SHP-1 sensitizes MCF-7 cells to trichostatin A-induced apoptosis by modulating PI3K-dependent events.
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Analysis of Variance↗
Biomedical subjects
D Banville
Publications and source records attributed to D Banville.
Transcriptional activity of the SHP-1 gene in MCF7 cells is differentially regulated by binding of NF-Y factor to two distinct CCAAT-elements.
Our previous studies have shown that SHP-1, a SH2 domain-containing protein-tyrosine phosphatase, is expressed not only in cells of hematopoietic lineages, but also in many non-hematopoietic cells under the control of an alternative tissue-specific promoter, P1. In this study, the activity of the P1 promoter was analyzed in a region spanning 3.5 kb upstream of the major transcription start site in non-hematopoietic MCF-7 cells. Using DNA footprinting, gel retardation assays and mutational analysis, we have characterized cis-regulatory elements that are essential to confer the P1 promoter activity. An upstream Sp1 element (-126 to -118) positively regulated this TATA-box-lacking promoter. Two inverted CCAAT-elements (-332 to -328 and -66 to -62) played important roles in regulating the SHP-1 gene expression, and transcription factor NF-Y predominantly bound to the two CCAAT-elements. Binding of NF-Y to the distal CCAAT-element enhanced the transcriptional activity of the P1 promoter. In contrast, binding of NF-Y to the proximal CCAAT-element and interacting with repressor(s) inhibited the promoter activity. Furthermore, incubation of MCF7 cells with 100 ng/ml trichostatin A, an inhibitor of histone deacetylase, significantly increased the activity of the P1 promoter. Mutation in the proximal CCAAT-element, however, eliminated the activating effect of trichostatin A on the promoter. Together, our data suggest that NF-Y factor can function either as a specific positive or negative regulator of P1 promoter activity in non-hematopoietic MCF7 cells.
Base Sequence↗
Identification and characterization of S2V, a novel putative siglec that contains two V set Ig-like domains and recruits protein-tyrosine phosphatases SHPs.
We describe the molecular cloning and characterization of S2V, a novel sialic acid binding immunoglobulin-like lectin. The cDNA of S2V encodes a type 1 transmembrane protein with four extracellular immunoglobulin-like (Ig-like) domains and a cytoplasmic tail bearing a typical immunoreceptor tyrosine-based inhibitory motif (ITIM) and an ITIM-like motif. A unique feature of S2V is the presence of two V-set Ig-like domains responsible for the binding to sialic acid, whereas all other known siglecs possess only one. S2V is predominantly expressed in macrophage. In vivo S2V was tyrosine-phosphorylated when co-expressed with exogenous c-Src kinase. Upon tyrosine phosphorylation, S2V recruits both Src homology 2 (SH2) domain-containing protein-tyrosine phosphatases SHP-1 and SHP-2, two important inhibitory regulators of immunoreceptor signal transduction. These findings suggest that S2V is involved in the negative regulation of the signaling in macrophage by functioning as an inhibitory receptor. When expressed in COS-7 cells, S2V was able to mediate sialic acid-dependent binding to human red blood cells, suggesting that S2V may function through cell-cell interaction.
Amino Acid Sequence↗
mSiglec-E, a novel mouse CD33-related siglec (sialic acid-binding immunoglobulin-like lectin) that recruits Src homology 2 (SH2)-domain-containing protein tyrosine phosphatases SHP-1 and SHP-2.
The sialic acid-binding immunoglobulin-like lectins (siglecs) represent a recently defined distinct subset of the immunoglobulin superfamily. By using the Src homology 2 (SH2)-domain-containing protein tyrosine phosphatase SHP-1 as bait in a yeast two-hybrid screen, we have identified a new member of the mouse siglec family, mSiglec-E. The mSiglec-E cDNA encodes a protein of 467 amino acids that contains three extracellular immunoglobulin-like domains, a transmembrane region and a cytoplasmic tail bearing two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). mSiglec-E is highly expressed in mouse spleen, a tissue rich in leucocytes. The ITIMs of mSiglec-E can recruit SHP-1 and SHP-2, two inhibitory regulators of immunoreceptor signal transduction. This suggests that the function of mSiglec-E is probably an involvement in haematopoietic cells and the immune system as an inhibitory receptor. When expressed in COS-7 cells, mSiglec-E was able to mediate sialic acid-dependent binding to human red blood cells, suggesting that mSiglec-E may function through cell-cell interactions. In comparison with the known members of the siglec family, mSiglec-E exhibits a high degree of sequence similarity to both human siglec-7 and siglec-9. The gene encoding mSiglec-E is localized in the same chromosome as that encoding mouse CD33. Phylogenetic analysis reveals that neither mouse mSiglec-E nor CD33 shows a clear relationship with any human siglecs so far identified.
Amino Acid Sequence↗
PILRalpha, a novel immunoreceptor tyrosine-based inhibitory motif-bearing protein, recruits SHP-1 upon tyrosine phosphorylation and is paired with the truncated counterpart PILRbeta.
SHP-1-mediated dephosphorylation of protein tyrosine residues is central to the regulation of several cell signaling pathways, the specificity of which is dictated by the intrinsic affinity of SH2 domains for the flanking sequences of phosphotyrosine residues. By using a modified yeast two-hybrid system and SHP-1 as bait, we have cloned a human cDNA, PILRalpha, encoding a 303-amino acid immunoglobulin-like transmembrane receptor bearing two cytoplasmic tyrosines positioned within an immunoreceptor tyrosine-based inhibitory motif. Substrate trapping in combination with pervanadate treatment of 293T cells confirms that PILRalpha associates with SHP-1 in vivo upon tyrosine phosphorylation. Mutation of the tyrosine residues in PILRalpha indicates the pivotal role of the Tyr-269 residue in recruiting SHP-1. Surface plasmon resonance analysis further suggests that the association between PILRalpha-Tyr-269 and SHP-1 is mediated primarily via the amino-terminal SH2 domain of the latter. Polymerase chain reaction amplification of cDNA in combination with genomic sequence analysis revealed a second gene, PILRbeta, coding for a putative activating receptor as suggested by a truncated cytoplasmic tail and a charged lysine residue in its transmembrane region. The PILRalpha and PILRbeta genes are localized to chromosome 7 which is in contrast with the mapping of known members of the inhibitory receptor superfamily.
Amino Acid Sequence↗
ZRP-1, a zyxin-related protein, interacts with the second PDZ domain of the cytosolic protein tyrosine phosphatase hPTP1E.
Protein-protein interactions play an important role in the specificity of cellular signaling cascades. By using the yeast two-hybrid system, a specific interaction was identified between the second PDZ domain of the cytosolic protein tyrosine phosphatase hPTP1E and a novel protein, which was termed ZRP-1 to indicate its sequence similarity to the Zyxin protein family. The mRNA encoding this protein is distributed widely in human tissues and contains an open reading frame of 1428 base pairs, predicting a polypeptide of 476 amino acid residues. The deduced protein displays a proline-rich amino-terminal region and three double zinc finger LIM domains at its carboxyl terminus. The specific interaction of this novel protein with the second PDZ domain of hPTP1E was demonstrated both in vitro, using bacterially expressed proteins, and in vivo, by co-immunoprecipitation studies. Deletion analysis indicated that an intact carboxyl terminus is required for its interaction with the second PDZ domain of hPTP1E in the yeast two-hybrid system and suggested that other sequences, including the LIM domains, also participate in the interaction. The genomic organization of the ZRP-1 coding sequence is identical to that of the lipoma preferred partner gene, another Zyxin-related protein, suggesting that the two genes have evolved from a recent gene duplication event.
ATPases Associated with Diverse Cellular Activitie↗
Fusion proteins could generate false positives in peptide phage display.
Fusion proteins are frequently used in the functional characterization of newly discovered proteins and to identify interacting partners. In our study of hPTP1E, a cytosolic protein tyrosine phosphatase, we used glutathione S-transferase (GST)-fusion protein of the second PDZ domain to identify interacting peptide motifs by peptide phage display. A consensus motif G X X V W L G was identified and found to be specific for binding to GST-PDZ2 as determined by ELISA, peptide displacement and by protein overlay. However, using nuclear magnetic resonance (NMR), no interaction of the peptide was observed with PDZ2 alone. In co-precipitation experiments using the consensus peptide cross-linked to Affi-Gel, only GST-PDZ2 (but not PDZ2 or GST alone) could be precipitated. These data suggest that there is a potential for identification of artifacts when using fusion proteins in peptide phage display, and one should exercise caution in interpreting these results. It is critical that the interaction be verified using a second, independent system.
Amino Acid Sequence↗
Epitope mapping of SHP-1 monoclonal antibodies using peptide phage display.
We have characterized the binding epitopes of four monoclonal antibodies for SHP-1, an SH2 domain containing protein tyrosine phosphatase, using two phage displayed random peptide libraries. Three of the antibodies are directed against the phosphatase domain of the molecule and the fourth is toward the NH2-terminal part of the second SH2 domain. The first two antibodies recognize the sequence NANY, amino acid 305 to amino acid 308, numbered in the non haematopoietic form of human SHP-1 sequence. The third antibody binds the sequence P Y W P (amino acids 365 to 368) located toward the middle of the phosphatase domain of the enzyme. The fourth antibody is directed against the first two amino acids, W Y (amino acids 112 and 113), of the second SH2 domain. The specificities of these antibodies are demonstrated by ELISA and western blot using different protein constructs expressed in bacteria. All the antibodies can detect wild type SHP-1, expressed in 293 cells, by western blot analysis, both under denaturing conditions as well as following renaturation. The data presented here show that the antibodies characterized in this study are raised against linear epitopes and suggest that these epitopes are accessible from the outside in the native SHP-1 molecule.
Amino Acid Sequence↗
SHP-1 associates with both platelet-derived growth factor receptor and the p85 subunit of phosphatidylinositol 3-kinase.
The Src homology 2 (SH2)-containing protein tyrosine phosphatase 1, SHP-1, is highly expressed in all hematopoietic cells as well as in many non-hematopoietic cells, particularly in some malignant epithelial cell lines. In hematopoietic cells, SHP-1 negatively regulates multiple cytokine receptor pathways. The precise function and the targets of SHP-1 in non-hematopoietic cells, however, are largely unknown. Here we demonstrate that SHP-1 associates with both the tyrosine-phosphorylated platelet-derived growth factor (PDGF) receptor and the p85 subunit of phosphatidylinositol 3-kinase in MCF-7 and TRMP cells. Through the use of mutant PDGF receptors and performing peptide competition for immunoprecipitation, it was determined that SHP-1 independently associates with the PDGF receptor and p85 and that its N-terminal SH2 domain is directly responsible for the interactions. Overexpression of SHP-1 in TRMP cells transfected with the PDGF receptor markedly inhibited PDGF-induced c-fos promoter activation, whereas the expression of three catalytically inactive SHP-1 mutants increased the c-fos promoter activation in response to PDGF stimulation. These results indicate that SHP-1 might negatively regulate PDGF receptor-mediated signaling in these cells. Identification of the association of SHP-1 with the PDGF receptor and p85 in MCF-7 and TRMP cells furthers our understanding of the function of SHP-1 in non-hematopoietic cells.
Animals↗
Main-chain signal assignment for the PDZ2 domain from human protein tyrosine phosphatase hPTP1E and its complex with a C-terminal peptide from the Fas receptor.
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Amino Acid Sequence↗
Effect of peptide binding on amide proton exchange rates in the PDZ2 domain from human phosphatase hPTP1E.
Amide hydrogen-deuterium exchange rates were measured in the PDZ2 domain from human phosphatase hPTPIE by 1H-15N heteronuclear NMR spectroscopy. Protection factors were calculated for the slowly exchanging hydrogens in both the free PDZ2 domain and its complex with an octapeptide peptide, R-N-E-I-Q-S-L-V, derived from the C-terminus of the Fas receptor. Aside from a short alpha-helical region alpha1 (amino acids A-45 to D-49), the pattern of highly protected amides correlated well with the presence of hydrogen bonds in elements of the secondary structure. Hydrogen-bonded amides showed relatively fast exchange rates with half-lives of less than 9 h at pD 7.6 and 8 degrees C. Protection factors, calculated as the ratio of theoretical (denatured) and observed exchange rates, showed less dispersion in maximal values than did the actual exchange rates. This behavior and the large pH dependence of the exchange rates suggest that amide exchange is close to the EX2 limit. In this limit, exchange of the most protected amides occurs through a global unfolding mechanism. The free energy of the unfolding calculated from the largest protection factors is 4.8 +/- 0.4 kcal/mol (1 cal = 4.184 J). This deltaG(o) closely matches the value measured by experiments with guanidine hydrochloride and fluorescence emission spectroscopy. Peptide binding to PDZ2 resulted in mostly global effects and stabilized the folded domain by 1.4 kcal/mol.
Deuterium↗
Protein-tyrosine phosphatase SHP2 is positively linked to proteinase-activated receptor 2-mediated mitogenic pathway.
Proteinase-activated receptor-2 (PAR2), a new member of family of the G protein-coupled receptors, is activated by proteolytic cleavage of its extracellular amino terminus, a mechanism similar to that used by the thrombin receptor. It has been suggested that PAR2 has a potential role in the late phases of the acute inflammatory response and in tissue repair and/or skin-related disorders. Here we demonstrate that the agonist peptide (SLIGRL) stimulated c-fos-mediated mitogenic activation and tyrosine phosphorylation of cellular proteins. One of the tyrosine-phosphorylated proteins was identified as an Src homology-2 domain-containing protein-tyrosine phosphatase, SHP2. The stimulatory effect of the agonist peptide on early gene transcription was markedly blocked by pertussis toxin treatment whereas the induced tyrosine phosphorylation of SHP2 was completely abolished by the drug. More importantly, while expression of wild-type SHP2 enhanced the agonist-stimulatory mitogenic activity, overexpression of a catalytically inactive mutant of SHP2 strongly suppressed the stimulatory effect of the agonist peptide on both early gene transcription and DNA synthesis. These results suggest that SHP2 acts as a positive regulator linked to the PAR2-mediated mitogenic pathway coupled to a pertussis toxin-sensitive heterotrimeric G protein. Demonstration of SHP2 as a positive mediator in a G protein-coupled, receptor-mediated signaling adds to our understanding of the function of both SHP2 and PAR2 in the signaling pathway.
Animals↗
Positive effect of overexpressed protein-tyrosine phosphatase PTP1C on mitogen-activated signaling in 293 cells.
PTP1C, an SH2 domain-containing protein-tyrosine phosphatase, is predominantly expressed in hematopoietic cells, in which it negatively regulates cellular signaling. However, this enzyme is also expressed in many non-hematopoietic cells. We demonstrate here that in non-hematopoietic 293 cells, overexpression of a catalytically inactive mutant of PTP1C strongly suppressed the stimulatory effects of the epidermal growth factor or serum on cell proliferation, early gene transcription, and DNA synthesis. Similarly, the phosphorylation of the mitogen-activated protein kinase and mitogen-activated protein kinase kinase activity was markedly inhibited by overexpression of mutant PTP1C. The inhibitory effect of mutant PTP1C was overcome by cotransfection with wild-type PTP1C, but not with the structurally related PTP2C. Furthermore, expression of the mutant phosphatase resulted in hyperphosphorylation on tyrosine of a 95-kDa protein that was co-immunoprecipitated with the mutant, but not with the wild-type protein. These results suggest that, unlike in hematopoietic cells, PTP1C in 293 cells plays a positive role in epidermal growth factor- or serum-activated mitogenesis. Thus, PTP1C participates in multiple signaling pathways, where the enzyme, depending on its target molecules, may function as either a positive or negative mediator.
Cell Division↗
Pseudocontact shifts used in the restraint of the solution structures of electron transfer complexes.
The geometry of the ferricytochrome b5-ferricytochrome c complex has been analysed using long-range interprotein paramagnetic dipolar shifts. Heteronuclear filtered NMR spectra of samples containing 15N-labelled cytochrome b5 in complex with unlabelled cytochrome c allowed unambiguous assessment of pseudocontact shifts relative to diamagnetic reference states. Because pseudocontact shifts can be observed for protons as much as 20 A from the paramagnetic centre, this approach allows study of electron transfer proteins in fast exchange. Our findings provide the first physical evidence confirming hypotheses presented in previous theoretical studies. This absence of certain predicted shifts that are expected based on the best fit to a static model of the complex suggests that cytochrome b5 is more dynamic in solution than in the crystal, in agreement with molecular dynamics simulations.
Animals↗
Differential promoter usage in prolactin receptor gene expression: hepatocyte nuclear factor 4 binds to and activates the promoter preferentially active in the liver.
Characterization of the rat PRL receptor (PRLR) gene has revealed three separate untranslated exon 1 sequences, each associated with a different transcription start site and 5'-flanking sequence. We show by RT-PCR that exon 1A is expressed primarily in liver but is also detectable in ovary and mammary gland. Exon 1B expression is observed exclusively in the ovary, whereas exon 1C is expressed in all three tissues. Transient transfection of luciferase reporter constructs containing parts of the 5'-flanking regions (0.3-1.1 kb) of exon 1A, 1B, and 1C, respectively, showed activity of the 1A promoter in Chinese hamster ovary (CHO) cells, the human hepatoma cell line, HepG2, and the rat hepatoma cell line, H4II, which was 10- to 14-fold increased compared with the activity of the promoter-less luciferase vector. No activity of the 1A promoter was detected in the human mammary cell line, T-47D. Relative to a vector containing the Simian virus 40 (SV40) promoter, the 1A promoter had 20% activity in H4II cells and 1-3% activity in CHO and HepG2 cells. The 1B promoter produced a 6.1-fold increase of luciferase activity in CHO cells (approximately 2% of the SV40 promoter), whereas no significant activity was detected in HepG2, H4II, and T-47D cells. The 1C promoter was strongly active in T-47D cells (approximately 64-fold over control) and moderately active in the other cell lines tested (9- to 13-fold over control). 5'-Deletion analysis of the 1A promoter revealed that a fragment containing -83/ +81 bp, relative to the transcription start site, was sufficient to drive transcription in hepatoma cells, whereas this construct was inactive in CHO cells. Cotransfection of CHO cells with the -83/+81 construct and an expression vector encoding the liver-enriched transcription factor, hepatocyte nuclear factor 4 (HNF4), revealed a dose-dependent transactivation of the proximal 1A promoter with a maximal stimulation of approximately 10-fold. Electrophoretic mobility shift assays showed binding of HNF4 to the sequence -14/+24 of the 1A promoter, and mutational analysis revealed that the sequence GGGCAAAGTCA at position +11/+21 is required for this binding. We conclude that the 1A, 1B, and 1C promoters of the PRLR gene are used in a cell type- dependent way that may play a role in differential hormonal regulation of the gene. In particular, we have shown that HNF4 operates on the proximal 1A promoter and may be responsible, in combination with other factors, for the increased activity of this promoter in adult female liver.
Animals↗
Human protein tyrosine phosphatase 1C (PTPN6) gene structure: alternate promoter usage and exon skipping generate multiple transcripts.
PTPN6, a protein tyrosine phosphatase with two Src homology-2 domains, is expressed predominantly in cells of the hematopoietic lineage. In addition, cDNAs encoding isoforms of the enzyme differing from the latter at their N-terminal have been detected in normal and transformed epithelial cells. To understand further the regulation and expression of the PTPN6 gene, we have isolated and characterized overlapping human genomic lambda clones containing the entire locus. The gene, located on chromosome 12, consists of 17 exons spanning 17 kb of DNA. Three nonhematopoietic PTPN6 transcripts were identified in a variety of cell lines and were shown to be transcribed from a common promoter. Alternate splicing and exon skipping result in transcripts encoding the two SH2 domains and minor transcripts from which part of or the entire N-terminal SH2 domain coding sequences are missing. The hematopoietic form of PTPN6 transcript is initiated at a downstream promoter separated by 7 kb from the first. This promoter is active exclusively in cells of the hematopoietic lineage. Characterization of the 5' ends of the PTPN6 mRNAs by RT-PCR and analysis of the flanking genomic sequences identified putative initiation sites within the two promoters.
Amino Acid Sequence↗
Structure, characterization, and expression of the rat oxytocin receptor gene.
The multiple hormonal and neurotransmitter functions of the nonapeptide oxytocin are mediated by specific oxytocin receptors (OTRs). In most target tissues, the number of OTRs is strongly regulated. Specifically, in the uterus, a dramatic OTR upregulation precedes the onset of parturition. To study the molecular mechanisms underlying OTR regulation, we have isolated and characterized recombinant bacteriophage lambda EMBL3 genomic clones containing the rat OTR gene, using sequence information derived from a human myometrial OTR cDNA. The rat OTR gene spans > 20 kb and contains three exons. A 97-bp intron is in the 5' untranslated region and a > 12-kb intron interrupts the coding region between transmembrane domains 6 and 7. The promoter region lacks an apparent TATA or CCAAT box but contains multiple putative interleukin-response elements [six NF-IL6 (C/EBP beta) and four APRF (STAT3) binding motifs], supporting the notion that interleukins may mediate labor induction via transcriptional activation of the OTR gene. The predicted amino acid sequence is 93% identical to the human OTR sequence but only 48% and 38% identical to the rat V1 and V2 vasopressin receptor sequences, respectively. At parturition, the OTR gene is highly expressed in the rat uterus and gives rise to at least three transcripts (2.9, 4.8, and 6.7 kb) which differ in the length of their 3' untranslated regions.
Amino Acid Sequence↗
A sensitive method for detection of mutations--a PCR-based RNase protection assay.
Several techniques are currently available for detecting point mutations in DNA. The most widely used methods either use hazardous chemicals (chemical mismatch cleavage) or can detect mutations only in short (200- to 500-bp) fragments (single-stranded conformational polymorphism and denaturing gradient gel electrophoresis). In an effort to develop a sensitive and reliable method for the detection of mutations in large segments of DNA, a novel RNase protection assay using RNase I was developed. In this method, the desired portion of the gene is amplified by the polymerase chain reaction (PCR) using specific oligonucleotides and hybridized to a 32P-labeled RNA probe containing the wild-type sequence. The RNA/DNA hybrid is subsequently digested with RNase I, which cleaves the RNA at the mismatch sites. The protected RNA fragments are separated on a denaturing polyacrylamide-urea gel and detected by autoradiography. Four different RNA probes from two protein tyrosine phosphatases (PTP1C and PTP2C) were assayed using this procedure. Several mutants of the two enzymes were tested using wild-type RNA probes. Single-base changes involving all four bases at the mismatch site could be detected efficiently. The ability of this method to detect insertions and single-base deletions was also demonstrated. Using a PCR-based RNase protection assay, a single-base deletion in PTP1C in the motheaten mutation in mice could be detected. Using fragments amplified from genomic DNA, mice that were heterozygous for the motheaten mutation could be distinguished from wild type and homozygotes for this mutation.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗