, the mechanism by which SMR induces the enhancement in SMR-stimulated DA release is apparent. It remains to be determined whether the effect of SMR on DA function is important in its therapeutic use in the treatment of drug addiction.     

Differential activation of four specific MAPK pathways by distinct elicitors

Plant cells respond to elicitors by inducing a variety of defense responses. Some of these reactions are dependent on the activity of protein kinases. Recently, mitogen-activated protein kinases (MAPKs) have been identified to be activated by fungal and bacterial elicitors as well as by pathogen infection. In gel kinase assays of alfalfa cells treated with yeast cell wall-derived elicitor (YE) revealed that 44- and 46-kDa MAPKs are rapidly and transiently activated. Immunokinase assays with specific MAPK antibodies revealed that YE mainly activated the 46-kDa SIMK and the 44-kDa MMK3 and to a lesser extent the 44-kDa MMK2 and SAMK. When cells were treated with chemically defined elicitors potentially contained in the YE (chitin and N-acetylglucosamine oligomers, beta-glucan, and ergosterol), the four MAPKs were found to be activated to different levels and with different kinetics. Whereas SIMK and SAMK have been found to be activated by a number of diverse stimuli, MMK3 is activated during mitosis and was therefore assumed to participate in cell division (). No physiological process could be associated with MMK2 activity so far. This is the first report that MMK2 and MMK3 can be activated by external stimuli. Overall, our findings indicate that plant cells can sense different cues of a given microorganism through the activation of multiple MAPKs.     

A homologue of the MAP/ERK family of protein kinase genes is expressed in vegetative and in female reproductive organs of Petunia hybrida

The mitogen activated protein (MAP) kinase pathway of eukaryotes is stimulated by many growth factors and is required for the integration of multiple cellular signals. In order to study the function of MAP kinases during plant ovule development we have synthesized a Petunia hybrida ovule-specific cDNA library and screened for MAP protein kinase-related sequences using a DNA probe obtained by PCR. A full-length cDNA clone was identified (PMEK for Petunia hybrida MAP/ERK-related protein kinase) and shown to encode a protein related to the family of MAP/ERK protein kinases. Southern blot analysis showed that PMEK is a member of a small multigene family in P. hybrida. The cDNA codes for a protein (PMEK1) of 44.4 kDa with an overall sequence identity of 44% to the products of the mammalian ERK/MAP kinase gene, and the budding yeast KSS1 and FUS3 genes. PMEK1 displays 96 and 80% identity respectively with the tobacco NTF3 and Arabidopsis ATMPK1 kinases, and only 50% to the more distantly related plant MAP kinase MsERK1 from alfalfa. The two phosphorylation sites found in the loop between subdomain VII and VIII in all the other MAP kinases are also present in PMEK1. RNA gel blot and RT-PCR analyses demonstrated that PMEK1 is expressed in vegetative organs and preferentially accumulated in female reproductive organs of P. hybrida. In situ hybridization experiments showed that in the reproductive organs PMEK1 is expressed only in the ovary and not in the stamen.