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D Banville

Publications and source records attributed to D Banville.

At least 37 records · Page 2Linked to original sources
PubMed1994

Identification of a novel protein tyrosine phosphatase with sequence homology to the cytoskeletal proteins of the band 4.1 family.

Use of the polymerase chain reaction (PCR) in conjunction with Southern hybridization, using probes corresponding to known phosphatase sequences, resulted in the identification of rat cDNA clones encoding a novel protein tyrosine phosphatase which was termed rPTP2E. The cDNAs comprise 5,543 bp and predict a polypeptide of 1175 amino acids possessing a single catalytic domain at its C-terminus. The N-terminal region of the deduced polypeptide displays high sequence homology to the cytoskeleton-associated proteins of the band 4.1 family. A variant form, termed rPTP2E1, was also identified which contains the catalytic domain only. rPTP2E and rPTP2E1 were expressed in various rat tissues, particularly abundantly in adrenal glands. The catalytic domain of PTP2E was expressed in Escherichia coli and was shown to possess specific protein tyrosine phosphatase activity. The identification of rPTP2E suggests the existence of a subfamily of band 4.1 domain-containing PTPs which may play an important role in signalling pathway and control of cytoskeletal integrity.

Amino Acid Sequence↗
PubMed1994

A novel protein-tyrosine phosphatase with homology to both the cytoskeletal proteins of the band 4.1 family and junction-associated guanylate kinases.

Reversible phosphorylation of proteins on tyrosine residues is critical in several cellular activities. The removal of the phosphate groups from tyrosine-phosphorylated proteins is accomplished by a class of enzymes which constitute a large family of related proteins, the protein tyrosine phosphatases. We report here the isolation of a complementary DNA encoding a novel protein tyrosine phosphatase, which we termed hPTP1E. Several overlapping cDNA clones were isolated to reconstruct a sequence of 8301 nucleotides. Northern blot analysis of poly(A)+ RNA from different human tissues revealed the presence of a mRNA of 8.2-8.5 kilobases suggesting the near full-length sequence had been cloned. The composite hPTP1E cDNA contains an open reading frame encoding 2490 amino acid residues. The predicted protein (M(r) = 277,567) does not contain a signal peptide or a membrane-spanning region and possesses a single catalytic domain (amino acids 2241-2470). The recombinant phosphatase domain has been expressed in Escherichia coli, purified to near homogeneity and showed to possess specific protein tyrosine phosphatase activity. The primary structure of hPTP1E also displays homology to cytoskeleton- and membrane junction-associated proteins. Thus, the region encompassing amino acids 568-1053 is related to the cytoskeletal proteins of the band 4.1 family. In addition, hPTP1E contains five imperfect repeats possessing significant homology to the GLGF repeats of the junction-associated guanylate kinases such as the Drosophila discs-large tumor suppressor gene (dlg-1). The structural features of hPTP1E suggest that it localizes at the junction between the plasma membrane and the cytoskeleton where it may regulate signal transduction and cytoskeletal integrity.

Alternative Splicing↗
PubMed1994

Phosphorylation and identification of a major tyrosine phosphorylation site in protein tyrosine phosphatase 1C.

Protein tyrosine phosphatase 1C (PTP1C) was the first member of the protein tyrosine phosphatase family demonstrated to contain the src homology 2 (SH2) domain. This enzyme is believed to play a role in regulating downstream signaling in hematopoietic cells since it was predominantly expressed in these cells. However, recent studies have revealed that the protein is expressed in other tissues as well. This report describes both the phosphorylation of PTP1C in non-hematopoietic cells treated with growth factors (in vivo) and incubation of purified PTP1C with a variety of protein kinases (in vitro). PTP1C was transiently phosphorylated in A431 and 293 cells and also when the purified enzyme was incubated with receptor protein tyrosine kinases. In vitro, the tyrosine-phosphorylated PTP1C underwent rapid auto-dephosphorylation, an effect which could be blocked by the addition of sodium vanadate. On the other hand, cells containing a PTP1C in which the catalytic site had been inactivated through mutagenesis, stably phosphorylated the phosphatase. These results suggested that PTP1C was responsible for its own auto-dephosphorylation. The sites of tyrosine phosphorylation were characterized from purified enzyme following treatment with insulin receptor kinase and from PTP1C expressed in 293 cells which had been stimulated with platelet-derived growth factor. Through the techniques of peptide mapping and microsequencing, Tyr538 was determined to be the major phosphorylation site. This result was confirmed in vivo through site-specific mutagenesis of PTP1C expressed in 293 cells; changing Tyr538 to Phe538 completely abolished tyrosine phosphorylation of the molecule. In addition, Tyr538 lies within the sequence ESEYGNI which can be correlated with the consensus sequence pYXNX associated with GRB2 binding. These results suggest that PTP1C plays a prominent role in growth factor receptor-mediated signal transduction within both hematopoietic cells and tissues of non-lymphoid origin.

Amino Acid Sequence↗
PubMed1994

14-kDa ubiquitin-conjugating enzyme: structure of the rat gene and regulation upon fasting and by insulin.

Upon fasting, an increase in proteolysis occurs in rat skeletal muscle and is associated with increased levels of ubiquitin-protein conjugates. As this suggests that formation of conjugates may be activated upon fasting, we studied the expression of the gene encoding the 14-kDa ubiquitin-conjugating enzyme (E2(14k)). A cDNA encoding rat E2(14k) was isolated and used to probe Northern blots of RNA from extensor digitorum longus muscles of fed, fasted, and refed rats. Two mRNA transcripts of 1.2 and 1.8 kb were observed. Isolation and sequencing of a genomic clone determined that these transcripts arise from differential sites of polyadenylation. The 1.2-kb transcript increased threefold upon fasting at 2 days and returned to normal with refeeding. Northern analysis of RNA from various tissues of fed and fasted rats showed that E2(14k) mRNA was expressed at high levels in testes, moderate levels in muscle, heart, and brain, but low levels in liver and kidney. Upon fasting, increases in mRNA levels were seen in muscle, heart, liver, and kidney. In vitro, in rat L6 myotubes, insulin lowered levels of E2(14k) mRNA. Because E2s catalyze the first irreversible reaction in the pathway and E2(14k) gene expression appears to change in parallel with the changes in levels of ubiquitinated proteins and rates of proteolysis, conjugation mediated by this E2 may be a rate-limiting step in the pathway. This is the first demonstration of direct hormonal regulation of a gene in the ubiquitin system and argues strongly for a role of the ubiquitin system in the metabolic response to fasting in skeletal muscle.

Animals↗
PubMed1993

Inhibition of the activity of protein tyrosine phosphate 1C by its SH2 domains.

Full-length protein tyrosine phosphatase 1C (PTP1C), the catalytic domain of PTP1C (delta PTP1C), and the N-terminal SH2 domain truncated PTP1C (delta NPTP1C) were overexpressed in Escherichia coli and purified to near homogeneity. Various phosphorylated states of the synthetic phosphotyrosyl peptide TRDIYETDYYRK (IRP), corresponding to the major insulin receptor autophosphorylation sites, were used as substrates for the PTPs. There was no indication for selective dephosphorylation of any of the three phosphotyrosyl residues from the triphosphotyrosyl IRP. Kinetic studies were carried out using all seven different phosphotyrosyl IRPs. Saturation kinetics were observed for PTP1C using the triphosphotyrosyl IRP only. In contrast, for delta PTP1C, saturation was achieved for all seven phosphotyrosyl IRPs. The best substrate for delta PTP1C was the triphosphotyrosyl IRP possessing a Km of approximately 1.6 microM, about 3-4-fold lower than either the mono- or diphosphotyrosyl IRPs. However, in contrast to delta PTP1C, PTP1C had a 22-fold lower affinity for triphosphotyrosyl IRP. Furthermore, deletion of a single N-terminal SH2 domain increased the affinity of the enzyme for the triphosphotyrosyl IRP to a value similar to that obtained with delta PTP1C. The pH optima for all three enzyme constructs were very similar and could not account for the observed change in substrate affinity between the three enzymes. These results suggest that the SH2 domain of PTP1C exerts an inhibitory effect on its PTP activity.

Amino Acid Sequence↗
PubMed1993

Human alpha and beta parvalbumins. Structure and tissue-specific expression.

alpha and beta parvalbumins are Ca(2+)-binding proteins of the EF-hand type. We determined the protein sequence of human brain alpha parvalbumin by mass spectrometry and cloned human beta parvalbumin (or oncomodulin) from genomic DNA and preterm placental cDNA. beta parvalbumin differs in 54 positions from alpha parvalbumin and lacks the C-terminal amino acid 109. From MS analyses of alpha and beta parvalbumins we conclude that parvalbumins generally lack posttranslational modifications. alpha and beta parvalbumins were differently expressed in human tissues when analyzed by immunoblotting and polymerase-chain-reaction techniques. Whereas alpha parvalbumin was found in a number of adult human tissues, beta parvalbumin was restricted to preterm placenta. The pattern of alpha parvalbumin expression also differs in man compared to other vertebrates. For example, in rat, alpha parvalbumin was found in extrafusal and intrafusal skeletal-muscle fibres whereas, in man, alpha parvalbumin was restricted to the muscle spindles. Different functions for alpha and beta parvalbumins are discussed.

Amino Acid Sequence↗
PubMed1993

A widely expressed human protein-tyrosine phosphatase containing src homology 2 domains.

A cDNA encoding a nontransmembrane protein-tyrosine phosphatase (PTP; EC 3.1.3.48), termed PTP2C, was isolated from a human umbilical cord cDNA library. The enzyme contains a single phosphatase domain and two adjacent copies of the src homology 2 (SH2) domain at its amino terminus. A variant of PTP2C (PTP2Ci) which has four extra amino acid residues within the catalytic domain has been identified also. PTP2C is widely expressed in human tissues and is particularly abundant in heart, brain, and skeletal muscle. The catalytic domain of PTP2C was expressed as a recombinant enzyme in Escherichia coli and purified to near homogeneity by two chromatographic steps. The recombinant enzyme was totally specific toward phosphotyrosine residues. The structural similarity between PTP2C and the previously described PTP1C suggests the existence of a subfamily of SH2-containing PTPs; these may play an important role in signal transduction through interaction of their SH2 domains with phosphotyrosine-containing proteins.

Amino Acid Sequence↗
PubMed1993

A study of sensitized lanthanide luminescence in an engineered calcium-binding protein.

In this study, the CD loop of the Ca(2+)-binding protein oncomodulin was replaced by a high-affinity, metal-binding sequence that was found to reverse the order of fill of the two sites in the protein. A cysteine was included at position 7 of this sequence, i.e., DKNADGCIEFEE. The cysteine allowed covalent attachment of chromophores to the loop that could subsequently be tested for their ability to sensitize the luminescence of Tb3+ or Eu3+ bound in the loop. 7-Diethylamino-3-((4'-iodoacetylamino)phenyl)-4-methylcoumarin was the most efficient Eu3+ sensitizer studied, consistent with a mechanism of energy transfer that involves the triplet state of the donor. 4-Iodoacetamidosalicylic acid was the most efficient Tb3+ donor tested. Levels of lanthanide ion and labeled C3 as low as 5 x 10(-10) mol/liter could be detected. This protein chelator system has potential to be a useful, flexible complement to the organic chelators currently used in lanthanide-based, time-resolved luminescence immunoassays.

Amino Acid Sequence↗
PubMed1992

Comparison of terbium (III) luminescence enhancement in mutants of EF hand calcium binding proteins.

The luminescent isomorphous Ca2+ analogue, Tb3+, can be bound in the 12-amino acid metal binding sites of proteins of the EF hand family, and its luminescence can be enhanced by energy transfer from a nearby aromatic amino acid. Tb3+ can be used as a sensitive luminescent probe of the structure and function of these proteins. The effect of changing the molecular environment around Tb3+ on its luminescence was studied using native Cod III parvalbumin and site-directed mutants of both oncomodulin and calmodulin. Titrations of these proteins showed stoichiometries of fill corresponding to the number of Ca2+ binding loops present. Tryptophan in binding loop position 7 best enhanced Tb3+ luminescence in the oncomodulin mutant Y57W, as well as VU-9 (F99W) and VU-32 (T26W) calmodulin. Excitation spectra of Y57F, F102W, Y65W oncomodulin, and Cod III parvalbumin revealed that the principal Tb3+ luminescence donor residues were phenylalanine or tyrosine located in position 7 of a loop, despite the presence of other nearby donors, including tryptophan. Spectra also revealed conformational differences between the Ca2+- and Tb(3+)-bound forms. An alternate binding loop, based on Tb3+ binding to model peptides, was inserted into the CD loop of oncomodulin by cassette mutagenesis. The order of fill of Tb3+ in this protein reversed, with the mutated loop binding Tb3+ first. This indicates a much higher affinity for the consensus-based mutant loop. The mutant loop inserted into oncomodulin had 32 times more Tb3+ luminescence than the identical synthetic peptide, despite having the same donor tryptophan and metal binding ligands. In this paper, a ranking of sensitivity of luminescence of bound Tb3+ is made among this subset of calcium binding proteins. This ranking is interpreted in light of the structural differences affecting Tb3+ luminescence enhancement intensity. The mechanism of energy transfer from an aromatic amino acid to Tb3+ is consistent with a short-range process involving the donor triplet state as described by Dexter (Dexter, D. L. (1953) J. Chem. Phys. 21, 836). This cautions against the use of the Förster equation in approximating distances in these systems.

Amino Acid Sequence↗
PubMed1992

Characterization of a prolyl endopeptidase from Flavobacterium meningosepticum. Complete sequence and localization of the active-site serine.

A prolyl endopeptidase was purified from Flavobacterium meningosepticum. It was digested with trypsin. Two oligonucleotides, based on tryptic peptide sequences and used in PCR experiments, amplified a 300-base pair (bp) fragment. A 2.4-kilobase EcoRI fragment that hybridized to the 300-bp probe was cloned in lambda ZAP and sequenced from both strands. It contains a reading frame of 2115 bp, encoding the complete protein sequence of 705 amino acids. Ion-spray mass spectrometry experiments demonstrated the presence of an NH2-terminal signal peptide: the periplasmic mature protease is 685 residues in length for a molecular mass of 76784 Da. The prolyl endopeptidase showed no general sequence homology with known protein sequences except with that of porcine brain prolyl endopeptidase. In order to identify the active-site serine, the prolyl endopeptidase was labeled with [3H]diisopropyl fluorophosphate. One labeled peptide was purified and sequenced. The active-site serine was located in position 536 within the sequence GRSNGG. This sequence is different from the active-site sequence of the trypsin (GDSGGP) and subtilisin (GTSMAS) families.

Amino Acid Sequence↗
PubMed1992

A rabbit reticulocyte ubiquitin carrier protein that supports ubiquitin-dependent proteolysis (E214k) is homologous to the yeast DNA repair gene RAD6.

The two isoforms of the 14-kDa ubiquitin carrier protein (E2(14k)) are unique among rabbit E2s in efficiently supporting ubiquitin-protein ligase (E3)-mediated ubiquitination of proteins destined for degradation. To begin determining the structural basis for this property, we have isolated a cDNA encoding the predominant reticulocyte isoform of the E2 from a rabbit skeletal muscle library. The sequence predicts a protein of 152 amino acids with a molecular weight of 17,293. Expression of the cDNA in Escherichia coli and purification of the recombinant protein revealed an E2 with high affinity for E3 and ubiquitin activating enzyme (E1). The latter high affinity interaction appears to be between the ubiquitin charged form of E1 and the uncharged form of E2 and does not result in a stable complex between these two enzymes. The predicted sequence shows regions of strong homology with other sequenced E2s, suggesting that these regions may be involved in binding to E1 and/or in ubiquitin transfer from E1, functions common to all E2s. Surprisingly, the E2(14k)) sequence is markedly more similar to Saccharomyces cerevisiae RAD6 (69% identity) than to its proposed homologs UBC4/UBC5 (38% identity). The sequence is identical to that recently reported for a human 17-kDa E2 which can complement rad6 mutants thereby identifying rabbit E2(14k) as a RAD6 homologue. The biochemical properties of this previously uncharacterized human 17-kDa E2 are now defined and its misassignment as a homologue of rabbit E2(17k) is corrected. Our findings resolve current confusion regarding relationships among E2s and define yeast RAD6, rabbit E2(14k), and the human 17-kDa E2 as a subclass of E2s which biochemically support E3-mediated conjugation and ubiquitin-dependent proteolysis and physiologically play a role in DNA repair.

Amino Acid Sequence↗
PubMed1992

The intracisternal A particle derived solo LTR promoter of the rat oncomodulin gene is not present in the mouse gene.

The rat gene encoding oncomodulin, a small calcium-binding protein related to parvalbumin, is under the control of a solo long terminal repeat (LTR) derived from an endogenous intracisternal A-particle (IAP). This gene was the first example of a mammalian gene regulated in normal cells by a promoter of retroviral origin (see also article by D. Robins and L. Samuelson in this volume). We show here that the oncomodulin LTR is a member of a small subset of sequence related solo LTR elements present in the rat genome and that a full length IAP genome containing LTRs of this type is no longer present in the rat genome. We have assayed the transcriptional activity of the oncomodulin LTR coupled to the human growth hormone gene as a reporter. Transfections in both Hela cells and 293 cells indicate the oncomodulin LTR promoter is sufficient to efficiently initiate transcription. In 293 cells (human embryo kidney cells transformed with human adenovirus type 5 DNA), the oncomodulin LTR is a strong promoter, capable of bidirectional transcription. Finally, we have determined the structure and the sequence of the mouse oncomodulin gene. Our results suggest that the integration of the IAP particle genome within the rat oncomodulin gene occurred after the rat and the mouse became distinct species.

Amino Acid Sequence↗
PubMed1991

Metal-induced changes in the fluorescence properties of tyrosine and tryptophan site-specific mutants of oncomodulin.

Oncomodulin is a 108-residue, oncodevelopmental protein containing two calcium-binding sites identified as the CD- and EF-loops. The protein contains no tryptophan and only two tyrosine residues, one which is a calcium ligand in the CD-loop (Tyr-57) and one which lies in the flanking D-helix of this loop (Tyr-65). Site-specific mutagenesis was performed to yield five mutants, two with phenylalanine substituted for tyrosine in positions 57 and 65 and three with tryptophan substituted into position 57 in the CD-loop, position 65 in the D-helix, and position 96 in the EF-loop. The single Tyr-containing mutants demonstrated that position 57 was perturbed to a significantly greater extent than position 65 upon calcium binding. Although both tyrosine residues responded to decalcification, the fluorescence intensity changes were in opposite directions, with the more dominant Tyr-57 accounting for the majority of the intrinsic fluorescence observed in native oncomodulin. The substitution of tryptophan for each tyrosyl residue revealed that in both positions the tryptophan resided in polar, conformationally heterogeneous environments. The environment of Trp-57 was affected by Ca2+ binding to a much greater extent compared to that of Trp-65. Only 1 equiv of Ca2+ was required to produce greater than 70% of the Trp fluorescence changes in positions 57 and 65, indicating that Ca2+ binding to the higher affinity EF-loop had a pronounced effect on the protein structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1991

Regulation of parathyroid hormone-like peptide in cultured normal human keratinocytes. Effect of growth factors and 1,25 dihydroxyvitamin D3 on gene expression and secretion.

We have examined the expression and secretion of endogenous parathyroid hormone-like peptide (PLP) in primary cultures of normal human keratinocytes. In response to growth factors and fetal bovine serum, PLP mRNA expression and immunoreactive PLP release into conditioned medium was rapidly increased (within hours) whereas these effects were inhibited by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. These early responses were not influenced by raising the medium calcium concentration from 0.15 to 1.0 mM. In contrast, increasing the medium calcium concentration to 1.0 mM, addition of 1,25(OH)2D3, or a combination of both, resulted in a delayed augmentation (after several days) in PLP production which was associated with an increase in cellular differentiation as assessed by production of high molecular weight keratin. To investigate whether these factors were acting at the level of transcription of the PLP gene, a series of vectors were prepared by fusing segments of the 5' flanking region of the rat PLP gene to a growth hormone reporter gene. Transient transfection of these constructs into cultured keratinocytes and measurement of immunoreactive growth hormone in the medium showed that a region stimulated by growth factors is located in a 1.9-kb fragment of the 5' flanking region and that a PLP gene promoter region less than 1.2 kb and greater than 0.3 kb upstream of the cap site contains cis-acting elements which respond positively to serum, and negatively to 1,25(OH)2D3. These combined studies demonstrate that, in normal human keratinocytes, growth factors may acutely stimulate PLP mRNA levels and PLP release, whereas 1,25(OH)2D3 inhibits these responses. At least part of these effects are at the level of gene transcription. Additionally, PLP synthesis and release are enhanced under conditions in which keratinocyte differentiation is induced.

Animals↗
PubMed1991

The growth hormone/prolactin receptor gene family.

The identification of cDNAs encoding receptors for growth hormone and prolactin have allowed the identification of the primary structure of these receptors. The expression of these receptors can be measured in various tissues by estimation of mRNA levels using cDNA probes and measurements of protein levels by radioimmunoassay or immunoblot analysis using monoclonal or polyclonal antibodies to the receptors. Site-directed mutagenesis of ligand-binding domains will lead to an understanding of the residues important for hormone binding. In addition, mutagenesis of the cytoplasmic domains of the receptors performed together with functional tests should help identify regions of the receptor involved in the process of signal transduction. Finally, the identification of the enlarged receptor family, including receptors for cytokines, should shed some light on additional functions of growth hormone prolactin, especially in regulating immune functions.

Animals↗
PubMed1990

An amino-terminal fragment of urokinase isolated from a prostate cancer cell line (PC-3) is mitogenic for osteoblast-like cells.

A peptide mitogen for cultured osteoblast-like cells was purified from serum-free conditioned culture medium of a human prostatic cancer cell line, PC-3. Based on amino acid sequencing and estimated molecular weight, this peptide was identified as an NH2-terminal fragment of urokinase-type plasminogen activator (uPA). Recombinant high molecular weight (HMW) uPA and the NH2-terminal growth factor domain (GFD) of uPA, but not low molecular weight (LMW) uPA (lacking the NH2-terminal region) stimulated [3H] thymidine incorporation and proliferation in osteoblast-like cells, and specific, competitive binding sites for HMW, but not LMW, uPA were demonstrable. These studies demonstrate the production of a mitogenic NH2-terminal fragment of uPA by a human prostatic cancer cell line which may be of importance in the pathogenesis of osteoblastic metastases.

Adenocarcinoma↗