Biomedical subjects

B G Neel

Publications and source records attributed to B G Neel.

At least 73 records · Page 4Linked to original sources

PubMed1994

The DNA binding domain of retinoic acid receptor beta is required for ligand-dependent suppression of proliferation. Application of general purpose mammalian coexpression vectors.

We have developed a family of mammalian coexpression vectors that permit identification of living or fixed cells overexpressing a gene of interest by surrogate detection of a coexpressed marker protein. Using these 'pMARK' vectors, a fluorescence-based, single cell proliferation assay was developed and used to study the effect of retinoic acid receptor beta (RAR-beta) on cell cycling. We demonstrate that transient overexpression of RAR-beta in the presence, but not absence, of all-trans retinoic acid results in a dramatic suppression of cell proliferation. We further show that this effect requires the DNA binding (C) domain of RAR-beta. It has been previously shown that RAR-beta expression is markedly altered in a variety of neoplasms and cell lines. Our data support the hypothesis that loss of RAR-beta may contribute to tumor progression by removing normal restraints on proliferation. The pMARK vectors should be useful for studying other genes that putatively suppress or enhance proliferation.

Base Sequence↗

PubMed1993

Growth factor-inducible alternative splicing of nontransmembrane phosphotyrosine phosphatase PTP-1B pre-mRNA.

PTP-1B is a major nontransmembrane phosphotyrosine phosphatase in human cell lines and tissues, but its physiological function(s) and mechanism(s) of regulation are largely unknown. We have found that in human diploid fibroblasts a novel PTP-1B mRNA isoform is produced upon stimulation of quiescent cells with a variety of growth factors. Generation of this isoform requires protein synthesis, suggesting that the product(s) of an immediate early response gene(s) is required. RNase protection and reverse transcription polymerase chain reaction analysis demonstrate that the isoform arises as a consequence of alternative pre-mRNA splicing, leading to retention of the last intron. The novel isoform is found on polyribosomes, indicating that it is actively translated, and is variably expressed in human tissues. Sequence analysis indicates that the isoform encodes a PTP-1B protein with an altered C terminus. To our knowledge, this is the first example of growth factor-regulated alternative splicing.

Alternative Splicing↗

PubMed1993

Expression, purification, and characterization of SH2-containing protein tyrosine phosphatase, SH-PTP2.

A human protein tyrosine phosphatase containing two src homology 2 (SH2) domains (SH-PTP2) was expressed in Escherichia coli under T7 promoter control and purified to near homogeneity. The purified protein, with molecular mass of 68 kDa on SDS-polyacrylamide gel electrophoresis, was identified as SH-PTP2 by its protein tyrosine phosphatase activity and N-terminal amino acid sequence analysis. Its protein tyrosine phosphatase activity was sensitive to pH and salt concentration. Whereas its optimum pH for the low molecular weight substrate para-nitrophenyl phosphate is 5.6, the pH optima for peptide substrates were shifted toward neutral. With the artificial protein substrate reduced, carboxyamidomethylated, and maleylated lysozyme, it displays 2000-fold lower Km (1.7 microM) and 2.4-fold higher kcat (0.11 s-1) than with para-nitrophenyl phosphate. Among the phosphopeptides from autophosphorylation sites of receptors for epidermal growth factor and platelet-derived growth factor, SH-PTP2 displayed high activity toward phosphopeptides corresponding to pY992 of the epidermal growth factor receptor and pY1009 and pY1021 of the platelet-derived growth factor receptor. In further enzymatic studies with phosphopeptides corresponding to pY1009, SH-PTP2 showed nonlinear Line-weaver-Burk double-reciprocal plots, suggesting that the phosphopeptide corresponding to pY1009 may have a substrate and allosteric effect.

Amino Acid Sequence↗

PubMed1993

Activation of the SH2-containing phosphotyrosine phosphatase SH-PTP2 by its binding site, phosphotyrosine 1009, on the human platelet-derived growth factor receptor.

Much progress has been made in elucidating early events in signal transduction by growth factor receptors with intrinsic tyrosine kinase activity. Upon ligand addition, these receptors dimerize and activate, becoming phosphorylated at a number of tyrosyl residues. These phosphorylation sites serve as docking points for proteins containing src homology-2 (SH2) domains. However, little is known about how phosphotyrosine phosphatases (PTPs), participate in these events. Recently, we and others molecularly cloned a ubiquitously expressed SH2 domain-containing PTP, SH-PTP2 (Syp, PTP1D, PTP2C), and found that it interacts directly with several activated growth factor receptors via its SH2 domains. Using a peptide competition assay, we now demonstrate that the major binding site for SH-PTP2 on the platelet-derived growth factor receptor is phosphotyrosine 1009. Immunoprecipitation studies indicate that SH-PTP2 is the previously unidentified "64-kDa" protein known to bind at this site. Addition of a phosphotyrosyl peptide comprising the region around Tyr-1009 stimulates SH-PTP2 activity 5-10-fold, whereas other phosphotyrosyl peptides from the platelet-derived growth factor receptor have no stimulatory effect. Our data suggest that binding of SH-PTP2 to the activated receptor in vivo should result in stimulation of SH-PTP2 activity.

Animals↗

PubMed1993

Tyrosyl phosphorylation and growth factor receptor association of the human corkscrew homologue, SH-PTP2.

The pivotal role of tyrosine kinases in signal transduction is well established, but the role of tyrosine phosphatases remains obscure. The discovery of src homology 2 domain-containing protein tyrosine phosphatases suggested roles for these molecules in growth factor signaling pathways, since src homology 2 domains direct association of downstream signaling molecules with activated growth factor receptors and other phosphotyrosyl proteins. We have found that SH-PTP2, a putative homologue of Drosophila corkscrew, associates in vivo with the ligand-activated epidermal growth factor and platelet-derived growth factor receptors. The N-terminal src homology 2 domain of SH-PTP2 directly associates with activated receptors. SH-PTP2 itself is a phosphoprotein, and it becomes tyrosyl phosphorylated upon growth factor activation. These findings suggest several possible models for SH-PTP2 signaling.

3T3 Cells↗

PubMed1993

Overexpression, purification, and characterization of SHPTP1, a Src homology 2-containing protein-tyrosine-phosphatase.

A protein-tyrosine-phosphatase (PTPase; EC 3.1.3.48) containing two Src homology 2 (SH2) domains, SHPTP1, was previously identified in hematopoietic and epithelial cells. By placing the coding sequence of the PTPase behind a bacteriophage T7 promoter, we have overexpressed both the full-length enzyme and a truncated PTPase domain in Escherichia coli. In each case, the soluble enzyme was expressed at levels of 3-4% of total soluble E. coli protein. The recombinant proteins had molecular weights of 63,000 and 45,000 for the full-length protein and the truncated PTPase domain, respectively, as determined by SDS/PAGE. The recombinant enzymes dephosphorylated p-nitrophenyl phosphate, phosphotyrosine, and phosphotyrosyl peptides but not phosphoserine, phosphothreonine, or phosphoseryl peptides. The enzymes showed a strong dependence on pH and ionic strength for their activity, with pH optima of 5.5 and 6.3 for the full-length enzyme and the catalytic domain, respectively, and an optimal NaCl concentration of 250-300 mM. The recombinant PTPases had high Km values for p-nitrophenyl phosphate and exhibited non-Michaelis-Menten kinetics for phosphotyrosyl peptides.

Amino Acid Sequence↗

PubMed1993

Calpain-catalyzed cleavage and subcellular relocation of protein phosphotyrosine phosphatase 1B (PTP-1B) in human platelets.

The non-transmembrane phosphotyrosine phosphatase 1B (PTP-1B) is an abundant enzyme, normally localized to the cytosolic face of the endoplasmic reticulum via a C-terminal targeting sequence. We have found that agonist-induced platelet activation results in proteolytic cleavage of PTP-1B at a site upstream from this targeting sequence, causing subcellular relocation of its catalytic domain from membranes to the cytosol. PTP-1B cleavage is catalyzed by the calcium-dependent neutral protease calpain and is a general feature of platelet agonist-induced aggregation. Moreover, PTP-1B cleavage correlates with the transition from reversible to irreversible platelet aggregation in platelet-rich plasma. Engagement of gpIIb-IIIa is necessary for inducing PTP-1B cleavage, suggesting that integrins regulate tyrosine phosphatases as well as tyrosine kinases. PTP-1B cleavage is accompanied by a 2-fold stimulation of its enzymatic activity, as measured by immune complex phosphatase assay, and correlates with discrete changes in the pattern of tyrosyl phosphorylation. Cleavage and subcellular relocation of PTP-1B represents a novel mechanism for altering tyrosyl phosphorylation that may have important physiological implications in cell types other than platelets.

Blood Platelets↗

PubMed1993

Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins.

The pGEX glutathione S-transferase (GST) fusion protein system is used extensively for high level expression and rapid purification of fusion proteins from bacterial and eukaryotic cell lysates. Unfortunately, many GST fusion proteins are partially or completely insoluble, and thus cannot be purified efficiently from a crude lysate. We have adapted a protocol, previously used to solubilize actin, for the purification of otherwise insoluble GST fusion proteins. Using a GST fusion of the nontransmembrane protein tyrosine phosphatase 1B, we demonstrate that tyrosine phosphatase enzymatic activity is maintained during the purification process. We provide methods for the purification of GST fusion proteins at analytical and preparative scales, and demonstrate that saturation of glutathione agarose is dependent on fusion protein molecular weight. Finally, we present strategies for eluting purified fusion proteins from glutathione agarose beads, for storing eluted protein, and for preparing covalently coupled affinity matrices.

Animals↗

PubMed1993

Structure and function of SH2-domain containing tyrosine phosphatases.

The importance of tyrosyl phosphorylation in regulating growth factor-receptor-mediated signal transduction is firmly established, but the roles of protein tyrosine phosphatases (PTPases) in these pathways are unclear. PTPases that contain src-homology 2 (SH2) domains, which mediate interactions with specific phosphotyrosyl proteins, probably play an important role in early signaling events following growth factor stimulation. In this review, the two mammalian SH2-containing PTPases, SH-PTP1 and SH-PTP2, are described and their possible roles in signal transduction discussed. In addition, the implications of recent genetic studies in the mouse and Drosophila, which shed light on the actions of these PTPases in physiology and pathology, will be addressed.

Animals↗

PubMed1993

Use of a general purpose mammalian expression vector for studying intracellular protein targeting: identification of critical residues in the nuclear lamin A/C nuclear localization signal.

We have constructed a general purpose mammalian expression vector for the study of intracellular protein targeting. The vector, p3PK, facilitates construction of N- and/or C-terminal fusions of an amino acid sequence of interest to the normally cytosolic protein chicken muscle pyruvate kinase (CMPK). The vector has been engineered such that any fusion construct can be subcloned into the versatile pJx omega family of mammalian expression vectors and into pGEX bacterial expression vectors, for the generation of affinity reagents. In this paper, we demonstrate the general utility of p3PK by redirecting CMPK to mitochondria (using the twelve amino acid pre-sequence of yeast cytochrome c oxidase subunit IV) and to the nucleus (using a putative eight amino acid nuclear localization signal from human nuclear lamins A and C). We also report that, contrary to the predictions of previously published work, substitution of a critical residue in the nuclear lamin A/C nuclear localization signal (the equivalent of lysine 128 in the SV40 large T nuclear localization signal) retains nuclear localization, and discuss how amino acid context might affect targeting to the nucleus.

Amino Acid Sequence↗

PubMed1992

Identification of a human src homology 2-containing protein-tyrosine-phosphatase: a putative homolog of Drosophila corkscrew.

src homology 2 (SH2) domains direct binding to specific phosphotyrosyl proteins. Recently, SH2-containing protein-tyrosine-phosphatases (PTPs) were identified. Using degenerate oligonucleotides and the PCR, we have cloned a cDNA for an additional PTP, SH-PTP2, which contains two SH2 domains and is expressed ubiquitously. When expressed in Escherichia coli, SH-PTP2 displays tyrosine-specific phosphatase activity. Strong sequence similarity between SH-PTP2 and the Drosophila gene corkscrew (csw) and their similar patterns of expression suggest that SH-PTP2 is the human corkscrew homolog. Sequence comparisons between SH-PTP2, SH-PTP1, corkscrew, and other SH2-containing proteins suggest the existence of a subfamily of SH2 domains found specifically in PTPs, whereas comparison of the PTP domains of the SH2-containing PTPs with other tyrosine phosphatases suggests the existence of a subfamily of PTPs containing SH2 domains. Since corkscrew, a member of the terminal class signal transduction pathway, acts in concert with D-raf to positively transduce the signal generated by the receptor tyrosine kinase torso, these findings suggest several mechanisms by which SH-PTP2 may participate in mammalian signal transduction.

Amino Acid Sequence↗

PubMed1992

Molecular cloning of cDNAs encoding a guanine-nucleotide-releasing factor for Ras p21.

The stimulation of a variety of cell surface receptors promotes the accumulation of the active, GTP-bound form of Ras proteins in cells. This is a critical step in signal transduction because inhibition of Ras activation by anti-Ras antibodies or dominant inhibitory Ras mutants blocks many of the effects of these receptors on cellular function. To reach the active GTP-bound state, Ras proteins must first release bound GDP. This rate-limiting step in GTP binding is thought to be catalysed by a guanine-nucleotide-releasing factor (GRF). Here we report the cloning of complementary DNAs from a rat brain library that encode a approximately 140K GRF for Ras p21 (p140Ras-GRF). Its carboxy-terminal region is similar to that of CDC25, a GRF for Saccharomyces cerevisiae RAS. This portion of Ras-GRF accelerated the release of GDP from RasH and RasN p21 in vitro, but not from the related RalA, or CDC42Hs GTP-binding proteins. A region in the amino-terminal end of Ras-GRF is similar to both the human breakpoint cluster protein, Bcr, and the dbl oncogene product, a guanine-nucleotide-releasing factor for CDC42Hs. An understanding of Ras-GRF function will enhance our knowledge of the many signal transduction pathways mediated by Ras proteins.

Amino Acid Sequence↗

PubMed1992

The nontransmembrane tyrosine phosphatase PTP-1B localizes to the endoplasmic reticulum via its 35 amino acid C-terminal sequence.

We report the first intracellular characterization of an endogenous nontransmembrane protein tyrosine phosphatase (PTP). Using affinity-purified polyclonal antibodies, we have identified PTP-1B as a 50 kd serine phosphoprotein in immunoprecipitation and immunoblotting assays. Surprisingly, indirect immunofluorescence experiments indicate that PTP-1B is localized predominantly in the endoplasmic reticulum (ER). Subcellular fractionation is consistent with this localization and establishes that PTP-1B is tightly associated with microsomal membranes, with its phosphatase domain oriented towards the cytoplasm. The C-terminal 35 amino acids of PTP-1B are both necessary and sufficient for targeting to the ER. The finding of a tyrosine phosphatase on the ER suggests new possibilities for cellular events controlled by tyrosine phosphorylation.

Animals↗

PubMed1992

Isolation of a src homology 2-containing tyrosine phosphatase.

Tyrosine phosphorylation is controlled by the opposing actions of tyrosine kinases and phosphotyrosine phosphatases (PTPs). src homology 2 domains (SH2) are found in several types of signaling proteins, including some tyrosine kinases. These domains bind phosphotyrosyl proteins and thus help promote signal transduction. Using mixed oligonucleotide-directed polymerase chain reactions, two previously undescribed rat PTP cDNA fragments were generated. Through subsequent screening of rat megakaryocyte and human erythroleukemia libraries, we obtained a full-length coding sequence for one of these fragments. This cDNA, SH-PTP1, encodes a tyrosine phosphatase containing two highly conserved SH2 domains. SH-PTP1, with a 2.4-kilobase mRNA, a predicted open reading frame of 595 amino acids, and a structure suggesting a nontransmembrane protein, is expressed primarily in hematopoietic and epithelial cells. When expressed in Escherichia coli, SH-PTP1 possesses PTP activity. The structure of SH-PTP1 establishes an additional branch of the tyrosine phosphatase family and suggests mechanisms through which tyrosine phosphatases might participate in signal transduction pathways.

Amino Acid Sequence↗

PubMed1992

Chromosomal localization of an SH2-containing tyrosine phosphatase (PTPN6).

We have used panels of somatic cell hybrids and fluorescent in situ hybridization to determine the chromosomal localization of the novel nontransmembrane tyrosine phosphatase PTPN6 (protein tyrosine phosphatase, nonreceptor type 6), which contains two SH2 domains. PTPN6 maps to 12p13, a region commonly involved in leukemia-associated chromosomal abnormalities. Since PTPN6 is expressed at high levels in hematopoietic cells of all lineages and its expression is induced early in hematopoietic differentiation, altered expression and/or structure of PTPN6 may play a role in leukemogenesis.

Amino Acid Sequence↗

PubMed1992

Identification of an early-growth-response gene encoding a novel putative protein kinase.

Early-growth-response genes, also known as immediate-early genes, play important roles in regulating cell proliferation. We have identified a new type of early-growth-response gene product, a 77,811-Da putative serine/threonine kinase, which is highly inducible by serum and phorbol ester. mRNA encoding this putative kinase is markedly elevated within 1 h after treatment with mitogen, and this induction is synergistically increased by cycloheximide. Dexamethasone blocks serum induction of the kinase mRNA, as does transformation by v-Ki-ras. The kinase mRNA was detected in mouse brain, lung, and heart. This new putative kinase, which we term Snk, for serum-inducible kinase, showed similarity in its proposed catalytic domain to many other protein kinases; however, no other kinase showed enough sequence similarity with Snk to suggest the existence of a common function. Hence, Snk represents a new type of protein kinase involved in the early mitogenic response whose activity is transcriptionally and posttranscriptionally regulated.

3T3 Cells↗

PubMed1991

High frequency of retinoic acid receptor beta abnormalities in human lung cancer.

One of the three human retinoic acid receptors, RAR-beta, maps to a region on the short arm of chromosome 3 frequently deleted in lung cancer. Because retinoic acid is required for normal epithelial cell growth and regulation, and loss of a retinoic acid receptor might be expected to contribute to oncogenesis, we examined RAR-beta RNA and DNA in normal lung, 33 lung cancer cell lines and nine primary lung tumors. Normally, RAR-beta is expressed as two transcripts, of sizes 3.1 kb and 2.8 kb, which are strongly induced by retinoic acid. At least 50% of the cell lines and 30% of the tumor samples show altered RAR-beta expression and/or inducibility, including examples of absence or specific loss of one of the RAR-beta transcripts. Abnormalities in the expression patterns of RAR-alpha and RAR-gamma also are found, but at a lower frequency than RAR-beta abnormalities. Southern analysis reveals alteration of the RAR-beta gene in three of the cell lines. Our data suggest that abnormalities in structure and expression of the RAR-beta gene may be involved in the pathogenesis of lung cancer.

Carrier Proteins↗

PubMed1990

Cloning of a cDNA for a major human protein-tyrosine-phosphatase.

We have isolated a cDNA clone encoding the major protein-tyrosine-phosphatase (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) of human placenta. Degenerate oligonucleotides, based on the amino acid sequence of the protein, were used to amplify an internal fragment of the gene from human placental cDNA by the polymerase chain reaction. This fragment was then used to probe a human placental cDNA library. A 3.3-kilobase (kb) insert was isolated and sequenced. The insert has a single extended open reading frame that predicts a 435 amino acid protein of Mr approximately 50,000. From the amino terminus to residue 321, the deduced amino acid sequence is identical to that previously determined by peptide sequencing [Charbonneau, H., Tonks, N. K., Kumar, S., Diltz, C. D., Harrylock, M., Cool, D. E., Krebs, E. G., Fischer, E. H. & Walsh, K. A. (1989) Proc. Natl. Acad. Sci. USA 86, 5252-5256]; however, the sequence predicts that the protein contains an additional 114 amino acids not present in the reported peptide sequence. In vitro translation of the 3.3-kb insert produces a protein of Mr 56,000, in general agreement with the predicted size. The phosphatase gene appears to be present as a single copy in human genomic DNA and is transcribed into a 3.5-kb message in a variety of tissues.

Amino Acid Sequence↗