Characterization of an Arabidopsis cDNA encoding a subunit of serine palmitoyltransferase, the initial enzyme in sphingolipid biosynthesis.

Serine palmitoyltransferase (SPT; EC 2.3.1.50) catalyzes the condensation of serine with palmitoyl-CoA to form 3-ketosphinganine in the first step of de novo sphingolipid biosynthesis. In this study, we describe the cloning and functional characterization of a cDNA from Arabidopsis thaliana encoding the LCB2 subunit of SPT. The Arabidopsis LCB2 (AtLCB2) cDNA contains an open reading frame of 1,467 nucleotides, encoding 489 amino acids. The predicted polypeptide contains three transmembrane helices and a highly conserved motif involved in pyridoxal phosphate binding. Expression of this open reading frame in the Saccharomyces cerevisiae mutant strains defective in SPT activity resulted in the expression of a significant level of sphinganine, suggesting that AtLCB2 cDNA encodes SPT. Southern blot analysis and inspection of the complete Arabidopsis genome sequence database suggest that there is a second LCB2-like gene in Arabidopsis. Expression of a green fluorescent protein (GFP) fusion product in suspension-cultured tobacco BY-2 cells showed that AtLCB2 is localized to the endoplasmic reticulum. AtLCB2 cDNA may be used to study how sphingolipid synthesis is regulated in higher plants.     

Loss-of-function mutations and inducible RNAi suppression of Arabidopsis LCB2 genes reveal the critical role of sphingolipids in gametophytic and sporophytic cell viability

Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis, and downregulation of this enzyme provides a means for exploring sphingolipid function in cells. We have previously demonstrated that Arabidopsis SPT requires LCB1 and LCB2 subunits for activity, as is the case in other eukaryotes. In this study, we show that Arabidopsis has two genes ( AtLCB2a and AtLCB2b ) that encode functional isoforms of the LCB2 subunit. No alterations in sphingolipid content or growth were observed in T-DNA mutants for either gene, but homozygous double mutants were not recoverable, suggesting that these genes are functionally redundant. Reciprocal crosses conducted with Atlcb2a and Atlcb2b mutants indicated that lethality is associated primarily with the inability to transmit the lcb2 null genotype through the haploid pollen. Consistent with this, approximately 50% of the pollen obtained from plants homozygous for a mutation in one gene and heterozygous for a mutation in the second gene arrested during transition from uni-nucleate microspore to bicellular pollen. Ultrastructural analyses revealed that these pollen grains contained aberrant endomembranes and lacked an intine layer. To examine sphingolipid function in sporophytic cells, Arabidopsis lines were generated that allowed inducible RNAi silencing of AtLCB2b in an Atlcb2a mutant background. Studies conducted with these lines demonstrated that sphingolipids are essential throughout plant development, and that lethality resulting from LCB2 silencing in seedlings could be partially rescued by supplying exogenous long-chain bases. Overall, these studies provide insights into the genetic and biochemical properties of SPT and sphingolipid function in Arabidopsis.     

Purification of the serine palmitoyltransferase complex responsible for sphingoid base synthesis by using affinity peptide chromatography techniques

Serine palmitoyltransferase (SPT), a membrane-bound enzyme of the endoplasmic reticulum, catalyzes the condensation of palmitoyl coenzyme A (CoA) and L-serine to produce 3-ketodihydrosphingosine. This enzyme contains at least two different subunits, named the LCB1 and LCB2 proteins. In the present study, we expressed a FLAG- and His(6) peptide-tagged version of the hamster LCB1 protein in a Chinese hamster ovary cell mutant strain lacking the endogenous LCB1 subunit and purified SPT from the cells near to homogeneity by affinity peptide chromatography. The endogenous LCB2 protein was co-purified with the tagged LCB1 protein in purification of SPT. In various aspects, including optimum pH, acyl-CoA specificity, and sphingofungin sensitivity, the activity of purified SPT was consistent with the activity detected in lysates of wild-type Chinese hamster ovary cells. The optimum concentration of palmitoyl-CoA for 3-ketodihydrosphingosine formation by purified SPT was approximately 25 microM, and the apparent K(m) of L-serine was 0.28 mM. Competition analysis of the SPT reaction with various serine analogs showed that all of the amino, carboxyl, and hydroxyl groups of L-serine were responsible for the substrate recognition of the enzyme. SDS-polyacrylamide gel electrophoretic analysis of purified SPT, together with immunoprecipitation analysis of metabolically labeled LCB proteins, strongly suggested that the SPT enzyme consisted of the LCB1 and LCB2 proteins with a stoichiometry of 1:1.     

The ORMs interact with transmembrane domain 1 of Lcb1 and regulate serine palmitoyltransferase oligomerization,activity and localization

Serine palmitoyltransferase (SPT), an endoplasmic reticulum-localized membrane enzymecomposed of acatalytic LCB1/LCB2 heterodimer and a small activating subunit (Tsc3 in yeast; ssSPTs in mammals), is negatively regulated by the evolutionarily conserved family of proteins known as the ORMs. In yeast, SPT, the ORMs, and the PI4P phosphatase Sac1, copurify in the “SPOTs” complex. However, neither the mechanism of ORM inhibition of SPT nor details of the interactions of the ORMs and Sac1 with SPT are known. Here we report that the first transmembrane domain (TMD1) of Lcb1 is required for ORM binding to SPT. Loss of binding is not due to altered membrane topology of Lcb1 since replacing TMD1 with a heterologous TMD restores membrane topology but not ORM binding. TMD1 deletion also eliminates ORM-dependent formation of SPT oligomers as assessed by co-immunoprecipitation assays and in vivo imaging. Expression of ORMs lacking derepressive phosphorylation sites results in constitutive SPT oligomerization, while phosphomimetic ORMs fail to induce oligomerization under any conditions. Significantly, when LCB1–RFP and LCB1ΔTMD1–GFP were coexpressed, more LCB1ΔTMD1–GFP was in the peripheral ER, suggesting ORM regulation is partially accomplished by SPT redistribution. Tsc3 deletion does not abolish ORM inhibition of SPT, indicating the ORMs do not simply prevent activation by Tsc3. Binding of Sac1 to SPT requires Tsc3, but not the ORMs, and Sac1 does not influence ORM-mediated oligomerization of SPT. Finally, yeast mutants lacking ORM regulation of SPT require the LCB-P lyase Dpl1 to maintain long-chain bases at sublethal levels.     

Localization,topology, and function of the LCB1 subunit of serine palmitoyltransferase in mammalian cells

Serine palmitoyltransferase (SPT), the enzyme catalyzing the initial step in the biosynthesis of sphingolipids, comprises two different subunits, LCB1 and LCB2. LCB1 has a single highly hydrophobic domain near the N terminus. Chinese hamster ovary cell mutant LY-B cells are defective in SPT activity because of the lack of expression of an endogenous LCB1 subunit. Stable expression of LCB1 having an epitope tag at either the N or C terminus restored SPT activity of LY-B cells, suggesting that the epitope tag did not affect the localization or topology of LCB1. Indirect immunostaining showed that the N- and C-terminal epitopes are oriented toward the lumenal and cytosol side, respectively, at the endoplasmic reticulum. Interestingly, there was far less LCB2 in LY-B cells than in wild-type cells, and the amount of LCB2 in LY-B cells was restored to the wild-type level by transfection with LCB1 cDNA. In addition, overproduction of the LCB2 subunit required co-overproduction of the LCB1 subunit. These results indicated that the LCB1 subunit is most likely an integral protein having a single transmembrane domain with a lumenal orientation of its N terminus in the endoplasmic reticulum and that the LCB1 subunit is indispensable for the maintenance of the LCB2 subunit in mammalian cells.     

The essential nature of sphingolipids in plants as revealed by the functional identification and characterization of the Arabidopsis LCB1 subunit of serine palmitoyltransferase

Serine palmitoyltransferase (SPT) catalyzes the first step of sphingolipid biosynthesis. In yeast and mammalian cells, SPT is a heterodimer that consists of LCB1 and LCB2 subunits, which together form the active site of this enzyme. We show that the predicted gene for Arabidopsis thaliana LCB1 encodes a genuine subunit of SPT that rescues the sphingolipid long-chain base auxotrophy of Saccharomyces cerevisiae SPT mutants when coexpressed with Arabidopsis LCB2. In addition, homozygous T-DNA insertion mutants for At LCB1 were not recoverable, but viability was restored by complementation with the wild-type At LCB1 gene. Furthermore, partial RNA interference (RNAi) suppression of At LCB1 expression was accompanied by a marked reduction in plant size that resulted primarily from reduced cell expansion. Sphingolipid content on a weight basis was not changed significantly in the RNAi suppression plants, suggesting that plants compensate for the downregulation of sphingolipid synthesis by reduced growth. At LCB1 RNAi suppression plants also displayed altered leaf morphology and increases in relative amounts of saturated sphingolipid long-chain bases. These results demonstrate that plant SPT is a heteromeric enzyme and that sphingolipids are essential components of plant cells and contribute to growth and development.     

De novo ceramide accumulation due to inhibition of its conversion to complex sphingolipids in apoptotic photosensitized cells

The oxidative stress induced by photodynamic therapy (PDT) with the photosensitizer phthalocyanine 4 is accompanied by increases in ceramide mass. To assess the regulation of de novo sphingolipid metabolism during PDT-induced apoptosis, Jurkat human T lymphoma and Chinese hamster ovary cells were labeled with [14C]serine, a substrate of serine palmitoyltransferase (SPT), the enzyme catalyzing the initial step in the sphingolipid biosynthesis. A substantial elevation in [14C]ceramide with a concomitant decrease in [14C]sphingomyelin was detected. The labeling of [14C]ceramide was completely abrogated by the SPT inhibitor ISP-1. In addition, ISP-1 partly suppressed PDT-induced apoptosis. Pulse-chase experiments showed that the contribution of sphingomyelin degradation to PDT-initiated increase in de novo ceramide was absent or minor. PDT had no effect on either mRNA amounts of the SPT subunits LCB1 and LCB2, LCB1 protein expression, or SPT activity in Jurkat cells. Moreover in Chinese hamster ovary cells LCB1 protein underwent substantial photodestruction, and SPT activity was profoundly inhibited after treatment. We next examined whether PDT affects conversion of ceramide to complex sphingolipids. Sphingomyelin synthase, as well as glucosylceramide synthase, was inactivated by PDT in both cell lines in a dose-dependent manner. These results are the first to show that in the absence of SPT up-regulation PDT induces accumulation of de novo ceramide by inhibiting its conversion to complex sphingolipids.     

A water-soluble homodimeric serine palmitoyltransferase from Sphingomonas paucimobilis EY2395T strain. Purification, characterization, cloning, and overproduction

Serine palmitoyltransferase (SPT, EC ) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of l-serine and palmitoyl-coenzyme A to 3-ketodihydrosphingosine. We found that the Gram-negative obligatory aerobic bacteria Sphingomonas paucimobilis EY2395(T) have significant SPT activity and purified SPT to homogeneity. This enzyme is a water-soluble homodimeric protein unlike eukaryotic enzymes, known as heterodimers composed of tightly membrane-bound subunits, named LCB1 and LCB2. The purified SPT shows an absorption spectrum characteristic of a pyridoxal 5'-phosphate-dependent enzyme. The substrate specificity of the Sphingomonas SPT is less strict than the SPT complex from Chinese hamster ovary cells. We isolated the SPT gene encoding 420 amino acid residues (M(r) 45,041) and succeeded in overproducing the SPT protein in Escherichia coli, in which the product amounted to about 10-20% of the total protein of the cell extract. Sphingomonas SPT shows about 30% homology with the enzymes of the alpha-oxamine synthase family, and amino acid residues supposed to be involved in catalysis are conserved. The recombinant SPT was catalytically and spectrophotometrically indistinguishable from the native enzyme. This is the first successful overproduction of an active enzyme in the sphingolipid biosynthetic pathway. Sphingomonas SPT is a prototype of the eukaryotic enzyme and would be a useful model to elucidate the reaction mechanism of SPT.     

Characterization of enzymatic synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae: mutant strains exhibiting long-chain-base auxotrophy are deficient in serine palmitoyltransferase activity.

We have begun a biochemical-genetic analysis of the synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae and found evidence for the occurrence of serine palmitoyltransferase (SPT) and 3-ketosphinganine reductase, enzymes that catalyze the initial steps of the pathway in other organisms. SPT activity was demonstrated in vitro with crude membrane preparations from S. cerevisiae as judged by the formation of radiolabeled 3-ketosphinganine from the condensation of palmitoyl-coenzyme A (CoA) with radiolabeled serine. Shorter (C12 and C14) and longer (C18) acyl-CoAs sustain significant SPT activity, a result consistent with the finding of both C18 and C20 long-chain bases in the organism. Three products of the long-chain-base synthetic pathway, 3-ketosphinganine, erythrosphinganine, and phytosphingosine, neither directly inhibited the reaction in vitro nor affected the specific activity of the enzyme when these bases were included in the culture medium of wild-type cells. Thus, no evidence for either feedback inhibition or repression of enzyme synthesis could be found with these putative effectors. Mutant strains of S. cerevisiae that require a sphingolipid long-chain base for growth fall into two genetic complementation groups, LCB1 and LCB2. Membrane preparations from both lcb1 and lcb2 mutant strains exhibited negligible SPT activity when tested in vitro. Step 2 of the long-chain-base synthetic pathway was demonstrated by the stereospecific NADPH-dependent reduction of 3-ketosphinganine to erythrosphinganine. Membranes isolated from wild-type cells and from an lcb1 mutant exhibited substantial 3-ketosphinganine reductase activity. We conclude that the Lcb- phenotype of these mutants results from a missing or defective SPT, an activity controlled by both the LCB1 and LCB2 genes. These results and earlier work from this laboratory establish that SPT plays an essential role in sphingolipid synthesis in S. cerevisiae.     

Expression of a novel marine viral single-chain serine palmitoyltransferase and construction of yeast and mammalian single-chain chimera

The genus Coccolithovirus is a recently discovered group of viruses that infect the globally important marine calcifying microalga Emiliania huxleyi. Surprisingly, the viral genome contains a cluster of putative sphingolipid biosynthetic genes not found in other viral genus. To address the role of these genes in viral pathogenesis, the ehv050 gene predicted to encode a serine palmitoyltransferase (SPT), the first and rate-limiting enzyme of sphingolipid biosynthesis, was expressed and characterized in Saccharomyces cerevisiae. We show that the encoded protein is indeed a fully functional, endoplasmic reticulum-localized, single-chain SPT. In eukaryotes SPT is a heterodimer comprised of long chain base 1 (LCB1) and LCB2 subunits. Sequence alignment and mutational analysis showed that the N-terminal domain of the viral protein most closely resembled the LCB2 subunit and the C-terminal domain most closely resembled the LCB1 subunit. Regardless of whether the viral protein was expressed as a single polypeptide or as two independent domains, it exhibited an unusual preference for myristoyl-CoA rather than palmitoyl-CoA. This preference was reflected by the increased presence of C16-sphingoid bases in yeast cells expressing the viral protein. The occurrence of a single-chain SPT suggested to us that it might be possible to create other fusion SPTs with unique properties. Remarkably, when the two subunits of the yeast SPT were thus expressed, the single-chain chimera was functional and displayed a novel substrate preference. This suggests that expression of other multisubunit membrane proteins as single-chain chimera could provide a powerful approach to the characterization of integral membrane proteins.     

Topological and Functional Characterization of the ssSPTs,Small Activating Subunits of Serine Palmitoyltransferase

The topological and functional organization of the two isoforms of the small subunits of human serine palmitoyltransferase (hssSPTs) that activate the catalytic hLCB1/hLCB2 heterodimer was investigated. A variety of experimental approaches placed the N termini of the ssSPTs in the cytosol, their C termini in the lumen, and showed that they contain a single transmembrane domain. Deletion analysis revealed that the ability to activate the heterodimer is contained in a conserved 33-amino acid core domain that has the same membrane topology as the full-length protein. In combination with analysis of isoform chimera and site-directed mutagenesis, a single amino acid residue in this core (Met²⁵ in ssSPTa and Val²⁵ in ssSPTb) was identified which confers specificity for palmitoyl- or stearoyl-CoA, respectively, in both yeast and mammalian cells. This same residue also determines which isoform is a better activator of a mutant heterodimer, hLCB1^S331F/hLCB2a, which has increased basal SPT activity and decreased amino acid substrate selectivity. This suggests that the role of the ssSPTs is to increase SPT activity without compromising substrate specificity. In addition, the observation that the C-terminal domains of ssSPTa and ssSPTb, which are highly conserved within each subfamily but are the most divergent regions between isoform subfamilies, are not required for activation of the heterodimer or for acyl-CoA selectivity suggests that the ssSPTs have additional roles that remain to be discovered.     

A Disease-causing Mutation in the Active Site of Serine Palmitoyltransferase Causes Catalytic Promiscuity

The autosomal dominant peripheral sensory neuropathy HSAN1 results from mutations in the LCB1 subunit of serine palmitoyltransferase (SPT). Serum from patients and transgenic mice expressing a disease-causing mutation (C133W) contain elevated levels of 1-deoxysphinganine (1-deoxySa), which presumably arise from inappropriate condensation of alanine with palmitoyl-CoA. Mutant heterodimeric SPT is catalytically inactive. However, mutant heterotrimeric SPT has ∼10–20% of wild-type activity and supports growth of yeast cells lacking endogenous SPT. In addition, long chain base profiling revealed the synthesis of significantly more 1-deoxySa in yeast and mammalian cells expressing the heterotrimeric mutant enzyme than in cells expressing wild-type enzyme. Wild-type and mutant enzymes had similar affinities for serine. Surprisingly, the enzymes also had similar affinities for alanine, indicating that the major affect of the C133W mutation is to enhance activation of alanine for condensation with the acyl-CoA substrate. In vivo synthesis of 1-deoxySa by the mutant enzyme was proportional to the ratio of alanine to serine in the growth media, suggesting that this ratio can be used to modulate the relative synthesis of sphinganine and 1-deoxySa. By expressing SPT as a single-chain fusion protein to ensure stoichiometric expression of all three subunits, we showed that GADD153, a marker for endoplasmic reticulum stress, was significantly elevated in cells expressing mutant heterotrimers. GADD153 was also elevated in cells treated with 1-deoxySa. Taken together, these data indicate that the HSAN1 mutations perturb the active site of SPT resulting in a gain of function that is responsible for the HSAN1 phenotype.     

MPK6, sphinganine and the LCB2a gene from serine palmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in Arabidopsis

Long chain bases (LCBs) are sphingolipid intermediates acting as second messengers in programmed cell death (PCD) in plants. Most of the molecular and cellular features of this signaling function remain unknown. We induced PCD conditions in Arabidopsis thaliana seedlings and analyzed LCB accumulation kinetics, cell ultrastructure and phenotypes in serine palmitoyltransferase (spt), mitogen-activated protein kinase (mpk), mitogen-activated protein phosphatase (mkp1) and lcb-hydroxylase (sbh) mutants. The lcb2a-1 mutant was unable to mount an effective PCD in response to fumonisin B1 (FB1), revealing that the LCB2a gene is essential for the induction of PCD. The accumulation kinetics of LCBs in wild-type (WT) and lcb2a-1 plants and reconstitution experiments with sphinganine indicated that this LCB was primarily responsible for PCD elicitation. The resistance of the null mpk6 mutant to manifest PCD on FB1 and sphinganine addition and the failure to show resistance on pathogen infection and MPK6 activation by FB1 and LCBs indicated that MPK6 mediates PCD downstream of LCBs. This work describes MPK6 as a novel transducer in the pathway leading to LCB-induced PCD in Arabidopsis, and reveals that sphinganine and the LCB2a gene are required in a PCD process that operates as one of the more effective strategies used as defense against pathogens in plants.     

Serine palmitoyltransferase, a key enzyme for de novo synthesis of sphingolipids, is essential for male gametophyte development in Arabidopsis

Sphingolipids are important signaling molecules involved in various cellular activities. De novo sphingolipid synthesis is initiated by a rate-limiting enzyme, serine palmitoyltransferase (SPT), a heterodimer consisting of LONG-CHAIN BASE1 (LCB1) and LCB2 subunits. A mutation in the Arabidopsis thaliana LCB1 gene, lcb1-1, was found to cause embryo lethality. However, the underpinning molecular and cellular mechanisms remain largely unclear. Here, we report the identification of the fumonisin B(1) resistant11-2 (fbr11-2) mutant, an allele of lcb1-1. The fbr11-2 mutation, most likely an allele stronger than lcb1-1, was transmitted only through female gametophytes and caused the formation of abortive microspores. During the second pollen mitosis, fbr11-2 initiated apoptotic cell death in binucleated microspores characteristic of nuclear DNA fragmentation, followed by cytoplasm shrinkage and organelle degeneration at the trinucleated stage. In addition, a double mutant with T-DNA insertions in two homologous LCB2 genes showed a phenotype similar to fbr11-2. Consistent with these observations, the FBR11/LCB1 expression was confined in microspores during microgametogenesis. These results suggest that SPT-modulated programmed cell death plays an important role in the regulation of male gametophyte development.     

Arabidopsis LIP5, a Positive Regulator of Multivesicular Body Biogenesis,Is a Critical Target of Pathogen-Responsive MAPK Cascade in Plant Basal Defense

Multivesicular bodies (MVBs) play essential roles in many cellular processes. The MVB pathway requires reversible membrane association of the endosomal sorting complexes required for transports (ESCRTs) for sustained protein trafficking. Membrane dissociation of ESCRTs is catalyzed by the AAA ATPase SKD1, which is stimulated by LYST-INTERACTING PROTEIN 5 (LIP5). We report here that LIP5 is a target of pathogen-responsive mitogen-activated protein kinases (MPKs) and plays a critical role in plant basal resistance. Arabidopsis LIP5 interacts with MPK6 and MPK3 and is phosphorylated in vitro by activated MPK3 and MPK6 and in vivo upon expression of MPK3/6-activating NtMEK2^DD and pathogen infection. Disruption of LIP5 has little effects on flg22-, salicylic acid-induced defense responses but compromises basal resistance to Pseudomonas syringae. The critical role of LIP5 in plant basal resistance is dependent on its ability to interact with SKD1. Mutation of MPK phosphorylation sites in LIP5 does not affect interaction with SKD1 but reduces the stability and compromises the ability to complement the lip5 mutant phenotypes. Using the membrane-selective FM1–43 dye and transmission electron microscopy, we demonstrated that pathogen infection increases formation of both intracellular MVBs and exosome-like paramural vesicles situated between the plasma membrane and the cell wall in a largely LIP5-dependent manner. These results indicate that the MVB pathway is positively regulated by pathogen-responsive MPK3/6 through LIP5 phosphorylation and plays a critical role in plant immune system likely through relocalization of defense-related molecules.     

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.     

Murine protein serine/threonine kinase 38 activates apoptosis signal-regulating kinase 1 via Thr 838 phosphorylation

Murine protein serine/threonine kinase 38 (MPK38) is a member of the AMP-activated protein kinase-related serine/threonine kinase family that plays an important role in various cellular processes, including cell cycle, signaling pathways, and self-renewal of stem cells. Here we demonstrate a functional association between MPK38 and apoptosis signal-regulating kinase 1 (ASK1). The physical association between MPK38 and ASK1 was mediated through their carboxyl-terminal regulatory domains and was increased by H(2)O(2) or tumor necrosis factor alpha treatment. The use of kinase-dead MPK38 and ASK1 mutants revealed that MPK38-ASK1 complex formation was dependent on the activities of both kinases. Ectopic expression of wild-type MPK38, but not kinase-dead MPK38, stimulated ASK1 activity by Thr(838) phosphorylation and enhanced ASK1-mediated signaling to both JNK and p38 kinases. However, the phosphorylation of MKK6 and p38 by MPK38 was not detectable. In addition, MPK38-mediated ASK1 activation was induced through the increased interaction between ASK1 and its substrate MKK3. MPK38 also stimulated H(2)O(2)-mediated apoptosis by enhancing the ASK1 activity through Thr(838) phosphorylation. These results suggest that MPK38 physically interacts with ASK1 in vivo and acts as a positive upstream regulator of ASK1.     

Arabidopsis RNA polymerase II C-terminal domain phosphatase-like 1 targets mitogen-activated protein kinase cascades to suppress plant immunity

Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in plant defense against phytopathogens downstream of immune receptor complexes. The amplitude and duration of MAPK activation must be strictly controlled, but the underlying mechanism remains unclear. Here, we identified Arabidopsis CPL1 (C-terminal domain phosphatase-like 1) as a negative regulator of microbe-associated molecular pattern (MAMP)-triggered immunity via a forward-genetic screen. Disruption of CPL1 significantly enhanced plant resistance to Pseudomonas pathogens induced by the bacterial peptide flg22. Furthermore, flg22-induced MPK3/MPK4/MPK6 phosphorylation was dramatically elevated in cpl1 mutants but severely impaired in CPL1 overexpression lines, suggesting that CPL1 might interfere with flg22-induced MAPK activation. Indeed, CPL1 directly interacted with MPK3 and MPK6, as well as the upstream MKK4 and MKK5. A firefly luciferase-based complementation assay indicated that the interaction between MKK4/MKK5 and MPK3/MPK6 was significantly reduced in the presence of CPL1. These results suggest that CPL1 plays a novel regulatory role in suppressing MAMP-induced MAPK cascade activation and MAMP-triggered immunity to bacterial pathogens.     

The Arabidopsis protein kinase Pto-interacting 1-4 is a common target of the oxidative signal-inducible 1 and mitogen-activated protein kinases

In Arabidopsis thaliana, the serine/threonine protein kinase oxidative signal-inducible 1 (OXI1), mediates oxidative stress signalling. Its activity is required for full activation of the mitogen-activated protein kinases (MAPKs), MPK3 and MPK6, in response to oxidative stress. In addition, the serine/threonine protein kinase Pto-interacting 1-2 (PTI1-2) has been positioned downstream from OXI1, but whether PTI1-2 signals through MAPK cascades is unclear. Using a yeast two-hybrid screen we show that OXI1 also interacts with PTI1-4. OXI1 and PTI1-4 are stress-responsive genes and are expressed in the same tissues. Therefore, studies were undertaken to determine whether PTI1-4 is positioned in the OXI1/MAPK signalling pathway. The interaction between OXI1 and PTI1-4 was confirmed by using in vivo co-immunoprecipitation experiments. OXI1 and PTI1-4 were substrates of MPK3 and MPK6 in vitro. Although no direct interaction was detected between OXI1 and MPK3 or MPK6, in vitro binding studies showed interactions between MPK3 or MPK6 with PTI1-4. In addition, PTI1-4 and MPK6 were found in vivo in the same protein complex. These results demonstrate that PTI1-4 signals via OXI1 and MPK6 signalling cascades.