Stephen Randall - Academia.edu (original) (raw)
Papers by Stephen Randall
Identifying and characterizing cold responsive genes in Fragaria vesca associated with or respons... more Identifying and characterizing cold responsive genes in Fragaria vesca associated with or responsible for low temperature tolerance is a vital part of strawberry cultivar development. In this study we have investigated the transcript levels of eight genes, two dehydrin genes, three putative ABA-regulated genes, two cold–inducible CBF genes and the alcohol dehydrogenase gene, extracted from leaf and crown tissues of three F. vesca genotypes that vary in cold tolerance. Transcript levels of the CBF/DREB1 transcription factor FvCBF1E exhibited stronger cold up-regulation in comparison to FvCBF1B.1 in all genotypes. Transcripts of FvADH were highly up-regulated in both crown and leaf tissues from all three genotypes. In the ‘ALTA’ genotype, FvADH transcripts were significantly higher in leaf than crown tissues and more than 10 to 20-fold greater than in the less cold-tolerant ‘NCGR1363’ and ‘FDP817’ genotypes. FvGEM, containing the conserved ABRE promoter element, transcript was found t...
Plant science : an international journal of experimental plant biology, 2016
Soybean (Glycine max) is considered to be cold intolerant and is not able to significantly acclim... more Soybean (Glycine max) is considered to be cold intolerant and is not able to significantly acclimate to cold/freezing stress. In most cold tolerant plants, the C-repeat/DRE Binding Factors (CBF/DREBs) are critical contributors to successful cold-responses; rapidly increasing following cold treatment and regulating the induction of many cold responsive genes. In soybean vegetative tissue, we found strong, transient accumulation of CBF transcripts in response to cold stress; however, the soybean transcripts of typical cold responsive genes (homologues to Arabidopsis genes such as dehydrins, ADH1, RAP2.1, and LEA14) were not significantly altered. Soybean CBFs were found to be functional, as when expressed constitutively in Arabidopsis they increased the levels of AtCOR47 and AtRD29a transcripts and increased freezing tolerance as measured by a decrease in leaf freezing damage and ion leakage. Furthermore the constitutive expression of GmDREB1A;2 and GmDREB1B;1 in Arabidopsis led to st...
Isoprenylation is a posttranslational modification that is believed to be necessary, but not suff... more Isoprenylation is a posttranslational modification that is believed to be necessary, but not sufficient, for the efficient association of numerous eukaryotic cell proteins with membranes. Additional modifications have been shown to be required for proper intracellular targeting and function of certain isoprenylated proteins in mammalian and yeast cells. Although protein isoprenylation has been demonstrated in plants, postisoprenylation processing of plant proteins has not been described. Here we demonstrate that cultured tobacco (Nicotiana tabacum cv Bright Yellow-2) cells contain farnesylcysteine and geranylgeranylcysteine ␣-carboxyl methyltransferase activities with apparent Michaelis constants of 73 and 21 M for N-acetyl-S-trans,trans-farnesyl-L-cysteine and N-acetyl-S-alltrans-geranylgeranyl-L-cysteine, respectively. Furthermore, competition analysis indicates that the same enzyme is responsible for both activities. These results suggest that ␣-carboxyl methylation is a step in the maturation of isoprenylated proteins in plants.
Plant Molecular Biology, Sep 1, 1996
To identify isoprenylated plant GTP-binding proteins, Arabidopsis thaliana and Nicotiana tabacum ... more To identify isoprenylated plant GTP-binding proteins, Arabidopsis thaliana and Nicotiana tabacum cDNA expression libraries were screened for cDNA-encoded proteins capable of binding [32P]GTP in vitro. ATGB2, an Arabidopsis homologue of the GTP-binding protein Rab2, was found to bind GTP in vitro and to be a substrate for a geranylgeranyl:protein transferase (GGTase) present in plant extracts. The carboxyl terminus of this protein contains a -GCCG sequence, which has not previously been shown to be recognized by any prenyl:protein transferase (PTase), but which most closely resembles that isoprenylated by the type II GGTase (-XXCC, -XCXC, or -CCXX). In vitro geranylgeranylation of an Arabidopsis Rab1 protein containing a carboxyl-terminal-CCGQ sequence confirmed the presence of a type II GGTase-like activity in plant extracts. Several other proteins were also identified by in vitro GTP binding, including Arabidopsis and tobacco homologues of Rab11, ARF (ADP-ribosylation factor) and Sar proteins, as well as a novel 22 kDa Arabidopsis protein (ATG81). This 22 kDa protein had consensus GTP-binding motifs and bound GTP with high specificity, but its structure was not closely related to that of any known GTP-binding protein (it most resembled proteins within the ARF/Sar and G protein alpha-subunit superfamilies).
Plant Physiology, Sep 1, 1998
Isoprenylation is a posttranslational modification that is believed to be necessary, but not suff... more Isoprenylation is a posttranslational modification that is believed to be necessary, but not sufficient, for the efficient association of numerous eukaryotic cell proteins with membranes. Additional modifications have been shown to be required for proper intracellular targeting and function of certain isoprenylated proteins in mammalian and yeast cells. Although protein isoprenylation has been demonstrated in plants, postisoprenylation processing of plant proteins has not been described. Here we demonstrate that cultured tobacco (Nicotiana tabacum cv Bright Yellow-2) cells contain farnesylcysteine and geranylgeranylcysteine alpha-carboxyl methyltransferase activities with apparent Michaelis constants of 73 and 21 &mgr;M for N-acetyl-S-trans, trans-farnesyl-L-cysteine and N-acetyl-S-all-trans-geranylgeranyl-L-cysteine, respectively. Furthermore, competition analysis indicates that the same enzyme is responsible for both activities. These results suggest that alpha-carboxyl methylation is a step in the maturation of isoprenylated proteins in plants.
Cancer Lett, 1996
Many isoprenylated proteins are known to participate in signal transduction, but not all have bee... more Many isoprenylated proteins are known to participate in signal transduction, but not all have been identified. Using an in vitro prenylation screen, two human cDNAs (FI'PCAAXI and PTkIAAX2) homologous to the rat PRL-I and human OV-1 protein tyrosine phosphatase genes were identified. PTPCAAXI and PTPCAAXZ were famesylated in vitro by mammalian farnesyl:protein transferase, and epitope-tagged PW~AAXZ was prenylated in epithelial cells. Overexpression of PTPCAAXI and PTPCAAXZ in epithelial cells caused a transformed phenotype in culture and tumor growth in nude mice. Thus, PTPCAAXI and PTPCAAXZ represent a novel class of isoprenylated, oncogenic protein tyrosine phosphatases.
The Journal of Biological Chemistry, May 25, 1987
The purified tonoplast H*-ATPase from oat roots (Avena sativa L. var. Lang) consists of at least ... more The purified tonoplast H*-ATPase from oat roots (Avena sativa L. var. Lang) consists of at least three different polypeptides with masses 72,60, and 16 kDa. We have used covalent modifiers (inhibitors) and polyclonal antibodies to identify the catalytic subunit of the H+-pumping ATPase. The inactivation of ATPase activity by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (Nbd-C1, an adenine analog) was protected by MgATP or MgADP, and showed kinetic properties consistent with active site-directed inhibition. Under similar conditions, [14C]Nbd-C1 preferentially labeled the 72-kDa polypeptide of the purified ATPase. This binding was reduced by MgATP or 2' (3')-)0-(2,4,6trinitrophenyl) ATP. Nbd-C1 probably modified cysteinyl-SH or tyrosyl-OH groups, as dithiothreitol reversed both ATPase inactivation and I"C]Nbd-Cl binding to the 72-kDa subunit. The finding that Nethylmaleimide inhibition of ATPase activity was protectable by nucleotides is consistent with the idea of sulfhydryl groups in the ATP-binding site. Polyclonal antibody made to the 72-kDa polypeptide specifically reacted (Western blot) with a 72-kDa polypeptide from both tonoplast-enriched membranes and the purified tonoplast ATPase, but it did not cross-react with the mitochondrial or Escherichia coli F,-ATPase. The antibody inhibited tonoplast ATPase and H*-pumping activities. We conclude from these results that the 72-kDa polypeptide of the tonoplast H+-ATPase contains an ATP-(or nucleotide-) binding site that may constitute the catalytic domain.
Plant Physiology, Dec 1, 1985
The tonoplast ATPase of oat roots is composed of at least three polypeptides of 72, 60, and 16 kD... more The tonoplast ATPase of oat roots is composed of at least three polypeptides of 72, 60, and 16 kDa. The 16 kDA polypeptide covalently binds N,N'-dicyclohexylcarbodiimide and is postulated to be a component of the proton channel. Initial studies to identify other subunits indicate that both the 72 and 60 kDa subunits covalently bind ¹â´C)-7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and (¹â´C)N-ethylamleimide, inhibitors of the tonoplast ATPase. ATP prevents binding of these inhibitors suggesting that both the 72 and 60 kDa subunits are involved in substrate binding. Polyclonal antibody has been made to the 72 kDa subunit. Western blot analysis of tonoplast vesicles reveals single reactive polypeptide (72 kDa). The antibody shows no cross-reactivity towards either the mitochondrial Fâ-ATPase or the plasma membrane ATPase. This antibody specifically inhibits ATP hydrolysis and ATP-dependent H/sup +/ pumping in native tonoplast vesicles. The authors conclude that the 72 kDa subunit is intimately associated with the catalytic (or ATP-binding) site.
Methods in Enzymology, 1987
American journal of translational research, 2012
The PRL-1 and PRL-2 phosphatases have been implicated as oncogenic, however the involvement of th... more The PRL-1 and PRL-2 phosphatases have been implicated as oncogenic, however the involvement of these molecules in human neoplasms is not well understood. To increase understanding of the role PRL-1 and PRL-2 play in the neoplastic process, in situ hybridization was used to examine PRL-1 and PRL-2 mRNA expression in 285 normal, benign, and malignant human tissues of diverse origin. Immunohistochemical analysis was performed on a subset of these. PRL-1 and PRL-2 mRNA expression was also assessed in a small set of samples from a variety of diseases other than cancer. Where possible, associations with clinicopathological characteristics were evaluated. Alterations in PRL-1 or -2 expression were a frequent event, but the nature of those alterations was highly tumor type specific. PRL-1 was significantly overexpressed in 100% of hepatocellular and gastric carcinomas, but significantly under-expressed in 100% of ovarian, 80% of breast, and 75% of lung tumors. PRL-2 expression was significa...
The Journal of biological chemistry, Jan 25, 1986
Higher plant cells have one or more vacuoles important for maintaining cell turgor and for the tr... more Higher plant cells have one or more vacuoles important for maintaining cell turgor and for the transport and storage of ions and metabolites. One driving force for solute transport across the vacuolar membrane (tonoplast) is provided by an ATP-dependent electrogenic H+ pump. The tonoplast H+-pumping ATPase from oat roots has been solubilized with Triton X-100 and purified 16-fold by Sepharose 4B chromatography. The partially purified enzyme was sensitive to the same inhibitors (N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide (DCCD), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, and NO-3) as the native membrane-bound enzyme. The partially purified enzyme was stimulated by Cl- (Km(app) = 1.0 mM) and hydrolyzed ATP with a Km(app) of 0.25 mM. Thus, the partially purified tonoplast ATPase has retained the properties of the native membrane-bound enzyme. [14C]DCCD labeled a single polypeptide (14-18 kDa) in the purified tonoplast AT...
PLoS ONE, 2013
Background: The protein tyrosine phosphatase PRL-1 represents a putative oncogene with wide-rangi... more Background: The protein tyrosine phosphatase PRL-1 represents a putative oncogene with wide-ranging cellular effects. Overexpression of PRL-1 can promote cell proliferation, survival, migration, invasion, and metastasis, but the underlying mechanisms by which it influences these processes remain poorly understood. Methodology: To increase our comprehension of PRL-1 mediated signaling events, we employed transcriptional profiling (DNA microarray) and proteomics (mass spectrometry) to perform a thorough characterization of the global molecular changes in gene expression that occur in response to stable PRL-1 overexpression in a relevant model system (HEK293).
Trends in Plant Science, 1996
Proceedings of the National Academy of Sciences, 2002
A Saccharomyces cerevisae microarray expression study indicated that an ORF, YER044C, now designa... more A Saccharomyces cerevisae microarray expression study indicated that an ORF, YER044C, now designated ERG28, was strongly coregulated with ergosterol biosynthesis. Disruption of the ERG28 gene results in slow growth and accumulation of sterol intermediates similar to those observed in erg26 and erg27 null strains, suggesting that the Erg28p may interact with Erg26p and͞or Erg27p. In this study, a peptide from human hemagglutinin protein (HA) epitope tag was added to ERG26 and ERG27 genes, and a Myc tag was added to the ERG28 gene to detect interactions between Erg28p and Erg26p͞ Erg27p. Differential centrifugation showed that Erg26p, Erg27p, and Erg28p are all membrane-associated proteins. Green fluorescent protein-fusion protein localization studies showed that Erg26p, Erg27p, and Erg28p are all located in the endoplasmic reticulum. Solubilized membrane protein coimmunoprecipitation studies using rabbit anti-Erg25p indicated that Erg25p coimmunoprecipitates with both Erg27p and Erg28p. Erg28p was also shown to reciprocally coimmunoprecipitate with Erg27p. However, no coimmunoprecipitation was observed with Erg26p, most likely because of the poor solubilization of this protein. Sucrose gradient ultracentrifugation studies suggested that Erg25p͞Erg26p͞Erg27p͞Erg28p, along with other proteins in sterol biosynthesis, might form a complex between 66 and 200 kDa. Using an anti-HA column with Erg27p-HA and Erg26p-HA as target proteins, a complex containing Erg25p͞Erg26p͞ Erg27p͞Erg28p was identified. Thus, we suggest that Erg28p works as a transmembrane scaffold to tether Erg27p and possibly other C-4 demethylation proteins (Erg25p, Erg26p), forming a demethylation complex in the endoplasmic reticulum.
Plant, Cell and Environment, 2005
Skip to Main Content. Wiley Online Library will be disrupted 3 Sep from 10-12 BST for monthly mai... more Skip to Main Content. Wiley Online Library will be disrupted 3 Sep from 10-12 BST for monthly maintenance. ...
Plant Physiology and Biochemistry, 2013
Soybean (Glycine max) is a relatively cold intolerant plant. In most stress tolerant plants the r... more Soybean (Glycine max) is a relatively cold intolerant plant. In most stress tolerant plants the responsive expression of dehydrin proteins in vegetative tissues can be a significant contributor to protection against environmental stresses. The purpose of this study was to examine the expression of dehydrins in various organs and the cold-responses of dehydrin genes in vegetative tissues of soybean. Examination of the soybean genome indicated the presence of genes encoding ten distinct dehydrins. Levels of dehydrin proteins were probed with several antibodies specific to dehydrins or to the signature K-sequence. A single vegetatively expressed dehydrin protein was detected and the levels were insignificantly altered in response to cold, drought, or salt stress, nor was the transcript responsive to ABA. This SK2-type, acidic dehydrin family member (GmERD14) was purified, identified by mass spectroscopy, and shown to be in vivo phosphorylated; indicating characteristics similar to other known acidic dehydrins. The lack of cold stress-regulated acidic dehydrin expression may contribute to the inability of soybean to cold acclimate. While transcripts for all ten dehydrins could be detected in various tissues, only three accumulated to significant levels in vegetative tissues (two of the KS type and one of SK2 type). One of these transcripts, a KS dehydrin, was accumulated following cold treatments. The accumulation of the KS dehydrin was also responsive to exogenous ABA.
PLANT PHYSIOLOGY, 1992
The vacuole plays a major structural and biochemical role in the higher plant cell. Among the mos... more The vacuole plays a major structural and biochemical role in the higher plant cell. Among the most studied properties of the vacuole have been transport activities. One important aspect of vacuolar function is its participation in the regulation of cytosolic calcium levels. To identify the molecular entities involved in calcium regulation, a study of vacuole-associated, calcium-binding proteins (CaBs) was initiated. A competition assay was used, and it was observed that the majority of the total cellular membrane-associated, calcium-binding activity resided in low-density fractions enriched in vacuole membranes. Much of that calcium-binding activity was inactivated by a 0.5 M KI wash, and of the remaining activity, 77% was estimated to be peripherally associated with vacuolar membranes, whereas 23% was integrally associated with the vacuolar membrane. Calcium-ligand blots were used, and four major CaBs, with apparent molecular masses of 64, 58, 55, and 42 kD, were detected in purified vacuole membrane fractions. Two of these, the 58-and the 55-kD polypeptide, also appear to be present in significant amounts in endoplasmic reticulum-enriched fractions. However, the 64-and the 42-kD polypeptide are found primarily in vacuolar fractions. It is interesting that expression of the 42-kD polypeptide appears to be restricted to the heavily vacuolated cortical tissues (i.e. it is not found in vascular tissues). The localization of CaBs in the vacuole is consistent with the presence of calcium uptake (H'/Ca2' antiport) and releaseinechanisms (inositol trisphosphate sensitive) on vacuolar membranes. These vacuoleassociated CaBs, which may play a role in calcium buffering, together with the calcium transport systems, could mediate the vacuolar component of cellular calcium homeostasis.
PLANT PHYSIOLOGY, 1989
To determine whether the tonoplast-type H+-ATPase was differentially synthesized in various parts... more To determine whether the tonoplast-type H+-ATPase was differentially synthesized in various parts of the oat seedling, sections of 4-day-old oat (Avena satlva L. var Lang) seedlings were labeled in vivo with [3MS]methionine and ATPase subunits were precipitated with polyclonal antisera. ATPase subunits were detected in all portions of the seedling with the exception of the seed. Lesser amounts of the 60 and 72 kilodalton polypeptides of the ATPase were found in apical regions (0-5 millimeter) than in maturing regions (10-15, or 20-25 millimeter from the tip) of the roots or shoots. To initiate a study of the biosynthesis of the ATPase, the intracellular site of synthesis for two peripheral ATPase subunits was investigated. Poly(A) RNA from either free or membrane-bound polysomes was isolated and translated in vitro. Message encoding the 72 kilodalton (catalytic) subunit was found predominantly in mRNA isolated from membrane-bound polysomes. In contrast, the message for the 60 kilodalton (putative regulatory) subunit was found predominantly on free polysomes. Polypeptides synthesized in vivo or obtained from RNA translated in vitro exhibited no apparent size differences (limit of resolution, approximately I kilodalton), suggesting the absence of cleaved precursors for the 72 or 60 kilodalton subunits. These data suggest a complex mechanism for the synthesis and assembly of the tonoplast ATPase.
Identifying and characterizing cold responsive genes in Fragaria vesca associated with or respons... more Identifying and characterizing cold responsive genes in Fragaria vesca associated with or responsible for low temperature tolerance is a vital part of strawberry cultivar development. In this study we have investigated the transcript levels of eight genes, two dehydrin genes, three putative ABA-regulated genes, two cold–inducible CBF genes and the alcohol dehydrogenase gene, extracted from leaf and crown tissues of three F. vesca genotypes that vary in cold tolerance. Transcript levels of the CBF/DREB1 transcription factor FvCBF1E exhibited stronger cold up-regulation in comparison to FvCBF1B.1 in all genotypes. Transcripts of FvADH were highly up-regulated in both crown and leaf tissues from all three genotypes. In the ‘ALTA’ genotype, FvADH transcripts were significantly higher in leaf than crown tissues and more than 10 to 20-fold greater than in the less cold-tolerant ‘NCGR1363’ and ‘FDP817’ genotypes. FvGEM, containing the conserved ABRE promoter element, transcript was found t...
Plant science : an international journal of experimental plant biology, 2016
Soybean (Glycine max) is considered to be cold intolerant and is not able to significantly acclim... more Soybean (Glycine max) is considered to be cold intolerant and is not able to significantly acclimate to cold/freezing stress. In most cold tolerant plants, the C-repeat/DRE Binding Factors (CBF/DREBs) are critical contributors to successful cold-responses; rapidly increasing following cold treatment and regulating the induction of many cold responsive genes. In soybean vegetative tissue, we found strong, transient accumulation of CBF transcripts in response to cold stress; however, the soybean transcripts of typical cold responsive genes (homologues to Arabidopsis genes such as dehydrins, ADH1, RAP2.1, and LEA14) were not significantly altered. Soybean CBFs were found to be functional, as when expressed constitutively in Arabidopsis they increased the levels of AtCOR47 and AtRD29a transcripts and increased freezing tolerance as measured by a decrease in leaf freezing damage and ion leakage. Furthermore the constitutive expression of GmDREB1A;2 and GmDREB1B;1 in Arabidopsis led to st...
Isoprenylation is a posttranslational modification that is believed to be necessary, but not suff... more Isoprenylation is a posttranslational modification that is believed to be necessary, but not sufficient, for the efficient association of numerous eukaryotic cell proteins with membranes. Additional modifications have been shown to be required for proper intracellular targeting and function of certain isoprenylated proteins in mammalian and yeast cells. Although protein isoprenylation has been demonstrated in plants, postisoprenylation processing of plant proteins has not been described. Here we demonstrate that cultured tobacco (Nicotiana tabacum cv Bright Yellow-2) cells contain farnesylcysteine and geranylgeranylcysteine ␣-carboxyl methyltransferase activities with apparent Michaelis constants of 73 and 21 M for N-acetyl-S-trans,trans-farnesyl-L-cysteine and N-acetyl-S-alltrans-geranylgeranyl-L-cysteine, respectively. Furthermore, competition analysis indicates that the same enzyme is responsible for both activities. These results suggest that ␣-carboxyl methylation is a step in the maturation of isoprenylated proteins in plants.
Plant Molecular Biology, Sep 1, 1996
To identify isoprenylated plant GTP-binding proteins, Arabidopsis thaliana and Nicotiana tabacum ... more To identify isoprenylated plant GTP-binding proteins, Arabidopsis thaliana and Nicotiana tabacum cDNA expression libraries were screened for cDNA-encoded proteins capable of binding [32P]GTP in vitro. ATGB2, an Arabidopsis homologue of the GTP-binding protein Rab2, was found to bind GTP in vitro and to be a substrate for a geranylgeranyl:protein transferase (GGTase) present in plant extracts. The carboxyl terminus of this protein contains a -GCCG sequence, which has not previously been shown to be recognized by any prenyl:protein transferase (PTase), but which most closely resembles that isoprenylated by the type II GGTase (-XXCC, -XCXC, or -CCXX). In vitro geranylgeranylation of an Arabidopsis Rab1 protein containing a carboxyl-terminal-CCGQ sequence confirmed the presence of a type II GGTase-like activity in plant extracts. Several other proteins were also identified by in vitro GTP binding, including Arabidopsis and tobacco homologues of Rab11, ARF (ADP-ribosylation factor) and Sar proteins, as well as a novel 22 kDa Arabidopsis protein (ATG81). This 22 kDa protein had consensus GTP-binding motifs and bound GTP with high specificity, but its structure was not closely related to that of any known GTP-binding protein (it most resembled proteins within the ARF/Sar and G protein alpha-subunit superfamilies).
Plant Physiology, Sep 1, 1998
Isoprenylation is a posttranslational modification that is believed to be necessary, but not suff... more Isoprenylation is a posttranslational modification that is believed to be necessary, but not sufficient, for the efficient association of numerous eukaryotic cell proteins with membranes. Additional modifications have been shown to be required for proper intracellular targeting and function of certain isoprenylated proteins in mammalian and yeast cells. Although protein isoprenylation has been demonstrated in plants, postisoprenylation processing of plant proteins has not been described. Here we demonstrate that cultured tobacco (Nicotiana tabacum cv Bright Yellow-2) cells contain farnesylcysteine and geranylgeranylcysteine alpha-carboxyl methyltransferase activities with apparent Michaelis constants of 73 and 21 &mgr;M for N-acetyl-S-trans, trans-farnesyl-L-cysteine and N-acetyl-S-all-trans-geranylgeranyl-L-cysteine, respectively. Furthermore, competition analysis indicates that the same enzyme is responsible for both activities. These results suggest that alpha-carboxyl methylation is a step in the maturation of isoprenylated proteins in plants.
Cancer Lett, 1996
Many isoprenylated proteins are known to participate in signal transduction, but not all have bee... more Many isoprenylated proteins are known to participate in signal transduction, but not all have been identified. Using an in vitro prenylation screen, two human cDNAs (FI'PCAAXI and PTkIAAX2) homologous to the rat PRL-I and human OV-1 protein tyrosine phosphatase genes were identified. PTPCAAXI and PTPCAAXZ were famesylated in vitro by mammalian farnesyl:protein transferase, and epitope-tagged PW~AAXZ was prenylated in epithelial cells. Overexpression of PTPCAAXI and PTPCAAXZ in epithelial cells caused a transformed phenotype in culture and tumor growth in nude mice. Thus, PTPCAAXI and PTPCAAXZ represent a novel class of isoprenylated, oncogenic protein tyrosine phosphatases.
The Journal of Biological Chemistry, May 25, 1987
The purified tonoplast H*-ATPase from oat roots (Avena sativa L. var. Lang) consists of at least ... more The purified tonoplast H*-ATPase from oat roots (Avena sativa L. var. Lang) consists of at least three different polypeptides with masses 72,60, and 16 kDa. We have used covalent modifiers (inhibitors) and polyclonal antibodies to identify the catalytic subunit of the H+-pumping ATPase. The inactivation of ATPase activity by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (Nbd-C1, an adenine analog) was protected by MgATP or MgADP, and showed kinetic properties consistent with active site-directed inhibition. Under similar conditions, [14C]Nbd-C1 preferentially labeled the 72-kDa polypeptide of the purified ATPase. This binding was reduced by MgATP or 2' (3')-)0-(2,4,6trinitrophenyl) ATP. Nbd-C1 probably modified cysteinyl-SH or tyrosyl-OH groups, as dithiothreitol reversed both ATPase inactivation and I"C]Nbd-Cl binding to the 72-kDa subunit. The finding that Nethylmaleimide inhibition of ATPase activity was protectable by nucleotides is consistent with the idea of sulfhydryl groups in the ATP-binding site. Polyclonal antibody made to the 72-kDa polypeptide specifically reacted (Western blot) with a 72-kDa polypeptide from both tonoplast-enriched membranes and the purified tonoplast ATPase, but it did not cross-react with the mitochondrial or Escherichia coli F,-ATPase. The antibody inhibited tonoplast ATPase and H*-pumping activities. We conclude from these results that the 72-kDa polypeptide of the tonoplast H+-ATPase contains an ATP-(or nucleotide-) binding site that may constitute the catalytic domain.
Plant Physiology, Dec 1, 1985
The tonoplast ATPase of oat roots is composed of at least three polypeptides of 72, 60, and 16 kD... more The tonoplast ATPase of oat roots is composed of at least three polypeptides of 72, 60, and 16 kDa. The 16 kDA polypeptide covalently binds N,N'-dicyclohexylcarbodiimide and is postulated to be a component of the proton channel. Initial studies to identify other subunits indicate that both the 72 and 60 kDa subunits covalently bind ¹â´C)-7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and (¹â´C)N-ethylamleimide, inhibitors of the tonoplast ATPase. ATP prevents binding of these inhibitors suggesting that both the 72 and 60 kDa subunits are involved in substrate binding. Polyclonal antibody has been made to the 72 kDa subunit. Western blot analysis of tonoplast vesicles reveals single reactive polypeptide (72 kDa). The antibody shows no cross-reactivity towards either the mitochondrial Fâ-ATPase or the plasma membrane ATPase. This antibody specifically inhibits ATP hydrolysis and ATP-dependent H/sup +/ pumping in native tonoplast vesicles. The authors conclude that the 72 kDa subunit is intimately associated with the catalytic (or ATP-binding) site.
Methods in Enzymology, 1987
American journal of translational research, 2012
The PRL-1 and PRL-2 phosphatases have been implicated as oncogenic, however the involvement of th... more The PRL-1 and PRL-2 phosphatases have been implicated as oncogenic, however the involvement of these molecules in human neoplasms is not well understood. To increase understanding of the role PRL-1 and PRL-2 play in the neoplastic process, in situ hybridization was used to examine PRL-1 and PRL-2 mRNA expression in 285 normal, benign, and malignant human tissues of diverse origin. Immunohistochemical analysis was performed on a subset of these. PRL-1 and PRL-2 mRNA expression was also assessed in a small set of samples from a variety of diseases other than cancer. Where possible, associations with clinicopathological characteristics were evaluated. Alterations in PRL-1 or -2 expression were a frequent event, but the nature of those alterations was highly tumor type specific. PRL-1 was significantly overexpressed in 100% of hepatocellular and gastric carcinomas, but significantly under-expressed in 100% of ovarian, 80% of breast, and 75% of lung tumors. PRL-2 expression was significa...
The Journal of biological chemistry, Jan 25, 1986
Higher plant cells have one or more vacuoles important for maintaining cell turgor and for the tr... more Higher plant cells have one or more vacuoles important for maintaining cell turgor and for the transport and storage of ions and metabolites. One driving force for solute transport across the vacuolar membrane (tonoplast) is provided by an ATP-dependent electrogenic H+ pump. The tonoplast H+-pumping ATPase from oat roots has been solubilized with Triton X-100 and purified 16-fold by Sepharose 4B chromatography. The partially purified enzyme was sensitive to the same inhibitors (N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide (DCCD), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, and NO-3) as the native membrane-bound enzyme. The partially purified enzyme was stimulated by Cl- (Km(app) = 1.0 mM) and hydrolyzed ATP with a Km(app) of 0.25 mM. Thus, the partially purified tonoplast ATPase has retained the properties of the native membrane-bound enzyme. [14C]DCCD labeled a single polypeptide (14-18 kDa) in the purified tonoplast AT...
PLoS ONE, 2013
Background: The protein tyrosine phosphatase PRL-1 represents a putative oncogene with wide-rangi... more Background: The protein tyrosine phosphatase PRL-1 represents a putative oncogene with wide-ranging cellular effects. Overexpression of PRL-1 can promote cell proliferation, survival, migration, invasion, and metastasis, but the underlying mechanisms by which it influences these processes remain poorly understood. Methodology: To increase our comprehension of PRL-1 mediated signaling events, we employed transcriptional profiling (DNA microarray) and proteomics (mass spectrometry) to perform a thorough characterization of the global molecular changes in gene expression that occur in response to stable PRL-1 overexpression in a relevant model system (HEK293).
Trends in Plant Science, 1996
Proceedings of the National Academy of Sciences, 2002
A Saccharomyces cerevisae microarray expression study indicated that an ORF, YER044C, now designa... more A Saccharomyces cerevisae microarray expression study indicated that an ORF, YER044C, now designated ERG28, was strongly coregulated with ergosterol biosynthesis. Disruption of the ERG28 gene results in slow growth and accumulation of sterol intermediates similar to those observed in erg26 and erg27 null strains, suggesting that the Erg28p may interact with Erg26p and͞or Erg27p. In this study, a peptide from human hemagglutinin protein (HA) epitope tag was added to ERG26 and ERG27 genes, and a Myc tag was added to the ERG28 gene to detect interactions between Erg28p and Erg26p͞ Erg27p. Differential centrifugation showed that Erg26p, Erg27p, and Erg28p are all membrane-associated proteins. Green fluorescent protein-fusion protein localization studies showed that Erg26p, Erg27p, and Erg28p are all located in the endoplasmic reticulum. Solubilized membrane protein coimmunoprecipitation studies using rabbit anti-Erg25p indicated that Erg25p coimmunoprecipitates with both Erg27p and Erg28p. Erg28p was also shown to reciprocally coimmunoprecipitate with Erg27p. However, no coimmunoprecipitation was observed with Erg26p, most likely because of the poor solubilization of this protein. Sucrose gradient ultracentrifugation studies suggested that Erg25p͞Erg26p͞Erg27p͞Erg28p, along with other proteins in sterol biosynthesis, might form a complex between 66 and 200 kDa. Using an anti-HA column with Erg27p-HA and Erg26p-HA as target proteins, a complex containing Erg25p͞Erg26p͞ Erg27p͞Erg28p was identified. Thus, we suggest that Erg28p works as a transmembrane scaffold to tether Erg27p and possibly other C-4 demethylation proteins (Erg25p, Erg26p), forming a demethylation complex in the endoplasmic reticulum.
Plant, Cell and Environment, 2005
Skip to Main Content. Wiley Online Library will be disrupted 3 Sep from 10-12 BST for monthly mai... more Skip to Main Content. Wiley Online Library will be disrupted 3 Sep from 10-12 BST for monthly maintenance. ...
Plant Physiology and Biochemistry, 2013
Soybean (Glycine max) is a relatively cold intolerant plant. In most stress tolerant plants the r... more Soybean (Glycine max) is a relatively cold intolerant plant. In most stress tolerant plants the responsive expression of dehydrin proteins in vegetative tissues can be a significant contributor to protection against environmental stresses. The purpose of this study was to examine the expression of dehydrins in various organs and the cold-responses of dehydrin genes in vegetative tissues of soybean. Examination of the soybean genome indicated the presence of genes encoding ten distinct dehydrins. Levels of dehydrin proteins were probed with several antibodies specific to dehydrins or to the signature K-sequence. A single vegetatively expressed dehydrin protein was detected and the levels were insignificantly altered in response to cold, drought, or salt stress, nor was the transcript responsive to ABA. This SK2-type, acidic dehydrin family member (GmERD14) was purified, identified by mass spectroscopy, and shown to be in vivo phosphorylated; indicating characteristics similar to other known acidic dehydrins. The lack of cold stress-regulated acidic dehydrin expression may contribute to the inability of soybean to cold acclimate. While transcripts for all ten dehydrins could be detected in various tissues, only three accumulated to significant levels in vegetative tissues (two of the KS type and one of SK2 type). One of these transcripts, a KS dehydrin, was accumulated following cold treatments. The accumulation of the KS dehydrin was also responsive to exogenous ABA.
PLANT PHYSIOLOGY, 1992
The vacuole plays a major structural and biochemical role in the higher plant cell. Among the mos... more The vacuole plays a major structural and biochemical role in the higher plant cell. Among the most studied properties of the vacuole have been transport activities. One important aspect of vacuolar function is its participation in the regulation of cytosolic calcium levels. To identify the molecular entities involved in calcium regulation, a study of vacuole-associated, calcium-binding proteins (CaBs) was initiated. A competition assay was used, and it was observed that the majority of the total cellular membrane-associated, calcium-binding activity resided in low-density fractions enriched in vacuole membranes. Much of that calcium-binding activity was inactivated by a 0.5 M KI wash, and of the remaining activity, 77% was estimated to be peripherally associated with vacuolar membranes, whereas 23% was integrally associated with the vacuolar membrane. Calcium-ligand blots were used, and four major CaBs, with apparent molecular masses of 64, 58, 55, and 42 kD, were detected in purified vacuole membrane fractions. Two of these, the 58-and the 55-kD polypeptide, also appear to be present in significant amounts in endoplasmic reticulum-enriched fractions. However, the 64-and the 42-kD polypeptide are found primarily in vacuolar fractions. It is interesting that expression of the 42-kD polypeptide appears to be restricted to the heavily vacuolated cortical tissues (i.e. it is not found in vascular tissues). The localization of CaBs in the vacuole is consistent with the presence of calcium uptake (H'/Ca2' antiport) and releaseinechanisms (inositol trisphosphate sensitive) on vacuolar membranes. These vacuoleassociated CaBs, which may play a role in calcium buffering, together with the calcium transport systems, could mediate the vacuolar component of cellular calcium homeostasis.
PLANT PHYSIOLOGY, 1989
To determine whether the tonoplast-type H+-ATPase was differentially synthesized in various parts... more To determine whether the tonoplast-type H+-ATPase was differentially synthesized in various parts of the oat seedling, sections of 4-day-old oat (Avena satlva L. var Lang) seedlings were labeled in vivo with [3MS]methionine and ATPase subunits were precipitated with polyclonal antisera. ATPase subunits were detected in all portions of the seedling with the exception of the seed. Lesser amounts of the 60 and 72 kilodalton polypeptides of the ATPase were found in apical regions (0-5 millimeter) than in maturing regions (10-15, or 20-25 millimeter from the tip) of the roots or shoots. To initiate a study of the biosynthesis of the ATPase, the intracellular site of synthesis for two peripheral ATPase subunits was investigated. Poly(A) RNA from either free or membrane-bound polysomes was isolated and translated in vitro. Message encoding the 72 kilodalton (catalytic) subunit was found predominantly in mRNA isolated from membrane-bound polysomes. In contrast, the message for the 60 kilodalton (putative regulatory) subunit was found predominantly on free polysomes. Polypeptides synthesized in vivo or obtained from RNA translated in vitro exhibited no apparent size differences (limit of resolution, approximately I kilodalton), suggesting the absence of cleaved precursors for the 72 or 60 kilodalton subunits. These data suggest a complex mechanism for the synthesis and assembly of the tonoplast ATPase.