ABSTRACT
Naturally occurring echinocandin B and FR901379 are potent antifungal lipopeptides featuring a cyclic hexapeptide nucleus and a fatty acid side chain. They are the parent compounds of echinocandin drugs for the treatment of severe fungal infections caused by the Candida and Aspergilla species. To minimize hemolytic toxicity, the native fatty acid side chains in these drug molecules are replaced with designer acyl side chains. The deacylation of the N-acyl side chain is, therefore, a crucial step for the development and manufacturing of echinocandin-type antibiotics. Echinocandin E (ECE) is a novel echinocandin congener with enhanced stability generated via the engineering of the biosynthetic machinery of echinocandin B (ECB). In the present study, we report the discovery of the first echinocandin E acylase (ECEA) using the enzyme similarity tool (EST) for enzymatic function mining across protein families. ECEA is derived from Streptomyces sp. SY1965 isolated from a sediment collected from the Mariana Trench. It was cloned and heterologously expressed in S. lividans TK24. The resultant TKecea66 strain showed efficient cleavage activity of the acyl side chain of ECE, showing promising applications in the development of novel echinocandin-type therapeutics. Our results also provide a showcase for harnessing the essentially untapped biodiversity from the hadal ecosystems for the discovery of functional molecules.
Subject(s)
Antifungal Agents , Echinocandins , Streptomyces , Streptomyces/enzymology , Streptomyces/genetics , Echinocandins/chemistry , Antifungal Agents/pharmacology , Antifungal Agents/chemistry , Amidohydrolases/metabolism , Fungal ProteinsABSTRACT
The hydroxyl groups in the amino acid residues of echinocandin B were related to the biological activity, the instability, and the drug resistance. The modification of hydroxyl groups was expected to obtain the new lead compounds for next generation of echinocandin drug development. In this work one method for heterologous production of the tetradeoxy echinocandin was achieved. A reconstructed biosynthetic gene cluster for tetradeoxy echinocandins composed of ecdA/I/K and htyE was designed and successfully hetero-expressed in Aspergillus nidulans. The target product of echinocandin E (1) together with one unexpected derivative echinocandin F (2), were isolated from the fermentation culture of engineered strain. Both of compounds were unreported echinocandin derivatives and the structures were identified on the basis of mass and NMR spectral data analysis. Compared with echinocandin B, echinocandin E demonstrated superior stability and comparable antifungal activity.
Subject(s)
Aspergillus nidulans , Echinocandins , Echinocandins/pharmacology , Echinocandins/chemistry , Echinocandins/genetics , Antifungal Agents/pharmacology , Antifungal Agents/metabolism , Fungal Proteins/metabolism , Aspergillus nidulans/genetics , Aspergillus nidulans/metabolism , Multigene Family , Amino Acids/metabolism , Microbial Sensitivity TestsABSTRACT
Mammalian cells express two isoforms of type II phosphatidylinositol 4-kinase: PI4KIIα and PI4KIIß. PI4KIIα exists almost exclusively as a constitutively active integral membrane protein because of its palmitoylation (Barylko, B., Gerber, S. H., Binns, D. D., Grichine, N., Khvotchev, M., Südhof, T. C., and Albanesi, J. P. (2001) J. Biol. Chem. 276, 7705-7708). In contrast, PI4KIIß is distributed almost evenly between membranes and cytosol. Whereas the palmitoylated membrane-bound pool is catalytically active, the cytosolic kinase is inactive (Wei, Y. J., Sun, H. Q., Yamamoto, M., Wlodarski, P., Kunii, K., Martinez, M., Barylko, B., Albanesi, J. P., and Yin, H. L. (2002) J. Biol. Chem. 277, 46586-46593; Jung, G., Wang, J., Wlodarski, P., Barylko, B., Binns, D. D., Shu, H., Yin, H. L., and Albanesi, J. P. (2008) Biochem. J. 409, 501-509). In this study, we identify the molecular chaperone Hsp90 as a binding partner of PI4KIIß, but not of PI4KIIα. Geldanamycin (GA), a specific Hsp90 inhibitor, disrupts the Hsp90-PI4KIIß interaction and destabilizes PI4KIIß, reducing its half-life by 40% and increasing its susceptibility to ubiquitylation and proteasomal degradation. Cytosolic PI4KIIß is much more sensitive to GA treatment than is the integrally membrane-associated species. Exposure to GA induces a partial redistribution of PI4KIIß from the cytosol to membranes and, with brief GA treatments, a corresponding increase in cellular phosphatidylinositol 4-kinase activity. Stimuli such as PDGF receptor activation that also induce recruitment of the kinase to membranes disrupt the Hsp90-PI4KIIß interaction to a similar extent as GA treatment. These results support a model wherein Hsp90 interacts predominantly with the cytosolic, inactive pool of PI4KIIß, shielding it from proteolytic degradation but also sequestering it to the cytosol until an extracellular stimulus triggers its translocation to the Golgi or plasma membrane and subsequent activation.
Subject(s)
HSP90 Heat-Shock Proteins/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Animals , Benzoquinones/pharmacology , COS Cells , Chlorocebus aethiops , Cycloheximide/pharmacology , Electrophoresis, Polyacrylamide Gel , HEK293 Cells , HSP90 Heat-Shock Proteins/genetics , HeLa Cells , Humans , Immunoprecipitation , Lactams, Macrocyclic/pharmacology , Mass Spectrometry , Microscopy, Fluorescence , Minor Histocompatibility Antigens , Phosphotransferases (Alcohol Group Acceptor)/genetics , Protein Binding/genetics , Protein Binding/physiology , Protein Stability/drug effects , RatsABSTRACT
TRAIL selectively kills diseased cells in vivo, spurring interest in this death ligand as a potential therapeutic. However, many cancer cells are resistant to TRAIL, suggesting the mechanism mediating TRAIL-induced apoptosis is complex. Here we identify PACS-2 as an essential TRAIL effector, required for killing tumor cells in vitro and virally infected hepatocytes in vivo. PACS-2 is phosphorylated at Ser437 in vivo, and pharmacologic and genetic studies demonstrate Akt is an in vivo Ser437 kinase. Akt cooperates with 14-3-3 to regulate the homeostatic and apoptotic properties of PACS-2 that mediate TRAIL action. Phosphorylated Ser437 binds 14-3-3 with high affinity, which represses PACS-2 apoptotic activity and is required for PACS-2 to mediate trafficking of membrane cargo. TRAIL triggers dephosphorylation of Ser437, reprogramming PACS-2 to promote apoptosis. Together, these studies identify the phosphorylation state of PACS-2 Ser437 as a molecular switch that integrates cellular homeostasis with TRAIL-induced apoptosis.
Subject(s)
14-3-3 Proteins/metabolism , Apoptosis/physiology , Cell Membrane/metabolism , Homeostasis , Proto-Oncogene Proteins c-akt/metabolism , TNF-Related Apoptosis-Inducing Ligand/metabolism , Vesicular Transport Proteins/metabolism , 14-3-3 Proteins/genetics , Animals , BH3 Interacting Domain Death Agonist Protein/genetics , BH3 Interacting Domain Death Agonist Protein/metabolism , Caspases/metabolism , Cell Line, Tumor , Cells, Cultured , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Mice , Mice, Knockout , Neoplasms/metabolism , Neoplasms/pathology , Proto-Oncogene Proteins c-akt/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Serine/metabolism , TNF-Related Apoptosis-Inducing Ligand/genetics , Vesicular Transport Proteins/geneticsABSTRACT
Lysosomes can trigger the mitochondrial apoptotic pathway by releasing proteases. Here we report that a 25-kDa protein purified from rat liver lysosomes possesses a long standing potent Bid cleavage activity at neutral pH, and the truncated Bid can in turn induce rapid mitochondrial release of cytochrome c. This protease was revealed as chymotrypsin B by biochemical and mass spectrometric analysis. Although it was long recognized as a digestive protease exclusively secreted by the exocrine pancreas, our data support that it also expresses and intracellularly resides in rat liver lysosomes. Translocation of lysosomal chymotrypsin B into cytosol was triggered by apoptotic stimuli such as tumor necrosis factor-alpha, and intracellular delivery of chymotrypsin B protein induced apoptotic cell death with a potency comparable with cathepsin B, suggestive of a lysosomal-mitochondrial pathway to apoptosis regulated by chymotrypsin B following its release. Noteworthily, either knockdown of chymotrypsin B expression by RNA interference or pretreatment with chymotrypsin B inhibitor N-p-tosyl-L-phenylalanine chloromethyl ketone significantly reduced tumor necrosis factor-alpha-induce apoptosis. These results demonstrate for the first time that chymotrypsin B is not only restricted to the pancreas but can function intracellularly as a pro-apoptotic protease.
Subject(s)
Apoptosis , Chymotrypsin/metabolism , Liver/enzymology , Lysosomes/enzymology , Mitochondria/enzymology , Signal Transduction , Animals , Cell Line , Chymotrypsin/genetics , Gene Expression Regulation, Enzymologic , Hepatocytes/enzymology , RNA, Messenger/genetics , Rats , Rats, Sprague-DawleyABSTRACT
Mammalian cells contain two isoforms of the type II PI4K (phosphoinositol 4-kinase), PI4KIIalpha and beta. These 55 kDa proteins have highly diverse N-terminal regions (approximately residues 1-90) but conserved catalytic domains (approximately from residue 91 to the C-termini). Nearly the entire pool of PI4KIIalpha behaves as an integral membrane protein, in spite of a lack of a transmembrane domain. This integral association with membranes is due to palmitoylation of a cysteine-rich motif, CCPCC, located within the catalytic domain. Although the CCPCC motif is conserved in PI4KIIbeta, only 50% of PI4KIIbeta is membrane-associated, and approximately half of this pool is only peripherally attached to the membranes. Growth factor stimulation or overexpression of a constitutively active Rac mutant induces the translocation of a portion of cytosolic PI4KIIbeta to plasma membrane ruffles and stimulates its activity. Here, we demonstrate that membrane-associated PI4KIIbeta undergoes two modifications, palmitoylation and phosphorylation. The cytosolic pool of PI4KIIbeta is not palmitoylated and has much lower lipid kinase activity than the membrane-associated kinase. Although only membrane-associated PI4KIIbeta is phosphorylated in the unique N-terminal region, this modification apparently does not influence its membrane binding or activity. A series of truncation mutants and alpha/beta chimaeras were generated to identify regions responsible for the isoform-specific behaviour of the kinases. Surprisingly, the C-terminal approx. 160 residues, and not the diverse N-terminal regions, contain the sites that are most important in determining the different solubilities, palmitoylation states and stimulus-dependent redistributions of PI4KIIalpha and beta.
Subject(s)
Cell Membrane/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Animals , Binding Sites , COS Cells , Cells, Cultured , Chlorocebus aethiops , HeLa Cells , Humans , Lipoylation , Microscopy, Fluorescence , Minor Histocompatibility Antigens , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Phosphotransferases (Alcohol Group Acceptor)/genetics , Protein Processing, Post-Translational , Transfection , rac GTP-Binding Proteins/metabolismABSTRACT
Mammalian mitochondrial dihydrolipoamide dehydrogenase (DLDH, EC 1.8.1.4) catalyzes NAD(+)-dependent oxidation of dihydrolipoamide in vivo and can also act as a diaphorase catalyzing in vitro nicotinamide adenine dinucleotide (reduced form) (NADH)-dependent reduction of electron-accepting molecules such as ubiquinone and nitroblue tetrazolium (NBT). In this paper, we report a gel-based method for histochemical staining and quantification of DLDH diaphorase activity using blue native PAGE (BN-PAGE). Rat brain mitochondrial extracts, used as the source of DLDH, were resolved by nongradient BN-PAGE (9%), which was followed by diaphorase activity staining using NADH as the electron donor and NBT as the electron acceptor. It was shown that activity staining of DLDH diaphorase was both protein amount- and time-dependent. Moreover, this in-gel activity-staining method was demonstrated to be in good agreement with the conventional spectrophotometric method that measures DLDH dehydrogenase activity using dihydrolipoamide as the substrate. The method was applied to determine levels of DLDH diaphorase activity in several rat tissues other than the brain, and the results indicated a similar level of DLDH diaphorase activity for all the tissues examined. Finally, the effects of thiol-reactive reagents such as N-ethylmaleimide (NEM) and nitric oxide donors on DLDH diaphorase activity were evaluated, demonstrating that, with this method, DLDH diaphorase activity can be determined without having to remove these thiol-reactive reagents that may otherwise interfere with spectrophotometric measurement of DLDH dehydrogenase activity. The gel-based method can also be used as a means to isolate mitochondrial DLDH that is to be analyzed by mass spectral techniques in studying DLDH post-translational modifications.
Subject(s)
Dihydrolipoamide Dehydrogenase/metabolism , Mitochondrial Proteins/metabolism , Animals , Chromatography, Liquid , Dihydrolipoamide Dehydrogenase/analysis , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Ethylmaleimide/metabolism , Mitochondrial Proteins/analysis , NAD/metabolism , Nitroblue Tetrazolium/chemistry , Oxidation-Reduction , Rats , Rats, Sprague-Dawley , Spectrometry, Mass, Electrospray Ionization , Staining and Labeling , Tandem Mass Spectrometry , Thioctic Acid/analogs & derivatives , Thioctic Acid/metabolismABSTRACT
Members of the B56 family of protein phosphatase 2A (PP2A) regulatory subunits play crucial roles in Drosophila cell survival. Distinct functions of two B56 subunits were investigated using a combination of RNA interference, DNA microarrays, and proteomics. RNA interference-mediated knockdown of the B56-1 subunit (PP2A-B') but not the catalytic (mts) or B56-2 subunit (wdb) of PP2A resulted in increased expression of the apoptotic inducers reaper and sickle. Co-knockdown of B56-1 with reaper, but not with sickle, reduced the apoptosis caused by depletion of the B56 subunits. Two-dimensional gel electrophoresis and mass spectrometry identified proteins modified in cells depleted of PP2A subunits. These included generation of caspase-dependent cleavage products, increases in protein abundance, and covalent modifications. Results suggested that up-regulation of the ribosome-associated protein stubarista can serve as a sensitive marker of apoptosis. Up-regulation of transcripts for multiple glutathione transferases and other proteins suggested that loss of PP2A affected pathways involved in the response to oxidative stress. Knockdown of PP2A elevated basal JNK activity and substantially decreased activation of ERK in response to oxidative stress. The results reveal that the B56-containing isoform of PP2A functions within multiple signaling pathways, including those that regulate expression of reaper and the response to oxidative stress, thus promoting cell survival in Drosophila.
Subject(s)
Drosophila melanogaster/enzymology , Gene Expression Regulation, Enzymologic , Genome, Insect , Genomics/methods , Phosphoprotein Phosphatases/metabolism , Animals , Apoptosis , Cell Line , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Oligonucleotide Array Sequence Analysis , Oxidative Stress , Phosphoprotein Phosphatases/genetics , Protein Isoforms , Protein Phosphatase 2 , Proteomics , RNA Interference , Signal Transduction , Up-RegulationABSTRACT
Regulation of cytoskeletal dynamics is essential to neuronal plasticity during development and adulthood. Dysregulation of these mechanisms may contribute to neuropsychiatric and neurodegenerative diseases. The neuronal protein kinase, cyclin-dependent kinase 5 (Cdk5), is involved in multiple aspects of neuronal function, including regulation of cytoskeleton. A neuroproteomic search identified the tubulin-binding protein, stathmin, as a novel Cdk5 substrate. Stathmin was phosphorylated by Cdk5 in vitro at Ser25 and Ser38, previously identified as mitogen-activated protein kinase (MAPK) and p38 MAPKdelta sites. Cdk5 predominantly phosphorylated Ser38, while MAPK and p38 MAPKdelta predominantly phosphorylated Ser25. Stathmin was phosphorylated at both sites in mouse brain, with higher levels in cortex and striatum. Cdk5 knockout mice exhibited decreased phospho-Ser38 levels. During development, phospho-Ser25 and -Ser38 levels peaked at post-natal day 7, followed by reduction in total stathmin. Inhibition of protein phosphatases in striatal slices caused an increase in phospho-Ser25 and a decrease in total stathmin. Interestingly, the prefrontal cortex of schizophrenic patients had increased phospho-Ser25 levels. In contrast, total and phospho-Ser25 stoichiometries were decreased in the hippocampus of Alzheimer's patients. Thus, microtubule regulatory mechanisms involving the phosphorylation of stathmin may contribute to developmental synaptic pruning and structural plasticity, and may be involved in neuropsychiatric and neurodegenerative disorders.
Subject(s)
Cyclin-Dependent Kinase 5/metabolism , Mitogen-Activated Protein Kinases/metabolism , Stathmin/metabolism , Animals , DNA Primers , Mass Spectrometry , Molecular Sequence Data , Phosphorylation , Polymerase Chain Reaction , Rats , Stathmin/genetics , Tubulin/metabolismABSTRACT
Inhibitor-1 becomes a potent inhibitor of protein phosphatase 1 when phosphorylated by cAMP-dependent protein kinase at Thr(35). Moreover, Ser(67) of inhibitor-1 serves as a substrate for cyclin-dependent kinase 5 in the brain. Here, we report that dephosphoinhibitor-1 but not phospho-Ser(67) inhibitor-1 was efficiently phosphorylated by protein kinase C at Ser(65) in vitro. In contrast, Ser(67) phosphorylation by cyclin-dependent kinase 5 was unaffected by phospho-Ser(65). Protein kinase C activation in striatal tissue resulted in the concomitant phosphorylation of inhibitor-1 at Ser(65) and Ser(67), but not Ser(65) alone. Selective pharmacological inhibition of protein phosphatase activity suggested that phospho-Ser(65) inhibitor-1 is dephosphorylated by protein phosphatase 1 in the striatum. In vitro studies confirmed these findings and suggested that phospho-Ser(67) protects phospho-Ser(65) inhibitor-1 from dephosphorylation by protein phosphatase 1 in vivo. Activation of group I metabotropic glutamate receptors resulted in the up-regulation of diphospho-Ser(65)/Ser(67) inhibitor-1 in this tissue. In contrast, the activation of N-methyl-d-aspartate-type ionotropic glutamate receptors opposed increases in striatal diphospho-Ser(65)/Ser(67) inhibitor-1 levels. Phosphomimetic mutation of Ser(65) and/or Ser(67) did not convert inhibitor-1 into a protein phosphatase 1 inhibitor. On the other hand, in vitro and in vivo studies suggested that diphospho-Ser(65)/Ser(67) inhibitor-1 is a poor substrate for cAMP-dependent protein kinase. These observations extend earlier studies regarding the function of phospho-Ser(67) and underscore the possibility that phosphorylation in this region of inhibitor-1 by multiple protein kinases may serve as an integrative signaling mechanism that governs the responsiveness of inhibitor-1 to cAMP-dependent protein kinase activation.
Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Protein Kinase C/metabolism , Animals , Brain/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , In Vitro Techniques , Mice , Mice, Inbred C57BL , PC12 Cells , Phosphorylation , Proteins/metabolism , Rats , Serine , Substrate SpecificityABSTRACT
Loss of sister-chromatid cohesion triggers chromosome segregation in mitosis and occurs through two mechanisms in vertebrate cells: (1) phosphorylation and removal of cohesin from chromosome arms by mitotic kinases, including Plk1, during prophase, and (2) cleavage of centromeric cohesin by separase at the metaphase-anaphase transition. Bub1 and the MEI-S332/Shugoshin (Sgo1) family of proteins protect centromeric cohesin from mitotic kinases during prophase. We show that human Sgo1 binds to protein phosphatase 2A (PP2A). PP2A localizes to centromeres in a Bub1-dependent manner. The Sgo1-PP2A interaction is required for centromeric localization of Sgo1 and proper chromosome segregation in human cells. Depletion of Plk1 by RNA interference (RNAi) restores centromeric localization of Sgo1 and prevents chromosome missegregation in cells depleted of PP2A_Aalpha. Our findings suggest that Bub1 targets PP2A to centromeres, which in turn maintains Sgo1 at centromeres by counteracting Plk1-mediated chromosome removal of Sgo1.
Subject(s)
Cell Cycle Proteins/metabolism , Centromere/metabolism , Chromosome Segregation , Phosphoprotein Phosphatases/metabolism , Gene Expression , HeLa Cells , Humans , Mitosis , Mutation/genetics , Protein Binding , Protein Kinases/metabolism , Protein Phosphatase 2 , Protein Serine-Threonine Kinases/deficiency , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , Protein Subunits , Protein Transport , Proto-Oncogene Proteins/deficiency , Proto-Oncogene Proteins/metabolism , RNA Interference , Tumor Cells, Cultured , Polo-Like Kinase 1ABSTRACT
Reliable identification of posttranslational modifications is key to understanding various cellular regulatory processes. We describe a tool, InsPecT, to identify posttranslational modifications using tandem mass spectrometry data. InsPecT constructs database filters that proved to be very successful in genomics searches. Given an MS/MS spectrum S and a database D, a database filter selects a small fraction of database D that is guaranteed (with high probability) to contain a peptide that produced S. InsPecT uses peptide sequence tags as efficient filters that reduce the size of the database by a few orders of magnitude while retaining the correct peptide with very high probability. In addition to filtering, InsPecT also uses novel algorithms for scoring and validating in the presence of modifications, without explicit enumeration of all variants. InsPecT identifies modified peptides with better or equivalent accuracy than other database search tools while being 2 orders of magnitude faster than SEQUEST, and substantially faster than X!TANDEM on complex mixtures. The tool was used to identify a number of novel modifications in different data sets, including many phosphopeptides in data provided by Alliance for Cellular Signaling that were missed by other tools.
Subject(s)
Peptides/chemistry , Peptides/metabolism , Protein Processing, Post-Translational/physiology , Software , Tandem Mass Spectrometry/methods , Databases, Factual , I-kappa B Kinase/chemistry , Keratins/chemistry , Peptide Hydrolases/chemistry , Peptides/genetics , Sensitivity and SpecificityABSTRACT
Phosphorylation is a major regulatory mechanism controlling circadian clocks. In the Neurospora circadian clock, the PER-ARNT-SIM (PAS) domain-containing transcription factor, WHITE COLLAR (WC)-1, acts both as the blue light photoreceptor of the clock and as a positive element in the circadian negative feedback loop in constant darkness, by activating the transcription of the frequency (frq) gene. To understand the role of WC-1 phosphorylation, five in vivo WC-1 phosphorylation sites, located immediately downstream of the WC-1 zinc finger DNA binding domain, were identified by tandem mass spectrometry using biochemically purified endogenous WC-1 protein. Mutations of these phosphorylation sites suggest that they are major WC-1 phosphorylation sites under constant conditions but are not responsible for the light-induced hyperphosphorylation of WC-1. Although phosphorylation of these sites does not affect the light function of WC-1, strains carrying mutations of these sites show short period, low amplitude, or arrhythmic conidiation rhythms in constant darkness. Furthermore, normal or slightly higher levels of frq mRNA and FRQ proteins were observed in a mutant strain containing mutations of all five sites despite its low WC-1 levels. Together, these data suggest that phosphorylation of these sites negatively regulates the function of WC-1 in the circadian negative feedback loop and is important for the function of the Neurospora circadian clock.
Subject(s)
DNA-Binding Proteins/physiology , Feedback, Physiological , Gene Expression Regulation, Fungal , Light , Neurospora/metabolism , Transcription Factors/physiology , Amino Acid Sequence , Binding Sites , Blotting, Western , Circadian Rhythm , DNA-Binding Proteins/metabolism , Electrophoresis, Polyacrylamide Gel , Epitopes/chemistry , Fungal Proteins , Mass Spectrometry , Molecular Sequence Data , Mutation , Phosphorylation , Point Mutation , RNA/chemistry , RNA, Messenger/metabolism , Time Factors , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
To ensure the fidelity of chromosome segregation, the spindle checkpoint blocks the ubiquitin ligase activity of APC/C(Cdc20) in response to a single chromatid not properly attached to the mitotic spindle. Here we show that HeLa cells depleted for Bub1 by RNA interference are defective in checkpoint signaling. Bub1 directly phosphorylates Cdc20 in vitro and inhibits the ubiquitin ligase activity of APC/C(Cdc20) catalytically. A Cdc20 mutant with all six Bub1 phosphorylation sites removed is refractory to Bub1-mediated phosphorylation and inhibition in vitro. Upon checkpoint activation, Bub1 itself is hyperphosphorylated and its kinase activity toward Cdc20 is stimulated. Ectopic expression of the nonphosphorylatable Cdc20 mutant allows HeLa cells to escape from mitosis in the presence of spindle damage. Therefore, Bub1-mediated phosphorylation of Cdc20 is required for proper checkpoint signaling. We speculate that inhibition of APC/C(Cdc20) by Bub1 in a catalytic fashion may partly account for the exquisite sensitivity of the spindle checkpoint.
Subject(s)
Cell Cycle Proteins/metabolism , Protein Kinases/physiology , Spindle Apparatus/metabolism , Ubiquitin-Conjugating Enzymes/antagonists & inhibitors , Ubiquitin-Protein Ligase Complexes/antagonists & inhibitors , Anaphase-Promoting Complex-Cyclosome , Catalysis , Cdc20 Proteins , Genes, cdc/physiology , HeLa Cells , Humans , Mitosis , Mutagenesis, Site-Directed , Phosphorylation , Protein Serine-Threonine Kinases , RNA Interference , Serine/metabolism , Threonine/metabolism , Ubiquitin/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligase Complexes/metabolismABSTRACT
WNK (with no lysine [K]) protein kinases were named for their unique active site organization. Mutations in WNK1 and WNK4 cause a familial form of hypertension by undefined mechanisms. Here, we report that WNK1 selectively binds to and phosphorylates synaptotagmin 2 (Syt2) within its calcium binding C2 domains. Endogenous WNK1 and Syt2 coimmunoprecipitate and colocalize on a subset of secretory granules in INS-1 cells. Phosphorylation by WNK1 increases the amount of Ca2+ required for Syt2 binding to phospholipid vesicles; mutation of threonine 202, a WNK1 phosphorylation site, partially prevents this change. These findings suggest that phosphorylation of Syts by WNK1 can regulate Ca2+ sensing and the subsequent Ca2+-dependent interactions mediated by Syt C2 domains. These findings provide a biochemical mechanism that could lead to the retention or insertion of proteins in the plasma membrane. Interruption of this regulatory pathway may disturb membrane events that regulate ion balance.
Subject(s)
Cell Membrane/metabolism , Nerve Tissue Proteins/metabolism , Protein Binding/physiology , Protein Serine-Threonine Kinases/metabolism , Animals , Binding Sites/physiology , Calcium Signaling/genetics , Calcium-Binding Proteins/metabolism , Cell Line , Humans , Intracellular Signaling Peptides and Proteins , Minor Histocompatibility Antigens , Mutation/genetics , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein Structure, Tertiary/physiology , Secretory Vesicles/metabolism , Synaptotagmin II , Threonine/metabolism , Two-Hybrid System Techniques , WNK Lysine-Deficient Protein Kinase 1 , Water-Electrolyte BalanceABSTRACT
A major goal of the Alliance for Cellular Signaling is to elaborate the components of signal transduction networks in model cell systems, including murine B lymphocytes. Due to the importance of protein phosphorylation in many aspects of cell signaling, the initial efforts have focused on the identification of phosphorylated proteins. In order to identify serine- and threonine-phosphorylated proteins on a proteome-wide basis, WEHI-231 cells were treated with calyculin A, a serine/threonine phosphatase inhibitor, to induce high levels of protein phosphorylation. Proteins were extracted from whole-cell lysates and digested with trypsin. Phosphorylated peptides were then enriched using immobilized metal affinity chromatography and identified by liquid chromatography-tandem mass spectrometry. A total of 107 proteins and 193 phosphorylation sites were identified using these methods. Forty-two of these proteins have been reported to be phosphorylated, but only some of them have been detected in B cells. Fifty-four of the identified proteins were not previously known to be phosphorylated. The remaining 11 phosphoproteins have previously only been characterized as novel cDNA or genomic sequences. Many of the identified proteins were phosphorylated at multiple sites. The proteins identified in this study significantly expand the repertoire of proteins known to be phosphorylated in B cells. The number of newly identified phosphoproteins indicates that B cell signaling pathways utilizing protein phosphorylation are likely to be more complex than previously appreciated.
Subject(s)
Lymphoma, B-Cell/chemistry , Phosphoproteins/chemistry , Phosphoserine/chemistry , Phosphothreonine/chemistry , Proteomics , Animals , Cell Line , Chromatography, Affinity , Chromatography, Liquid , Enzyme Inhibitors/pharmacology , Lymphoma, B-Cell/metabolism , Lymphoma, B-Cell/pathology , Marine Toxins , Mice , Oxazoles/pharmacology , Peptide Fragments/chemistry , Phosphoproteins/metabolism , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Phosphorylation , Signal Transduction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Xanthomonas campestris pathovar vesicatoria (Xcv) uses the type III secretion system (TTSS) to inject effector proteins into cells of Solanaceous plants during pathogenesis. A number of Xcv TTSS effectors have been identified; however, their function in planta remains elusive. Here, we provide direct evidence for a functional role for a phytopathogenic bacterial TTSS effector in planta by demonstrating that the Xcv effector XopD encodes an active cysteine protease with plant-specific SUMO substrate specificity. XopD is injected into plant cells by the TTSS during Xcv pathogenesis, translocated to subnuclear foci and hydrolyses SUMO-conjugated proteins in vivo. Our studies suggest that XopD mimics endogenous plant SUMO isopeptidases to interfere with the regulation of host proteins during Xcv infection.
Subject(s)
Bacterial Proteins/metabolism , Cysteine Endopeptidases/metabolism , Plants/microbiology , SUMO-1 Protein/metabolism , Xanthomonas campestris/enzymology , Biological Transport, Active , Protein Transport/physiology , Recombinant Proteins/pharmacology , Xanthomonas campestris/metabolismABSTRACT
A small molecule, alpha-(trichloromethyl)-4-pyridineethanol (PETCM), was identified by high-throughput screening as an activator of caspase-3 in extracts of a panel of cancer cells. PETCM was used in combination with biochemical fractionation to identify a pathway that regulates mitochondria-initiated caspase activation. This pathway consists of tumor suppressor putative HLA-DR-associated proteins (PHAP) and oncoprotein prothymosin-alpha (ProT). PHAP proteins promoted caspase-9 activation after apoptosome formation, whereas ProT negatively regulated caspase-9 activation by inhibiting apoptosome formation. PETCM relieved ProT inhibition and allowed apoptosome formation at a physiological concentration of deoxyadenosine triphosphate. Elimination of ProT expression by RNA interference sensitized cells to ultraviolet irradiation-induced apoptosis and negated the requirement of PETCM for caspase activation. Thus, this chemical-biological combinatory approach has revealed the regulatory roles of oncoprotein ProT and tumor suppressor PHAP in apoptosis.
Subject(s)
Apoptosis , Neuropeptides , Nuclear Proteins/metabolism , Protein Precursors/metabolism , Proteins/metabolism , Pyridines/pharmacology , Thymosin/analogs & derivatives , Thymosin/metabolism , Amino Acid Sequence , Apoptotic Protease-Activating Factor 1 , Caspase 3 , Caspase 9 , Caspases/metabolism , Cell Extracts , Cytochrome c Group/metabolism , Deoxyadenine Nucleotides/metabolism , Deoxyadenine Nucleotides/pharmacology , Enzyme Activation , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins , Mitochondria/metabolism , Molecular Sequence Data , Nuclear Proteins/chemistry , Nuclear Proteins/isolation & purification , Nuclear Proteins/pharmacology , Protein Precursors/chemistry , Protein Precursors/isolation & purification , Protein Precursors/pharmacology , Proteins/chemistry , Proteins/isolation & purification , Proteins/pharmacology , Pyridines/chemistry , RNA Interference , RNA-Binding Proteins , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Signal Transduction , Thymosin/chemistry , Thymosin/isolation & purification , Thymosin/pharmacology , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/isolation & purification , Tumor Suppressor Proteins/metabolismABSTRACT
Rapid phagocytosis of apoptotic cells is thought to limit the development of inflammation and autoimmune disease. Serum enhances macrophage phagocytosis of apoptotic cells. Here we identified protein S as the factor responsible for serum-stimulated phagocytosis of apoptotic cells. Protein S is best known for its anti-thrombotic activity, serving as a cofactor for protein C. Purified protein S was equivalent to serum in its ability to stimulate macrophage phagocytosis of apoptotic lymphoma cells, and immunodepletion of protein S eliminated the prophagocytic activity of serum. Protein S acted by binding to phosphatidylserine expressed on the apoptotic cell surface. Protein S is thus a multifunctional protein that can facilitate clearance of early apoptotic cells in addition to regulating blood coagulation.
Subject(s)
Apoptosis/immunology , Phagocytosis/immunology , Phosphatidylserines/blood , Protein S/metabolism , Humans , Macrophages/immunology , Protein Binding , Protein S/immunology , Tumor Cells, CulturedABSTRACT
The Alliance for Cellular Signaling has chosen the mouse B lymphocyte as a model system to understand basic principles that govern cellular signalling. Progress to that end has focused initially on establishing a reproducible experimental cell system and characterizing essential signalling responses. Although unravelling this complex network will take years, findings revealed in the interim will prove immensely useful to the scientific community at large.