Fang-jen Lee - Academia.edu (original) (raw)
Papers by Fang-jen Lee
Journal of Biological Chemistry
ABSTRACT
Methods in Enzymology, 2001
ABSTRACT This chapter describes a method to prepare recombinant y adenosine diphospahte (ADP) rib... more ABSTRACT This chapter describes a method to prepare recombinant y adenosine diphospahte (ADP) ribosylation factors (ARF)-like proteins (ARL)3 from Escherichia coli using a pET-based vector. Recombinant yARL3 protein is isolated in a functional form from the soluble fraction of the bacterial lysate using a rapid two-step procedure involving Ni2+-agarose chromatography followed by gel filtration. This chapter also describes assays to quantify guanine nucleotide binding and guanosine tri phosphate (GTP) hydrolysis. The ability of purified recombinant yARL3 to bind GTPTS is determined by a filter trapping method. GTP hydrolysis is determined by binding [α-32p]GTP to recombinant yARL3 protein.
AfCS-Nature Molecule Pages, 2010
PLoS ONE, 2013
Activated chemokine receptor initiates inside-out signaling to transiently trigger activation of ... more Activated chemokine receptor initiates inside-out signaling to transiently trigger activation of integrins, a process involving multiple components that have not been fully characterized. Here we report that GM-CSF/IL-3/IL-5 receptor common betachain-associated protein (CBAP) is required to optimize this inside-out signaling and activation of integrins. First, knockdown of CBAP expression in human Jurkat T cells caused attenuated CXC chemokine ligand-12 (CXCL12)-induced cell migration and integrin a4b1and aLb2-mediated cell adhesion in vitro, which could be rescued sufficiently upon expression of murine CBAP proteins. Freshly isolated CBAP-deficient primary T cells also exhibited diminution of chemotaxis toward CC chemokine ligand-21 (CCL21) and CXCL12, and these chemokines-induced T-cell adhesions in vitro. Adoptive transfer of isolated naive T cells demonstrated that CBAP deficiency significantly reduced lymph node homing ability in vivo. Finally, migration of T cell-receptor-activated T cells induced by inflammatory chemokines was also attenuated in CBAP-deficient cells. Further analyses revealed that CBAP constitutively associated with both integrin b1 and ZAP70 and that CBAP is required for chemokine-induced initial binding of the talin-Vav1 complex to integrin b1 and to facilitate subsequent ZAP70mediated dissociation of the talin-Vav1 complex and Vav1 phosphorylation. Within such an integrin signaling complex, CBAP likely functions as an adaptor and ultimately leads to activation of both integrin a4b1 and Rac1. Taken together, our data suggest that CBAP indeed can function as a novel signaling component within the ZAP70/Vav1/talin complex and plays an important role in regulating chemokine-promoted T-cell trafficking.
Journal of Biological Chemistry, 1994
ADP-ribosylation factors (ARFs) are highly conserved approximately 20-kDa guanine nucleotide-bind... more ADP-ribosylation factors (ARFs) are highly conserved approximately 20-kDa guanine nucleotide-binding proteins that enhance the ADP-ribosyltransferase activity of cholera toxin, and are believed to participate in vesicular transport in both exocytic and endocytic pathways. Based on size, phylogenetic analysis, amino acid sequence, and gene structure, mammalian ARFs fall into three classes (class I, ARFs 1, 2, 3; class II, ARFs 4, 5; class III, ARF6). Two ARF genes (yARF1, yARF2) are known in Saccharomyces cerevisiae and believed to participate in vesicular trafficking in the Golgi system; the double deletion mutant is not viable. A third yeast ARF (yARF3) cDNA has been cloned by polymerase chain reaction-based procedures. It contains an open reading frame of 549 bases encoding a protein of 183 amino acids, with a deduced amino acid sequence more identical (60%) to that of the class III mammalian ARF than to those of the other two classes (52-56%). The yARF3 protein, however, reacted poorly with antibodies against any of the three classes of mammalian ARFs. In the presence of GTP, recombinant yARF3 protein stimulated cholera toxin-catalyzed auto-ADP-ribosylation. yARF3 gene transcription, similar to that of yARF2, was repressed by glucose. As yARF3 was not essential for cell viability and was not required for endoplasmic reticulum to Golgi protein transport, it may provide an opportunity to define an ARF function in another kind of vesicular trafficking.
Journal of Biological Chemistry, 1992
Giardia lamblia is a protozoan intestinal parasite that has characteristics of both eukaryotes an... more Giardia lamblia is a protozoan intestinal parasite that has characteristics of both eukaryotes and prokaryotes. To determine whether genes for guanine nucleotide-binding proteins are present in Giardia, genomic DNA and cDNA libraries were screened by polymerase chain reaction and by hybridization with mixed oligonucleotide probes complementary to sequences encoding conserved GTP-binding domains. A gene with a high degree of sequence identity with mammalian ADP-ribosylation factors (ARFs), believed to be important in vesicular transport, was identified. The Giardia ARF gene had a 573-base open reading frame encoding 191 amino acids which are 63-70% identical with known mammalian and yeast ARFs. Sequence conservation among ARFs was greatest in putative GTP-binding domains. A single ARF mRNA species of approximately 750 bases was found in two different Giardia isolates. Primer extension and RNA sequencing of the Giardia ARF transcript revealed a short (6-base) 5'-untranslated region similar in size to those found in other Giardia transcripts. Giardia extracts contained ARF activity, as shown by stimulation of cholera toxin-catalyzed ADP-ribosylation and a Giardia ARF expressed in Escherichia coli as a fusion protein likewise exhibited biochemical activity. Its presence in Giardia is consistent with the view that ARF emerged before the divergence of this protozoan from other eukaryotes (approximately 1.5 billion years ago), and that an ARF-like protein may have been the ancestor of several other classes of signal-transducing guanine nucleotide-binding proteins, including the alpha subunits of the heterotrimeric G proteins.
Journal of Biological Chemistry, 1993
A gene, termed RNA-binding protein (RBP1), was cloned from Saccharomyces cerevisiae. RBP1 contain... more A gene, termed RNA-binding protein (RBP1), was cloned from Saccharomyces cerevisiae. RBP1 contains an open reading frame of 2016 nucleotides that encodes a 672-amino acid protein with a calculated M(r) of approximately 75,000. Southern blots of genomic DNA from wild-type and RBP1-disrupted strains were consistent with the presence of homologous genes. RNA blots revealed a major 2.7-kb RNA band and two minor bands of 1.5 and 1.1 kb. The sequence of the putative RBP1 protein contains two copies of an RNA recognition motif, two glutamine stretches, an asparagine-rich region, a methionine-rich region, and two long potential alpha-helixes. In addition, recombinant RBP1 fusion protein can bind to RNA and single-stranded DNA but not double-stranded DNA. RBP1 is a glucose-repressible gene. Disruption of RBP1 increased cell growth rate in the early log phase. Overexpression of RBP1 or reduction in its translation by expression of antisense RNA decreased or increased the cell growth rate, respectively. From these observations, we infer that RBP1 may be involved in growth regulation, possibly through its participation in RNA metabolism.
Journal of Bacteriology, 1989
Acetylation is the most frequently occurring chemical modification of the alpha-NH2 group of euca... more Acetylation is the most frequently occurring chemical modification of the alpha-NH2 group of eucaryotic proteins and is catalyzed by N alpha-acetyltransferase. The yeast enzyme is encoded by the AAA1 (amino-terminal alpha-amino acetyltransferase) gene. A null mutation (aaa1-1) created by gene replacement, while not lethal, slows cell growth and results in heterogeneous colony morphology. In comparison with wild-type cells, aaa1-1/aaa1-1 diploids cannot enter stationary phase, are sporulation defective, and are sensitive to heat shock. In addition, the aaa1-1 mutation specifically reduces mating functions of MATa cells. These results indicate that N alpha acetylation plays a crucial role in yeast cell growth and mating.
Journal of Free Radicals in Biology & Medicine, 1985
Growth of Saccharomyces cerevisiae in the presence of paraquat caused an increase the intracellul... more Growth of Saccharomyces cerevisiae in the presence of paraquat caused an increase the intracellular fluxof superoxide radicals and in superoxide dismutase biosynthesis. The addition of copper to the growth medium also elicited an increase in superoxide dismutase levels. A cytochrome c deficient mutant strain was found to be more responsive than the wild type strain to paraquat and/or copper by increasing its copper-zinc superoxide dismutase. Catalase activity in both strains was not significantly affected by paraquat and/or copper.
Journal of Biological Chemistry
ABSTRACT
Journal of Biological Chemistry
ABSTRACT
Methods in Enzymology, 2001
ABSTRACT This chapter describes a method to prepare recombinant y adenosine diphospahte (ADP) rib... more ABSTRACT This chapter describes a method to prepare recombinant y adenosine diphospahte (ADP) ribosylation factors (ARF)-like proteins (ARL)3 from Escherichia coli using a pET-based vector. Recombinant yARL3 protein is isolated in a functional form from the soluble fraction of the bacterial lysate using a rapid two-step procedure involving Ni2+-agarose chromatography followed by gel filtration. This chapter also describes assays to quantify guanine nucleotide binding and guanosine tri phosphate (GTP) hydrolysis. The ability of purified recombinant yARL3 to bind GTPTS is determined by a filter trapping method. GTP hydrolysis is determined by binding [α-32p]GTP to recombinant yARL3 protein.
AfCS-Nature Molecule Pages, 2010
PLoS ONE, 2013
Activated chemokine receptor initiates inside-out signaling to transiently trigger activation of ... more Activated chemokine receptor initiates inside-out signaling to transiently trigger activation of integrins, a process involving multiple components that have not been fully characterized. Here we report that GM-CSF/IL-3/IL-5 receptor common betachain-associated protein (CBAP) is required to optimize this inside-out signaling and activation of integrins. First, knockdown of CBAP expression in human Jurkat T cells caused attenuated CXC chemokine ligand-12 (CXCL12)-induced cell migration and integrin a4b1and aLb2-mediated cell adhesion in vitro, which could be rescued sufficiently upon expression of murine CBAP proteins. Freshly isolated CBAP-deficient primary T cells also exhibited diminution of chemotaxis toward CC chemokine ligand-21 (CCL21) and CXCL12, and these chemokines-induced T-cell adhesions in vitro. Adoptive transfer of isolated naive T cells demonstrated that CBAP deficiency significantly reduced lymph node homing ability in vivo. Finally, migration of T cell-receptor-activated T cells induced by inflammatory chemokines was also attenuated in CBAP-deficient cells. Further analyses revealed that CBAP constitutively associated with both integrin b1 and ZAP70 and that CBAP is required for chemokine-induced initial binding of the talin-Vav1 complex to integrin b1 and to facilitate subsequent ZAP70mediated dissociation of the talin-Vav1 complex and Vav1 phosphorylation. Within such an integrin signaling complex, CBAP likely functions as an adaptor and ultimately leads to activation of both integrin a4b1 and Rac1. Taken together, our data suggest that CBAP indeed can function as a novel signaling component within the ZAP70/Vav1/talin complex and plays an important role in regulating chemokine-promoted T-cell trafficking.
Journal of Biological Chemistry, 1994
ADP-ribosylation factors (ARFs) are highly conserved approximately 20-kDa guanine nucleotide-bind... more ADP-ribosylation factors (ARFs) are highly conserved approximately 20-kDa guanine nucleotide-binding proteins that enhance the ADP-ribosyltransferase activity of cholera toxin, and are believed to participate in vesicular transport in both exocytic and endocytic pathways. Based on size, phylogenetic analysis, amino acid sequence, and gene structure, mammalian ARFs fall into three classes (class I, ARFs 1, 2, 3; class II, ARFs 4, 5; class III, ARF6). Two ARF genes (yARF1, yARF2) are known in Saccharomyces cerevisiae and believed to participate in vesicular trafficking in the Golgi system; the double deletion mutant is not viable. A third yeast ARF (yARF3) cDNA has been cloned by polymerase chain reaction-based procedures. It contains an open reading frame of 549 bases encoding a protein of 183 amino acids, with a deduced amino acid sequence more identical (60%) to that of the class III mammalian ARF than to those of the other two classes (52-56%). The yARF3 protein, however, reacted poorly with antibodies against any of the three classes of mammalian ARFs. In the presence of GTP, recombinant yARF3 protein stimulated cholera toxin-catalyzed auto-ADP-ribosylation. yARF3 gene transcription, similar to that of yARF2, was repressed by glucose. As yARF3 was not essential for cell viability and was not required for endoplasmic reticulum to Golgi protein transport, it may provide an opportunity to define an ARF function in another kind of vesicular trafficking.
Journal of Biological Chemistry, 1992
Giardia lamblia is a protozoan intestinal parasite that has characteristics of both eukaryotes an... more Giardia lamblia is a protozoan intestinal parasite that has characteristics of both eukaryotes and prokaryotes. To determine whether genes for guanine nucleotide-binding proteins are present in Giardia, genomic DNA and cDNA libraries were screened by polymerase chain reaction and by hybridization with mixed oligonucleotide probes complementary to sequences encoding conserved GTP-binding domains. A gene with a high degree of sequence identity with mammalian ADP-ribosylation factors (ARFs), believed to be important in vesicular transport, was identified. The Giardia ARF gene had a 573-base open reading frame encoding 191 amino acids which are 63-70% identical with known mammalian and yeast ARFs. Sequence conservation among ARFs was greatest in putative GTP-binding domains. A single ARF mRNA species of approximately 750 bases was found in two different Giardia isolates. Primer extension and RNA sequencing of the Giardia ARF transcript revealed a short (6-base) 5'-untranslated region similar in size to those found in other Giardia transcripts. Giardia extracts contained ARF activity, as shown by stimulation of cholera toxin-catalyzed ADP-ribosylation and a Giardia ARF expressed in Escherichia coli as a fusion protein likewise exhibited biochemical activity. Its presence in Giardia is consistent with the view that ARF emerged before the divergence of this protozoan from other eukaryotes (approximately 1.5 billion years ago), and that an ARF-like protein may have been the ancestor of several other classes of signal-transducing guanine nucleotide-binding proteins, including the alpha subunits of the heterotrimeric G proteins.
Journal of Biological Chemistry, 1993
A gene, termed RNA-binding protein (RBP1), was cloned from Saccharomyces cerevisiae. RBP1 contain... more A gene, termed RNA-binding protein (RBP1), was cloned from Saccharomyces cerevisiae. RBP1 contains an open reading frame of 2016 nucleotides that encodes a 672-amino acid protein with a calculated M(r) of approximately 75,000. Southern blots of genomic DNA from wild-type and RBP1-disrupted strains were consistent with the presence of homologous genes. RNA blots revealed a major 2.7-kb RNA band and two minor bands of 1.5 and 1.1 kb. The sequence of the putative RBP1 protein contains two copies of an RNA recognition motif, two glutamine stretches, an asparagine-rich region, a methionine-rich region, and two long potential alpha-helixes. In addition, recombinant RBP1 fusion protein can bind to RNA and single-stranded DNA but not double-stranded DNA. RBP1 is a glucose-repressible gene. Disruption of RBP1 increased cell growth rate in the early log phase. Overexpression of RBP1 or reduction in its translation by expression of antisense RNA decreased or increased the cell growth rate, respectively. From these observations, we infer that RBP1 may be involved in growth regulation, possibly through its participation in RNA metabolism.
Journal of Bacteriology, 1989
Acetylation is the most frequently occurring chemical modification of the alpha-NH2 group of euca... more Acetylation is the most frequently occurring chemical modification of the alpha-NH2 group of eucaryotic proteins and is catalyzed by N alpha-acetyltransferase. The yeast enzyme is encoded by the AAA1 (amino-terminal alpha-amino acetyltransferase) gene. A null mutation (aaa1-1) created by gene replacement, while not lethal, slows cell growth and results in heterogeneous colony morphology. In comparison with wild-type cells, aaa1-1/aaa1-1 diploids cannot enter stationary phase, are sporulation defective, and are sensitive to heat shock. In addition, the aaa1-1 mutation specifically reduces mating functions of MATa cells. These results indicate that N alpha acetylation plays a crucial role in yeast cell growth and mating.
Journal of Free Radicals in Biology & Medicine, 1985
Growth of Saccharomyces cerevisiae in the presence of paraquat caused an increase the intracellul... more Growth of Saccharomyces cerevisiae in the presence of paraquat caused an increase the intracellular fluxof superoxide radicals and in superoxide dismutase biosynthesis. The addition of copper to the growth medium also elicited an increase in superoxide dismutase levels. A cytochrome c deficient mutant strain was found to be more responsive than the wild type strain to paraquat and/or copper by increasing its copper-zinc superoxide dismutase. Catalase activity in both strains was not significantly affected by paraquat and/or copper.
Journal of Biological Chemistry
ABSTRACT