De novo ceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells. Dependence on protein phosphatase-1.

Previous studies have demonstrated that several splice variants are derived from both the caspase 9 and Bcl-x genes in which the Bcl-x splice variant, Bcl-x(L) and the caspase 9 splice variant, caspase 9b, inhibit apoptosis in contrast to the pro-apoptotic splice variants, Bcl-x(s) and caspase 9. In a recent study, we showed that ceramide induces the dephosphorylation of SR proteins, a family of protein factors that regulate alternative splicing. In this study, the regulation of the alternative processing of pre-mRNA of both caspase 9 and Bcl-x(L) was examined in response to ceramide. Treatment of A549 lung adenocarcinoma cells with cell-permeable ceramide, D-e-C(6) ceramide, down-regulated the levels of Bcl-x(L) and caspase 9b mRNA and immunoreactive protein with a concomitant increase in the mRNA and immunoreactive protein levels of Bcl-x(s) and caspase 9 in a dose- and time-dependent manner. Pretreatment with calyculin A (5 nm), an inhibitor of protein phosphatase-1 (PP1) and protein phosphatase 2A (PP2A) blocked ceramide-induced alternative splicing in contrast to okadaic acid (10 nm), a specific inhibitor of PP2A at this concentrations in cells, demonstrating a PP1-mediated mechanism. A role for endogenous ceramide in regulating the alternative splicing of caspase 9 and Bcl-x was demonstrated using the chemotherapeutic agent, gemcitabine. Treatment of A549 cells with gemcitabine (1 microm) increased ceramide levels 3-fold via the de novo sphingolipid pathway as determined by pulse labeling experiments and inhibition studies with myriocin (50 nm), a specific inhibitor of serine palmitoyltransferase (the first step in de novo synthesis of ceramide). Treatment of A549 cells with gemcitabine down-regulated the levels of Bcl-x(L) and caspase 9b mRNA with a concomitant increase in the mRNA levels of Bcl-x(s) and caspase 9. Again, inhibitors of ceramide synthesis blocked this effect. We also demonstrate that the change in the alternative splicing of caspase 9 and Bcl-x occurred prior to apoptosis following treatment with gemcitabine. Furthermore, doses of D-e-C(6) ceramide that induce the alternative splicing of both caspase 9 and Bcl-x-sensitized A549 cells to daunorubicin. These data demonstrate a role for protein phosphatases 1 (PP1) and endogenous ceramide generated via the de novo pathway in regulating this mechanism. This is the first report on the dynamic regulation of RNA splicing of members of the Bcl-2 and caspase families in response to regulators of apoptosis.     

Arabidopsis casein kinase 1-like 6 contains a microtubule-binding domain and affects the organization of cortical microtubules,

Members of the casein kinase 1 (CK1) family are evolutionarily conserved eukaryotic protein kinases that are involved in various cellular, physiological, and developmental processes in yeast and metazoans, but the biological roles of CK1 members in plants are not well understood. Here, we report that an Arabidopsis (Arabidopsis thaliana) CK1 member named casein kinase 1-like 6 (CKL6) associates with cortical microtubules in vivo and phosphorylates tubulins in vitro. The unique C-terminal domain of CKL6 was shown to contain the signal that allows localization of CKL6 to the cortical microtubules. This domain on its own was sufficient to associate with microtubules in vivo and to bind tubulins in vitro. CKL6 was able to phosphorylate soluble tubulins as well as microtubule polymers, and its endogenous activity was found to associate with a tubulin-enriched subcellular fraction. Two major in vitro phosphorylation sites were mapped to serine-413 and serine-420 of tubulin beta. Ectopic expression of wild-type CKL6 or a kinase-inactive mutant form induced alterations in cortical microtubule organization and anisotropic cell expansion. Collectively, these results demonstrate that CKL6 is a protein kinase containing a novel tubulin-binding domain and plays a role in anisotropic cell growth and shape formation in Arabidopsis through the regulation of microtubule organization, possibly through the phosphorylation of tubulins.     

The gibberellin-induced, cysteine-rich protein GIP2 from Petunia hybrida exhibits in planta antioxidant activity

Numerous GAST-like genes have been identified in various plant species. All code for small proteins with a conserved C-terminal region in which 12 cysteines are located in exactly the same positions. We have previously identified five gibberellin (GA)-induced GAST1-like genes in petunia, GIP1-5. GIP2 is expressed in elongating zones, and its suppression in transgenic petunia plants inhibits stem elongation, suggesting a role for the protein in GA-induced cell growth. However, nothing is known about the biochemical activity of GIP2 or any other GAST-like protein. As all contain putative catalytic disulfide bonds (putative redox-active cysteines), we speculated that they might be involved in redox regulation. Expression analysis of GIP2, GIP4 and GIP5 revealed that they are induced by H(2)O(2). To study whether GIP2 modulates H(2)O(2) levels, we generated transgenic petunia plants expressing GIP2 under the regulation of the ubiquitous CaMV 35S promoter. The transgene reduced H(2)O(2) levels in leaves following wounding. It also reduced the levels of H(2)O(2) in guard cells following osmotic stress and ABA treatments, leading to the suppression of stomatal closure. In addition, the transgene promoted stem and corolla elongation. As reactive oxygen species (ROS) are involved in cell elongation, we suggest that GIP2 affects growth by regulating the levels of ROS. As all known GAST-like proteins contain putative redox-active cysteines, they may all act as antioxidants.     

Involvement of dihydroceramide desaturase in cell cycle progression in human neuroblastoma cells

The role of dihydroceramide desaturase as a key enzyme in the de novo pathway of ceramide generation was investigated in human neuroblastoma cells (SMS-KCNR). A novel assay using water-soluble analogs of dihydroceramide, dihydroceramidoids (D-erythro-dhCCPS analogs), was used to measure desaturase activity in situ. Conversion of D-erythro-2-N-[12'-(1'-pyridinium)-dodecanoyl]-4,5-dihydrosphingosine bromide (C(12)-dhCCPS) to its 4,5-desaturated counterpart, D-erythro-2-N-[12'-(1'-pyridinium)dodecanoyl]sphingosine bromide (C(12)-CCPS), was determined by liquid chromatography/mass spectrometry analysis. The validity of the assay was confirmed using C(8)-cyclopropenylceramide, a competitive inhibitor of dihydroceramide desaturase. A human homolog (DEGS-1) of the Drosophila melanogaster des-1 gene was recently identified and reported to have desaturase activity. Transfection of SMS-KCNR cells with small interfering RNA to DEGS-1 significantly blocked the conversion of C(12)-dhCCPS to C(12)-CCPS. The associated accumulation of endogenous dihydroceramides confirmed DEGS-1 as the main active dihydroceramide desaturase in these cells. The partial loss of DEGS-1 inhibited cell growth, with cell cycle arrest at G(0)/G(1). This was accompanied by a significant decrease in the amount of phosphorylated retinoblastoma protein. This hypophosphorylation was inhibited by tautomycin and not by okadaic acid, suggesting the involvement of protein phosphatase 1. Additionally, we found that treatment of SMS-KCNR cells with fenretinide inhibited desaturase activity in a dose-dependent manner. An increase in dihydroceramides (but not ceramides) paralleled this process as measured by liquid chromatography/mass spectrometry. There were no effects on the mRNA or protein levels of DEGS-1, suggesting that fenretinide acts at the post-translational level as an inhibitor of this enzyme. Tautomycin was also able to block the hypophosphorylation of the retinoblastoma protein observed upon fenretinide treatment. These findings suggest a novel biological function for dihydroceramides.     

Regulation of telomere length by fatty acid elongase 3 in yeast. Involvement of inositol phosphate metabolism and Ku70/80 function

In this study, we investigated the roles of very long-chain fatty acid (VLCFA) synthesis by fatty acid elongase 3 (ELO3) in the regulation of telomere length and life span in the yeast Saccharomyces cerevisiae. Loss of VLCFA synthesis via deletion of ELO3 reduced telomere length, and reconstitution of the expression of wild type ELO3, and not by its mutant with decreased catalytic activity, rescued telomere attrition. Further experiments revealed that alterations of phytoceramide seem to be dispensable for telomere shortening in response to loss of ELO3. Interestingly, telomere shortening in elo3Delta cells was almost completely prevented by deletion of IPK2 or KCS1, which are involved in the generation of inositol phosphates (IP4, IP5, and inositol pyrophosphates). Deletion of IPK1, which generates IP6, however, did not affect regulation of telomere length. Further data also suggested that elo3Delta cells exhibit accelerated chronologic aging, and reduced replicative life span compared with wild type cells, and deletion of KCS1 helped recover these biological defects. Importantly, to determine downstream mechanisms, epistasis experiments were performed, and data indicated that ELO3 and YKU70/80 share a common pathway for the regulation of telomere length. More specifically, chromatin immunoprecipitation assays revealed that the telomere binding and protective function of YKu80p in vivo was reduced in elo3Delta cells, whereas its non-homologues end-joining function was not altered. Deletion of KCS1 in elo3Delta cells recovered the telomere binding and protective function of Ku, consistent with the role of KCS1 mutation in the rescue of telomere length attrition. Thus, these findings provide initial evidence of a possible link between Elo3-dependent VLCFA synthesis, and IP metabolism by KCS1 and IPK2 in the regulation of telomeres, which play important physiological roles in the control of senescence and aging, via a mechanism involving alterations of the telomere-binding/protection function of Ku.     

Plasmodesmal-associated protein kinase in tobacco and Arabidopsis recognizes a subset of non-cell-autonomous proteins

Cell-to-cell communication in plants involves the trafficking of macromolecules through specialized intercellular organelles, termed plasmodesmata. This exchange of proteins and RNA is likely regulated, and a role for protein phosphorylation has been implicated, but specific components remain to be identified. Here, we describe the molecular characterization of a plasmodesmal-associated protein kinase (PAPK). A 34-kD protein, isolated from a plasmodesmal preparation, exhibits calcium-independent kinase activity and displays substrate specificity in that it recognizes a subset of viral and endogenous non-cell-autonomous proteins. This PAPK specifically phosphorylates the C-terminal residues of tobacco mosaic virus movement protein (TMV MP); this posttranslational modification has been shown to affect MP function. Molecular analysis of purified protein established that tobacco (Nicotiana tabacum) PAPK is a member of the casein kinase I family. Subcellular localization studies identified a possible Arabidopsis thaliana PAPK homolog, PAPK1. TMV MP and PAPK1 are colocalized within cross-walls in a pattern consistent with targeting to plasmodesmata. Moreover, Arabidopsis PAPK1 also phosphorylates TMV MP in vitro at its C terminus. These results strongly suggest that Arabidopsis PAPK1 is a close homolog of tobacco PAPK. Thus, PAPK1 represents a novel plant protein kinase that is targeted to plasmodesmata and may play a regulatory role in macromolecular trafficking between plant cells.     

Plant phenylacetaldehyde synthase is a bifunctional homotetrameric enzyme that catalyzes phenylalanine decarboxylation and oxidation

We have isolated and characterized Petunia hybrida cv. Mitchell phenylacetaldehyde synthase (PAAS), which catalyzes the formation of phenylacetaldehyde, a constituent of floral scent. PAAS is a cytosolic homotetrameric enzyme that belongs to group II pyridoxal 5'-phosphate-dependent amino-acid decarboxylases and shares extensive amino acid identity (approximately 65%) with plant L-tyrosine/3,4-dihydroxy-L-phenylalanine and L-tryptophan decarboxylases. It displays a strict specificity for phenylalanine with an apparent Km of 1.2 mM. PAAS is a bifunctional enzyme that catalyzes the unprecedented efficient coupling of phenylalanine decarboxylation to oxidation, generating phenylacetaldehyde, CO2, ammonia, and hydrogen peroxide in stoichiometric amounts.