Phosphotyrosyl Protein Phosphatase Research Articles

Structural and Functional Characterization of Novel Phosphotyrosine Phosphatase Protein from Drosophila melanogaster (Pupal Retina).

A novel pair of protein tyrosine phosphatases in Drosophila melanogaster (pupal retina) has been identified. Phosphotyrosyl protein phosphatases (PTPs) are structurally diverse enzymes increasingly recognized as having a fundamental role in cellular processes including effects on metabolism, cell proliferation, and differentiation. This study presents identification of novel sequences of PTPs and their comparative homology modeling from Drosophila melanogaster (Dr-PTPs) and complexation with the potent inhibitor HEPES. The 3D structure was predicted based on sequence homology with bovine heart low molecular weight PTPs (Bh-PTPs). The sequence homologies are approximately 50% identical to each other and to low molecular weight protein tyrosine phosphatases (PTPs) in other species. Comparison of the 3D structures of Bh-PTPs and Dr-PTPs (primo-2) reveals a remarkable similarity having a four stranded central parallel β sheet with flanking α helices on both sides, showing two right handed β-α-β motifs. The inhibitor shows similar binding features as seen in other PTPs. The study also highlights the key catalytic residues important for target recognition and PTPs' activation. The structure guided studies of both proteins clearly reveal a common mechanism of action and inhibitor binding at the active site and will be expected to contribute toward the basic understanding of functional association of this enzyme with other molecules.

Adverse cardiac remodeling due to maternal low protein diet is associated with alterations in expression of genes regulating glucose metabolism

Background and aimsWe have previously shown that a maternal low protein (LP) diet during pregnancy in the rat results in adverse ventricular remodeling and contractile deficiencies of the neonatal rat heart. Since pathological cardiac hypertrophy is associated with increased expression of genes involved in glucose handling, this study was undertaken to examine if maternal LP diet alters the expression of genes encoding for some key components of glucose metabolism and uptake, and of the insulin receptor (IR) signal transduction in the heart of male offspring. Methods and resultsWe determined the effect of maternal LP and normal diet (90 and 180 g/casein/kg respectively) on IR β-subunit, insulin receptor substrate (IRS)-1, phosphotyrosyl protein phosphatase (PTP) 1B, GLUT4 and phosphatidylinositol (PI) 3-kinase in male rat offspring at 24 h and at 1, 4 and 8 wks post-partum. Quantitative real-time RT-PCR revealed significant age-dependent increases in the expression of IR β-subunit, IRS-1, PTP1B, GLUT4 and PI3-kinase in the LP group with concomitant increases in corresponding protein abundance at 4 wks of age. These changes were associated with increases in left ventricular (LV) internal diameters as well as increases in LV wall thickness. ConclusionA maternal LP diet can induce increases in the gene expression and protein levels of key components of glucose metabolism and the IR signal transduction pathway in the neonatal rat heart, which may be related to accelerated energy supply, demand and utilization for ventricular remodeling due to compromised contractile performance during early life.

Suppressive Effect of Orthovanadate on Hepatic Stellate Cell Activation and Liver Fibrosis in Rats

Orthovanadate (OV), an inhibitor of protein tyrosine phosphatases, affects various biological processes in a cell-type-specific manner. In this study, we investigated the effect of OV on hepatic stellate cells (HSCs). When primary rat HSCs were cultured in the presence of 10% serum, they spontaneously lost characteristic stellate morphology, proliferated, and were transformed into an activated state with the formation of abundant stress fibers and increased expression of both alpha-smooth muscle actin and collagen type I mRNA. OV treatment inhibited proliferation and activation of HSCs and partially reversed the phenotype of activated HSCs. Among the signaling molecules investigated, phosphorylation of the Src protein at tyrosine 416 was the most striking in OV-treated HSCs. Treatment of cells with Src family inhibitors partially abrogated the effects of OV. Furthermore, transfection of v-Src into activated HSCs induced a stellate morphology similar to that in the quiescent state. We then examined whether OV could effectively suppress HSC activation in vivo after liver injury induced by either carbon tetrachloride or dimethylnitrosamine. OV significantly reduced the appearance of alpha-smooth muscle actin-positive cells and decreased collagen deposition, concomitant with an improvement in liver function. Our study showed for the first time that OV was able to suppress the activation of HSCs, possibly through the modulation of Src activity, and attenuated fibrosis after chronic liver injury.

Phosphatase activity in Amoeba proteus at low pH values

In free-living Amoeba proteus (strain B), three forms of tartrate-sensitive phosphatase were revealed by using PAGE of supernatant of the ameba homogenate obtained with 1% Triton X-100 or distilled water and subsequent staining of gels with 2-naphthyl phosphate as substrate (pH 4.0). The form with the highest mobility in the gel turned out to be sensitive to all tested phosphatase activity modulators. Two other forms with the lower mobilities were completely or significantly inactivated not only by sodium L-(+)-tartrate, but also by L-(+)-tartaric acid, sodium orthovanadate, ammonium vanadate, ammonium molybdate, EDTA, EGTA, O-phospho-L-tyrosine, DL-dithiothreitol, H2O2, 2-mercaptoethanol, and ions of heavy metals—Fe2+, Fe3+, and Cu2+. Based on results of inhibitory analysis, lysosomal location in ameba cells, and wide substrate specificity of these two forms, it was concluded that they belonged to non-specific acid phosphomonoesterases (AcP, EC 3.1.3.2). This AcP is suggested to have both phosphomonoesterase and phosphotyrosylprotein phosphatase activities. Two ecto-phosphatases were revealed in culture medium, in which amebae were cultivated. One of them was inhibited by the same reagent as the ameba tartrate-sensitive AcP and seemed to be the AcP released into the culture medium in the process of exocytosis of the content of food vacuoles. In the culture medium, apart from this AcP, another phosphatase was revealed; it was not affected by any tested inhibitors of AcP and alkaline phosphatase. It cannot be ruled out that this phosphatase belongs to the ecto-ATPases found in many protists; however, so far its ability to hydrolyze ATP has not yet been proven.

Modulation of bovine sperm signalling pathways: correlation between intracellular parameters and sperm capacitation and acrosome exocytosis

In the present study, the viability, intracellular pH (pHi), cAMP ([cAMP]i), calcium concentration and protein phosphotyrosine content were evaluated in relation to the acrosomal and capacitation status of freshly ejaculated bull spermatozoa. These parameters were evaluated before and after incubation with the capacitation inducer heparin, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), the phosphotyrosyl-protein phosphatase inhibitors phenylarsine oxide (PAO) and sodium orthovanadate, and hydrogen peroxide. The results obtained were integrated to address the physiological interactions between the different signalling events affecting sperm capacitation and acrosome reaction. As expected, heparin promoted the expression of the 'B' pattern of chlortetracycline binding, increased pHi, [cAMP]i and the phosphotyrosine content of sperm proteins. The effects of heparin were enhanced by IBMX. Both PAO and sodium orthovanadate stimulated protein phosphotyrosine content and acrosomal exocytosis, although only PAO affected pH, Ca2+ and cAMP levels. Intracellular pH was increased while both Ca2+ and [cAMP]i were decreased. Physiological concentrations of H2O2 increased [cAMP]i and promoted acrosomal exocytosis. A significant positive correlation was found between sperm capacitation, protein phosphotyrosine content and stored Ca2+ concentration, whereas the acrosome reaction was correlated with pHi and Ca2+ concentration. This study presents the first global analysis of the major elements individually described during sperm capacitation and acrosome reaction signalling pathways, supported by statistical correlations.

Origins of Growth Factors: NGF and EGF

Growing up on the streets of Brooklyn, I remember being interested in how things worked: taking apart an old telephone and the gears of my new 4-speed bicycle was most enjoyable. As a biology major at Brooklyn College in 1945, I was fascinated by embryology. How does an egg turn into a chicken or a frog or a person? My only insight into the problem was the thought that it was necessary to understand the chemical reactions inside the egg and embryo and not simply observe biological structures. I, therefore, became a double major in chemistry and biology (also with the hope of earning a living as a laboratory technician). My first job after graduating was working nights as a bacteriologist in a milk processing plant. One of my professors at Brooklyn College wrote asking whether I was interested in going to graduate school. The college had been sending one student a year to the Biology Department at Oberlin to work as a laboratory assistant while studying for a master's degree. Because Oberlin offered to pay my tuition plus some $300 a semester for living expenses, I jumped at the chance. School and science were both interesting and fun. Upon graduating, I continued my education toward a Ph.D. working as an assistant in the Biochemistry Department of the University of Michigan under Howard B. Lewis. My research involved studying the Krebs urea cycle in the common earthworms that I collected from the university campus. I had been told at Oberlin that the yellow cells surrounding the gut in the worm corresponded to the mammalian liver. I decided to check whether the enzymatic reactions in worms were similar to those in mammals. They were (1). My first “real job” was in the Pediatrics Department at the University of Colorado studying creatine metabolism in premature infants under Harry Gordon. I think he hired me because he was impressed by my ability to stomach tube earthworms. After several years, I decided I needed to learn the then new technique of using radioisotopes in metabolic studies, and I obtained an American Cancer Society fellowship to work in the Radiology Department of Washington University under Martin Kamen where I combined this new knowledge with my interest in embryology. Although fertilized frog eggs and early embryos are impermeable to small molecules (amino acids, phosphates, etc.), sufficient amounts of C14O2 were metabolized by intact eggs and embryos to permit the identification of the radioactive compounds present at various stages of development (2). Upon completion of my fellowship, Dr. Kamen recommended me for a position in the Zoology Department of Washington University in the laboratory of Viktor Hamburger and Rita Levi-Montalcini. This move was critical in determining the direction of my research for the next 40-some odd years: the isolation, structure, and function of the first of the “growth factors,” nerve growth factor (NGF) and epidermal growth factor (EGF).

Theoretical investigations of prostatic acid phosphatase

The phosphotyrosyl protein phosphatase activity of prostatic acid phosphatase (PAP) has been well established. It has also been suggested that PAP partly regulates the activity of growth factor receptors by dephosphorylating the autophosphorylysable tyrosines in them. We studied the binding of the peptides from epidermal growth factor receptor (EGFR) and its homolog (ErbB-2), corresponding to their autophosphorylation sites, to PAP using theoretical modeling and molecular dynamics (MD) simulation methods. Nine different peptides, each with a phosphotyrosine residue, were docked on human PAP. The binding energies of these peptide-PAP complexes were calculated theoretically and compared to experimentally obtained affinities. The peptide Ace--DNLpYYWD--NH2 from ErbB-2(1197-1203) showed the most favorable free energy of binding when estimated theoretically. The results demonstrate that the presence of another tyrosine residue proximate to C-terminal of autophosphorylysable Tyr enhances the binding affinity considerably. The presence of a bulky group instead prevents the binding, as is observed in case of peptide Ace--NLYpYWDQ--NH2 which failed to bind, both in theoretical calculations and experiments. Thus we demonstarted that PAP could potentially bind to EGFR and Erbb-2 and dephosphorylate them. Thus it could be involved in the regulation of the function of such receptors. In addition, complexes of a peptide from AngiotensinII and phosphotyrosine(pY) with human PAP were also modeled. The effects of different protonation states of the titratable active site residues on ligand (pY) binding have also been investigated. For a favorable binding His12 and Asp258 should be neutral, His257 should be positively charged and the phosphate group of the ligand should be in PO(4) (3-) state. Furthermore, the analysis of protein motion as observed during simulations suggests the loop-loop contact in the PAP dimer to be of importance in cooperativity.

Low‐molecular‐weight phosphotyrosyl protein phosphatase expression in brain of chicken and some lower vertebrates

The presence of immunologically and biochemically related low‐molecular‐weight phosphotyrosyl protein phosphatase forms in vertebrate brain was investigated. An affinity purified polyclon‐al antibody, directed against the bovine liver enzyme, recognizes, in whole chicken brain, an 18‐kDa protein band having the same electrophoretic mobility as the enzyme purified from the same source and from bovine liver used as reference. A similar electrophoretic pattern was found using brain extracts from an amphibian (Rana esculenta), a teleost (Esox lucius) and an elasmo‐branch species (Raya clavata). The immunocytochemical localization in chicken brain showed immunoreactivity in nerve cell cytoplasm and processes. The subcellular localization, carried out using mild homogenization of fresh chicken brain tissue and differential centrifugation, revealed that the enzyme is essentially associated with both cytosolic and nerve‐ending fractions. Using immunoblotting and biochemical methods, were found similar molecular mass and biochemical properties of low‐molecular‐weight phosphotyrosyl protein phosphatase in brain of all the animals studied, suggesting a similar physiological role in vertebrate phylogenesis.

Elevated Expression of the cdc25A Protein Phosphatase in Colon Cancer

The nuclear protein phosphatase cdc25A has been postulated to be a protooncogene. The total nuclear phosphotyrosyl protein phosphatase (PTP) activity and the expression of cdc25A were compared in normal and cancerous colon epithelial tissue. Nuclei derived from normal mucosal epithelium and tumors were analyzed for phosphotyrosyl protein phosphatase activity using the malachite green assay and a synthetic phosphotyrosyl peptide based on the sequence of cdc2, a known cdc25A phosphotyrosyl protein substrate. Tumorigenesis resulted in elevated nuclear PTP activity (343.0 ± 37.0% of normal epithelial PTP activity) in 52% (29 of 56) of colon tumors. In all cases elevated nuclear PTP activity correlated with an increase in the expression of cdc25A. The changes in PTP activity observed were not due to any increase in the rate of growth of the colonic mucosa as no corresponding changes occurred with PTP activity under conditions of rapid mucosal growth.

Subcellular distribution of protein phosphatases and abnormally phosphorylated tau in the temporal cortex from Alzheimer's disease and control brains.

Microtubule-associated protein tau is abnormally hyperphosphorylated in the brain of patients with Alzheimer's disease (AD). In vitro studies have shown that protein phosphatases PP-2A and PP-2B can convert Alzheimer like tau to its normal state and that the activities of PP-1, PP-2A, and phosphotyrosyl-protein phosphatase (PTP) are reduced in AD brain. However, to have a direct effect on the regulation of phosphorylation on tau, these enzymes have to exist in neurons. Using specific polyclonal antibodies the levels of protein phosphatases PP-1, PP-2A, and PP-2B were determined by indirect ELISA in superior temporal cortical gray matter of AD and control brains. The protein levels of PP-2A and PP-2B were significantly increased in postsynaptosomal supernatant 2 (S2) of the AD group, and this alteration showed a significant linear correlation with levels of hyperphosphorylated tau. PP-1 and PTP-1B levels were not significantly changed in any of the AD fractions. Because of the large variation from case to case, the activity levels of none of the phosphatases investigated were significantly different between the AD and control groups. However, the PP-2B specific activity (activity/protein) showed a significant linear inverse correlation with hyperphosphorylated tau. These studies suggest that any attempt by the AD brain to compensate for the decreased tau phosphatase activity remains unsuccessful and that the decrease in phosphatase activity might contribute to increased levels of abnormally phosphorylated tau.

Effects of inhibitors of signal transduction pathways on transforming growth factor beta1 and osteogenic protein-1-induced insulinlike growth factor binding protein-3 expression in human bone cells.

Signal transduction initiated by TGFbeta1 and OP-1 was studied in MG63 human osteosarcoma cells and in normal human bone cells (HBCs) in the presence of inhibitors of signal transduction events, using insulinlike growth factor binding protein-3 (IGFBP-3) production as an end point. Treatment of serum-free MG63 cells and normal HBCs with TGFbeta1 increased IGFBP-3 protein level several fold in the conditioned medium. This effect of TGFbeta1 was mediated by increased de novo synthesis because mRNA level increased to the same extent as protein level and TGFbeta1 treatment had very little effect on IGFBP-3 protease activity. The stimulatory effect of TGFbeta1 on IGFBP-3 production was inhibited in a dose-dependent manner by pretreatment with staurosporine, a protein kinase C inhibitor, or with vanadate, a phosphotyrosyl protein phosphatase inhibitor in both MG63 cells and normal HBCs. In addition, pretreatment with okadoic acid, an inhibitor of serine/threonine protein phosphatase, counteracted TGFbeta1 induction of IGFBP-3 production. Interestingly, pretreatment of MG63 cells or HBCs with staurosporine, vanadate, or okadoic acid augmented OP-1 stimulation of IGFBP3 production. Staurosporine- or vanadate-induced changes in IGFBP-3 protein levels in the presence of TGFbeta1 and OP-1 were associated with corresponding changes in IGFBP-3 mRNA levels in MG63 cells. These findings are consistent with the hypothesis that TGFbeta1 and OP-1 increase IGFBP-3 expression via distinct intracellular signal transduction pathways.

Enhancement by sodium orthovanadate of the formation and mineralization of bone nodules by chick osteoblasts in vitro

Orthovanadate is a known inhibitor of phosphotyrosyl protein phosphatase and is reported to stimulate osteogenic cell proliferation and differentiation when administered during the logarithmic growth phase and to potentiate the mitogenic effects of several growth factors. There is little information concerning the effects of orthovanadate on bone matrix deposition and mineralization, although there is some evidence that it increases collagen synthesis by osteogenic cells. To test the effects of orthovanadate on bone nodule formation and mineralization, osteogenic cells were exposed to 5–50 μM orthovanadate or 10 −7 M insulin-like growth factor-1 for 3, 7, and 21 days after plating. Exposure to orthovanadate produced differential effects on cellular proliferation and alkaline phosphatase activity following completion of the logarithmic growth phase, and on resultant bone nodule formation and mineralization by these populations. The effects of orthovanadate on osteogenic cultures were concentration dependent: 5 μM concentrations produced a relatively large quantity of poorly mineralized matrix, while 30–50 μM concentrations produced a smaller quantity of heavily mineralized matrix. Thus, orthovanadate could possibly be used as a growth factor for bone, if administered at the critical dosage at the proper stage of the life cycle of the osteoblast.

Phosphotyrosyl protein phosphatase from the liver ofTilapia mossambica: Activating the Src-phosphorylated DNA topoisomerase I

Phosphotyrosyl protein phosphatase from the liver ofTilapia mossambica: Activating the Src-phosphorylated DNA topoisomerase I

Fluoride at mitogenic concentrations increases the steady state phosphotyrosyl phosphorylation level of cellular proteins in human bone cells.

This study was designed to test the hypothesis that treatment of human bone cells with mitogenic concentrations of fluoride would lead to an increase in the steady state level of tyrosyl phosphorylation of specific cellular proteins. With an immunoblot assay method, it was found that mitogenic concentrations of fluoride (i.e. 50-200 mumol/L) induced a dose- and time-dependent increase in the level of tyrosyl phosphorylation of at least 13 cellular proteins in both normal human bone cells and human TE85 osteosarcoma cells. Time-course studies revealed that a statistically significant increase in tyrosyl phosphorylation of these 13 cellular proteins in human bone cells was observed after 3-6 h of fluoride treatment and was sustained for up to 24 h. This time course was not compatible with a direct activation of tyrosyl kinases, as epidermal growth factor, which activates tyrosyl kinase activity, induced an immediate and acute response that was rapidly reversible within 1 h. Although fluoride increased the steady state tyrosyl phosphorylation of the cellular proteins in human bone cells, the same micromolar doses of fluoride had no effect on human skin fibroblasts, which are fluoride-nonresponsive cells. The effects of fluoride were rapidly reversible in the absence of fluoride and could be acutely potentiated by pretreatment with epidermal growth factor. In summary, we have shown for the first time that mitogenic concentrations (i.e. 50-200 mumol/L) of fluoride increased the steady state level of tyrosyl phosphorylation of at least 13 cellular proteins in human bone cells, and that the increases were relatively show in onset and sustained. In conclusion, these findings are consistent with the hypothesis that the osteogenic actions of fluoride are mediated at least in part by an inhibition of the activity of one or more fluoride-sensitive phosphotyrosyl protein phosphatases in human bone cells.

Modification of DNA topoisomerase I enzymatic activity with phosphotyrosyl protein phosphatase and alkaline phosphatase from the hepatopancreas of the shrimp Penaeus japonicus (Crustacea: Decapoda)

DNA topoisomerase I was partially purified from the hepatopancreas of the shrimp Penaeus japonicus. The specific activity of the final preparation was 7,000,000 units/mg of protein with SV40 viral DNA as substrate. SDD-polyacrylamide gel electrophoresis of the final preparation yielded two major bands of proteins with M r 70,000 and M r 67,000, as well as less intense bands of proteins with M r 64,000 and M r 56,000. Incubation of the partially purified enzyme fraction with rabbit antiserum against human DNA topoisomerase I, allowed all these proteins except that of M r 56,000, to be positively reacted. Treatment of the partially purified DNA topoisomerase I with tyrosine kinase p43 v-abl resulted in phosphorylation of only the two major subunits. Phosphorylation by tyrosine kinase p43 v-abl or dephosphorylation by phosphotyrosyl protein phosphatase resulted in a decrease of the enzymatic activity. The treatment with shrimp alkaline phosphatase abolished the enzymatic activity of the purified DNA topoisomerase I in a dose-dependent manner. Thus, the DNA topoisomerase I was apparently isolated from the hepatopancreas of the shrimp P. japonicus in a phosphorylated form, and this phosphorylation was essential for expression of enzymatic activity in vitro. The activity of DNA topoisomerase I is inhibited by ZnCl 2, CuCl 2 and Pb(NH 3) 3 at millimolar concentrations, but less inhibition was observed with CaCl 2.

Involvement of a Putative Protein-tyrosine Phosphatase and IκB-α Serine Phosphorylation in Nuclear Factor κB Activation by Tumor Necrosis Factor

Inhibitors of phosphotyrosyl protein phosphatases, pervanadate and phenylarsine oxide, abrogate tumor necrosis factor (TNF)-induced nuclear factor kappa B (NF-kappa B) nuclear translocation in transformed cell lines (U-937 and Jurkat) and primary fibroblasts (MRC-5 and REF). The inhibitors also abrogate NF-kappa B activation by the phosphoseryl/threonyl protein phosphatase inhibitor okadaic acid in U-937 cells. Inhibition of NF-kappa B activation is not due to a general inhibitory effect since neither pervanadate nor phenylarsine oxide treatment affected the constitutive DNA-binding activity of the transcription factors octamer-1 and cAMP response element-binding protein in U-937 cells, nor did these compounds inhibit the TNF-induced phosphorylation of proteins, viz. hsp-27, eukaryotic initiation factor 4e, and pp19, in MRC-5 fibroblasts. Overexpression of the protein-tyrosine phosphatase HPTP alpha resulted in a constitutive nuclear NF-kappa B-like DNA-binding activity in REF cells. Conversely, treatment of human protein-tyrosine phosphatase alpha-overexpressing cells with phenylarsine oxide led to a loss of the constitutive NF-kappa B activity. The presence of a tyrosine phosphorylation site on the inhibitor of NF-kappa B (I kappa B-alpha) suggested that it could be a target for TNF/okadaic acid-induced tyrosine dephosphorylation. However, no tyrosine phosphorylation was detected on I kappa B-alpha fron unstimulated cells, while TNF/okadaic acid-treated cells showed increased phosphorylation of I kappa B-alpha exclusively at serine residue(s). Treatment of cells with pervanadate inhibited TNF-induced I kappa B-alpha phosphorylation and degradation, whereas the serine protease inhibitors tosylphenylalanyl chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone prevented TNF-induced I kappa B-alpha degradation and NF-kappa B nuclear translocation, but not the TNF-induced phosphorylation of I kappa B-alpha. The data suggest that TNF and okadaic acid induce the activation of a putative protein-tyrosine phosphatase(s), leading to I kappa B-alpha serine phosphorylation and degradation and NF-kappa B nuclear translocation.

Purification of the delta isoenzyme of protein kinase C from the hepatopancreas of the shrimp Penaeus monodon with phosphorylation on tyrosine residues.

The delta isoenzyme of protein kinase C (PKC-delta), purified from the plasma membrane of the hepatopancreas of the shrimp Penaeus monodon is specifically phosphorylated at tyrosine residues, as demonstrated by specific dephosphorylation by phosphotyrosyl protein phosphatase from the hepatopancreas of the shrimp Penaeus monodon. The specific activity of purified PKC-delta was 200 units/mg of protein. The subunits of M(r) 66,000, 62,000, and 58,000 of PKC-delta were not autophosphorylated after the addition of phosphatidylserine and diolein. However, the purified PKC-delta was active and catalyzed the phosphorylation of myelin basic protein. The kinase activity of the purified PKC-delta could be decreased after treatment with phosphotyrosyl protein phosphatase.

Fluorescence Resolution of the Intrinsic Tryptophan Residues of Bovine Protein Tyrosyl Phosphatase

Fluorescence steady-state and lifetime measurements have been performed that permit the differentiation of the 2 intrinsic tryptophan residues in bovine low molecular weight phosphotyrosyl protein phosphatase (BPTP). Spectral information was obtained by use of two single-tryptophan mutant proteins, W39F and W49F, and the double mutant protein W39,49F. Fluorescence measurements show that Trp39 is characterized by a large blue shift, a low quantum yield, and a shorter mean lifetime compared to Trp49. Solute fluorescence quenching studies of W39F reveal that Trp49 is highly exposed to the aqueous environment. In contrast, Trp39 is situated within a hydrophobic core and is only partially accessible to quenching agents such as acrylamide, iodide ion, and cesium ion. The fluorescence contributions of Trp39 and Trp49 are additive, and their sum is equivalent to that observed for wild type BPTP. Calculated intramolecular distances between Trp39 or Trp49 and a 5-[[(acetylamino)-ethyl]amino]naphthalene-1- sulfonate group covalently bound at Cys12 or Cys17 of the respective protein mutants, place Trp49 within 10 A and Trp39 at least 20 A from the active site. The fluorescence decay of the single tryptophan mutants and, surprisingly, wild type BPTP were each adequately fitted as biexponentials. The latter is a consequence of the imprecision involved in determining actual minima in a three- and four-exponential fitting. Comparison of quenching results of wild type BPTP with those of the single tryptophan mutant proteins indicates that minor fluorescence components, easily resolved using a biexponential fitting for the mutant proteins, are unresolvable for wild type BPTP. These minor components skewed the weighted magnitudes and induced perturbations in lifetimes for the tryptophan fluorescence of wild type BPTP, which directly influenced the calculated values of Ksv and kq.

Tyrosine phosphorylation regulates the DNA binding activity of a nuclear factor 1-like repressor protein.

We have previously identified an ubiquitous repressor binding site that binds a nuclear factor 1 (NF-1)-like transcription factor designated BEF-1. The DNA binding activity of BEF-1, a 98-kDa protein, is increased by the oncoproteins of adenovirus, the early region 1a proteins (E1a), which results in the induction of further repression. Using the prototype repressor sequence, first identified in the enhancer of the human polyoma virus BKV-P2 we have shown that phosphorylation of BEF-1 is required for its DNA binding activity. We demonstrate here that the inhibition of DNA binding by BEF-1 dephosphorylated with potato acid phosphatase or calf intestinal alkaline phosphatase was reversed by sodium orthovanadate, a specific inhibitor of phosphotyrosyl-protein phosphatases. In addition, BEF-1 binding activity, but not the binding of related factor NF-1, could be inhibited by dephosphorylation with a specific phosphotyrosine phosphatase. We found that both polyclonal and monoclonal phosphotyrosine-specific antibodies blocked binding of the repressor protein to the BEF-1 site. Moreover, BEF-1 activity could be adsorbed on an anti-phosphotyrosine antibody column and specially eluted with phosphotyrosine. In transfection studies in HeLa cells, which contain high levels of BEF-1, we show that E1a-induced repression mediated by BEF-1 was relieved with the tyrosine kinase inhibitors genistein and tyrphostin. Together, these results demonstrate that a phosphotyrosine on the BEF-1 repressor protein regulates DNA binding activity and thus regulates repression of the BKV-P2 enhancer. This report represents the first demonstration that the phosphorylated state of a tyrosine can control gene expression by altering the DNA binding activity of a transcription factor.

Characterization of insulin receptor from the muscle of the shrimp Penaeus japonicus (Crustacea: Decapoda)

The beta-subunit of the insulin receptor from the muscle of the shrimp Penaeus japonicus exists as multiple subtypes with M(r) of 79,000, 77,000 and 75,000. Only the subunit of M(r) 79,000 is autophosphorylated after the addition of insulin. The autophosphorylation occurred specifically at Tyr residues, as demonstrated by the specific subsequent dephosphorylation by the phosphotyrosyl protein phosphatase from the human placenta. The detergent, Triton X-100, and the metal ion, Mn2+, caused a noticeable enhancement of the autophosphorylation of shrimp insulin receptors from the muscle. Okadaic acid activated the kinase activity of the insulin-stimulated insulin receptor, but not the basal activity of the insulin receptor without the addition of insulin. Further studies comparing the insulin binding of the shrimp insulin receptor in the regulation of kinase activity of the multiple beta-subunit subtypes from the shrimp muscle are under way.