Biomedical subjects

E H Fischer

Publications and source records attributed to E H Fischer.

At least 37 records · Page 2Linked to original sources

PubMed1996

Cloning and characterization of rat density-enhanced phosphatase-1, a protein tyrosine phosphatase expressed by vascular cells.

We have cloned from cultured vascular smooth muscle cells a protein tyrosine phosphatase, rat density-enhanced phosphatase-1 (rDEP-1), which is a probable rat homologue of DEP-1/HPTP eta. rDEP-1 is encoded by an 8.7-kb transcript and is expressed as a 180- to 220-kD protein. The rDEP-1 gene is located on human chromosome 11 (region p11.2) and on mouse chromosome 2 (region 2E). The cDNA sequence predicts a transmembrane protein consisting of a single phosphatase catalytic domain in the intracellular region, a single transmembrane domain, and eight fibronectin type III repeats in the extracellular region (GenBank accession number U40790). In situ hybridization analysis demonstrates that rDEP-1 is widely expressed in vivo but that expression is highest in cells that form epithelioid monolayers. In cultured cells with epitheliod morphology, including endothelial cells and newborn smooth muscle cells, but not in fibroblast-like cells, rDEP-1 transcript levels are dramatically upregulated as population density increases. In vivo, quiescent endothelial cells in normal arteries express relatively high levels of rDEP-1. During repair of vascular injury, expression of rDEP-1 is downregulated in migrating and proliferating endothelial cells. In vivo, rDEP-1 transcript levels are present in very high levels in megakaryocytes, and circulating plates have high levels of the rDEP-1 protein. In vitro, initiation of differentiation of the human megakaryoblastic cell line CHRF-288-11 with phorbol 12-myristate 13-acetate leads to a very strong upregulation of rDEP-1 transcripts. The deduced structure and the regulation of expression of rDEP-1 suggest that it may play a role in adhesion and/or signaling events involving cell-cell and cell-matrix contact.

Amino Acid Sequence↗

PubMed1995

Association of mitogen-activated protein kinase with the microtubule cytoskeleton.

Using indirect immunofluorescence microscopy and biochemical techniques, we have determined that approximately one-third of the total mitogen-activated protein kinase (MAPK) is associated with the microtubule cytoskeleton in NIH 3T3 mouse fibroblasts. This population of enzyme can be separated from the soluble form that is found distributed throughout the cytosol and is also present in the nucleus after mitogen stimulation. The microtubule-associated enzyme pool constitutes half of all detectable MAPK activity after mitogenic stimulation. These findings extend the known in vivo associations of MAPK with microtubules to include the entire microtubule cytoskeleton of proliferating cells, and they suggest that a direct association of MAPK with microtubules may be in part responsible for the observed correlations between MAPK activities and cytoskeletal alteration.

3T3 Cells↗

PubMed1995

Altered expression of protein-tyrosine phosphatase 2C in 293 cells affects protein tyrosine phosphorylation and mitogen-activated protein kinase activation.

PTP2C, an SH2 domain-containing protein-tyrosine phosphatase, is recruited to the growth factor receptors upon stimulation of cells. To investigate its role in growth factor signaling, we have overexpressed by approximately 6-fold the native PTP2C and a catalytically inactive mutant of the enzyme in 293 human embryonic kidney cells. The native PTP2C was located entirely in the cytosol, while the inactive mutant was nearly equally distributed in cytsolic and membrane fractions. Expression of the latter caused hyperphosphorylation on tyrosine of a 43-kDa protein, which was coimmunoprecipitated and co-partitioned in the plasma membrane fraction with the inactive PTP2C mutant. This protein may represent a physiological substrate of PTP2C. Overexpression of the native PTP2C enhanced epidermal growth factor (EGF)-stimulated mitogen-activated protein (MAP) kinase activity by 30%, whereas expression of the inactive mutant reduced the stimulated activity by 50%. Similar effects were observed for the activation of MAP kinase as determined by activity assay, gel mobility shift, and tyrosine phosphorylation. The data suggest that the phosphatase activity of PTP2C is partly required for MAP kinase activation by EGF and that PTP2C may function by dephosphorylating the 43-kDa membrane protein.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1995

Functional impairment in depressed inpatients.

The Sickness Impact Profile (SIP) was administered to 95 patients with major depression. The SIP includes 12 subscales, each representing a specific area of sickness-related dysfunction. To relate these measures to psychiatric symptoms, patients also completed a measure of depression severity. Consistent with earlier findings, there were high levels of functional impairment. Impairment was correlated with symptoms but, as noted elsewhere, functional status does not always parallel symptom severity. The SIP, widely used with medical disorders but, to our knowledge, underutilized to assess psychiatric patients, may provide more precise assessment of the impact of psychiatric disorders on the individual's daily life.

Activities of Daily Living↗

PubMed1995

COOH-terminal sequence motifs target the T cell protein tyrosine phosphatase to the ER and nucleus.

The noncatalytic domain of the human T cell protein tyrosine phosphatase (TCPTP) is alternatively spliced to generate a 45-kD form, p45TC, and a 48-kD form, p48TC (Champion-Arnaud et al., 1991; Mosinger et al., 1992). This manuscript concerns structural motifs in the noncatalytic segment of the enzyme responsible for targeting the two forms to different subcellular compartments. Endogenous and transiently expressed p48TC associates with the ER, as determined by sucrose gradient fractionation and indirect immunofluorescence, respectively. By contrast, p45TC localizes in the nucleus even though upon cell lysis it is not retained and fractionates with markers for soluble enzymes. Using fusion proteins consisting of beta-galactosidase and COOH-terminal fragments of p48TC, two motifs necessary for ER retention within a 70-residue targeting segment have been identified. These include the terminal 19 hydrophobic residues which comprise a potential membrane-spanning segment and residues 346-358 which encompass a cluster of basic amino acids that may represent another type of ER retention motif. The sequence RKRKR, which immediately precedes the splice junction, functions as a nuclear localization signal for p45TC.

Alternative Splicing↗

PubMed1995

Functional capabilities of depressed patients one year after hospitalization.

During hospitalization and at one-year follow-up, 48 patients with a diagnosis of major depression completed the Inventory to Diagnose Depression and the Sickness Impact Profile (SIP). Overall change scores revealed statistically significant and clinically important improvements in depression and functioning. SIP subscale scores showed no deficits in some functions (communication and bodily care) at baseline, restoration of functioning in other activities at one-year follow-up, and continuing dysfunctions in emotionality, alertness, recreation, socialization, and work. The SIP appears useful for measuring disability and patterns of recovery in depressed patients.

Activities of Daily Living↗

PubMed1995

Validity of the diagnostic interview schedule for detecting alcoholism in psychiatric inpatients.

This study examined the accuracy of the Diagnostic Interview Schedule (DIS) for indicating alcohol use disorder in a sample of patients hospitalized for depression. The Michigan Alcoholism Screening Test (MAST), with its established validity, was considered a good criterion against which to evaluate the DIS, and preferable to the clinician-assigned diagnosis in this regard. The rates of alcoholism in the sample were 31, 33, and 22.5% as yielded by the DIS, MAST, and physician's diagnosis of alcohol disorder, respectively. (The lower rate for physician's diagnosis may be due to the physician's not applying this diagnosis to recovered or currently abstinent alcohol patients.) Using the MAST's standard cutoff of five points as indicative of alcoholism, agreement with the DIS occurred in 91% of the cases, corresponding to a product moment coefficient of .79. It was concluded that the DIS alcoholism scale could be used, with reasonable confidence in its validity, for assessing alcoholism in psychiatric settings.

Adolescent↗

PubMed1994

Identification and purification of a chicken brain neuroglia-associated protein.

The identification, purification, and biochemical characterization of specific markers for neuroglial cells in the central nervous system is an essential step toward a better understanding of the function of glial cells. This manuscript reports the identification and purification of a neuroglia-associated protein (NAP-185) with an apparent molecular mass of 185 kDa. While its expression is not restricted to the brain, it was first identified in a specific subpopulation of glial cells when chick brain stem sections were analyzed with an affinity-purified rabbit antiserum raised against the catalytic domain of the T-cell protein tyrosine phosphatase. This 185-kDa antigen was purified to apparent homogeneity and confirmed to be responsible for the neuroglial staining observed. In spite of its immunological relation to T-cell protein tyrosine phosphatase, purified NAP-185 failed to display tyrosine phosphatase activity. The primary sequence of five NAP-185-derived peptides shows that this protein has not yet been characterized and that it is possibly related to AP180, a clathrin-associated protein.

Amino Acid Sequence↗

PubMed1994

Overexpression of mitogen-activated protein kinase kinase (MAPKK) and its mutants in NIH 3T3 cells. Evidence that MAPKK involvement in cellular proliferation is regulated by phosphorylation of serine residues in its kinase subdomains VII and VIII.

Mitogen-activated protein kinase kinase (MAPKK) is a dual specificity protein kinase that exhibits a high degree of specificity toward its downstream target, mitogen-activated protein kinase (MAPK). In this study, we used stable overexpression of MAPKK and its mutants in NIH 3T3 cells to study effects on downstream components of the MAPK signaling cascade and to correlate them to physiological responses. We have mutated the potential regulatory serine residue 222 to alanine (S222A) or to glutamate (S222E) and serines 212 and 218 together to alanine residues (S212A,S218A). Lysine 97 was mutated to alanine (K97A) to provide an inactive enzyme. Overexpression of the wild type MAPKK had no effect on any of the parameters examined. The K97A and S222A mutants served as dominant negatives by suppressing MAPKK, MAPK, and p90rsk activation in vivo. S222E enhanced all of these activities, and S212A,S218A had a small inhibitory effect. A similar trend was observed when cellular proliferation was examined and the different effects were accompanied by altered cellular shape. Taken together, our results demonstrate a direct linkage between the MAPK signaling pathway and the control of cellular proliferation and morphology and also establish that phosphorylation of serine 222 is essential for MAPKK activation together with the phosphorylation of an additional serine(s) (probably serine 218).

3T3 Cells↗

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

Dephosphorylation of insulin receptor substrate 1 by the tyrosine phosphatase PTP2C.

The phosphotyrosine (Tyr(P)) form of insulin receptor substrate 1 (IRS-1) is a key component in insulin signaling. Our previous study revealed that Tyr(P) IRS-1 binds to the widely distributed tyrosine phosphatase PTP2C through the src homology 2 (SH2) domains of the latter. In the present study, we examined the activity of this enzyme and of a truncated form lacking the SH2 domains (delta PTP2C) toward IRS-1 and also toward the cytoplasmic domain of the insulin receptor. Tyr(P) IRS-1 was prepared by phosphorylation of recombinant IRS-1 with recombinant cytoplasmic insulin receptor kinase (CIRK). PTP2C rapidly dephosphorylated Tyr(P) IRS-1; dephosphorylation by delta PTP2C was approximately one-third as fast. Other substrates, including Tyr(P) CIRK, were not dephosphorylated as rapidly by PTP2C; moreover, delta PTP2C was at least 10 times more active than PTP2C toward CIRK and other substrates. These results indicate that the binding of Tyr(P) residues on IRS-1 to the SH2 domain(s) of PTP2C enhances its activity toward IRS-1 and suggest that PTP2C is the phosphatase responsible for the dephosphorylation of IRS-1 in vivo. In addition, with the expectation that a PTP2C-resistant form of IRS-1 will be useful in investigations of IRS-1 function, we determined that IRS-1 can be thiophosphorylated with adenosine 5'-O-(3-thiotriphosphate) and CIRK and that this form of IRS-1 is resistant to PTP2C.

Animals↗

PubMed1994

Protein-tyrosine-phosphatase 2C is phosphorylated and inhibited by 44-kDa mitogen-activated protein kinase.

Protein-tyrosine-phosphatase 2C (PTP2C, also named SHPTP2, SHPTP3, or PTP1D) is a cytosolic enzyme with two Src homology 2 domains. We have investigated its regulation by phosphorylation in PC12 rat pheochromocytoma cells. In untreated cells, PTP2C was phosphorylated predominantly on serine residues. A 5-min treatment with epidermal growth factor (EGF) induced an increase in phosphorylation on threonine and, to a lesser degree, on serine. After 45 min of exposure to EGF, PTP2C phosphorylation returned to basal levels. Using an in vitro kinase assay, we found that the 44-kDa mitogen-activated protein kinase, p44mapk, phosphorylated PTP2C on serine and threonine residues. This phosphorylation resulted in a pronounced inhibition of PTP2C enzyme activity measured with phosphorylated EGF receptors as substrate. Moreover, in intact PC12 cells, PTP2C was also inhibited following a short EGF treatment, but its activity returned to normal when the exposure to EGF was maintained for 45 min. The profile of this response to EGF can be inversely correlated to that of the stimulatory action of EGF on p44mapk. These data suggest that the EGF-induced regulation of PTP2C activity is mediated by p44mapk. These findings provide evidence for an additional role of the mitogen-activated protein kinase cascade--namely, the regulation of a PTP.

Animals↗

PubMed1994

Phorbol ester-induced expression, phosphorylation, and translocation of protein-tyrosine-phosphatase 1C in HL-60 cells.

PTP1C, a protein-tyrosine-phosphatase (PTP) containing two Src homology 2 domains, is expressed at high levels in HL-60 human promyelocytic leukemia cells. It represents 0.15% of total protein in a postnuclear extract and corresponds to approximately 70% of phosphatase activity measured with p-nitrophenyl phosphate at pH 5.0. Upon differentiation of HL-60 cells to macrophages induced by phorbol 12-myristate 13-acetate (PMA), the activity and expression levels of PTP1C increase 2- to 3-fold. Furthermore, PTP1C is phosphorylated on serine residues upon PMA treatment. While the enzyme localizes entirely in the cytosolic fraction in untreated cells, 30-40% is found in a particulate fraction following differentiation. Immunofluorescent staining of differentiated cells before and after digitonin treatment indicates that the bound enzyme localizes on the plasma membrane. The time courses of induction and translocation of PTP1C correlate with the differentiation process. The PMA-induced differentiation is inhibited by sodium orthovanadate, suggesting the PTPs are required for this process.

Biological Transport↗

PubMed1994

Multiple forms of the human tyrosine phosphatase RPTP alpha. Isozymes and differences in glycosylation.

Among all the receptor-linked protein-tyrosine-phosphatase RPTP alpha clones described from mammalian tissues, one differed in that it encoded a 9-amino-acid insert 3 residues upstream from the transmembrane segment (Kaplan, R., Morse, B., Huebner, K., Croce, C., Howk, R. Ravera, M., Ricca, G., Jaye, M., and Schlessinger, J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7000-7004). Using the polymerase chain reaction technique, simultaneous expression of both isoforms was demonstrated in human T-cell and vascular smooth muscle libraries, as well as in the A431 human epidermal cancer cell line. Following transient expression in COS-1 cells, each isoform gave rise to two proteins of 100 and 130 kDa, respectively. Endoglycosidase treatment showed that the 100-kDa species corresponded to a molecule exclusively glycosylated on N-residues, whereas the 130-kDa species contained both, N- and O-linked carbohydrates. Pulse-chase experiments demonstrated that the smaller RPTP alpha protein is a precursor of the larger one. A high affinity antibody was generated that recognizes the immature protein only; however, both proteins can be detected by Western blot analysis after a simple chemical hydrolysis. Following Superose 12 chromatography, the 100- and 130-kDa species of RPTP alpha emerged as 200- and 340-kDa proteins, respectively. Both species exhibited similar enzymatic activities as determined with a peptide substrate in immunoprecipitates.

Amino Acid Sequence↗

PubMed1994

Purification and characterization of PTP2C, a widely distributed protein tyrosine phosphatase containing two SH2 domains.

PTP2C, a widely distributed protein tyrosine phosphatase (PTP) containing two SH2 domains, was expressed as a recombinant enzyme in Escherichia coli and purified to near homogeneity. The purified enzyme and a truncated form lacking the SH2 domains (delta SH2-PTP2C) have been characterized with four commonly used substrates. Both forms showed pH optima of around neutrality for protein substrates but below 5.5 for a peptide substrate and para-nitrophenylphosphate. The dependence of the enzymes on ionic strength varied with the nature of the substrates involved. Like its analog PTP1C, PTP2C displayed a specific activity of less than 0.1% of that observed with other known PTPs toward protein substrates. Deletion of the SH2 domains increased its activity by 12-45-fold, depending on the substrates used. Limited trypsinolysis which cleaved about 4 kDa from the carboxyl terminus resulted in a 2-5-fold activation of the full-length enzyme but was essentially without effect on the truncated enzyme. Both forms showed similar responses to effectors including activators (e.g. anionic phospholipids) or inhibitors (e.g. vanadate, molybdate, or Zn2+). PTP2C and delta SH2-PTP2C were phosphorylated in vitro by mitogen-activated protein kinase, protein kinase C, and various protein tyrosine kinases; in the latter case, they underwent autodephosphorylation. No significant effect of the phosphorylation reactions on enzyme activity could be observed in vitro.

Amino Acid Sequence↗

PubMed1993

Differential activities of protein tyrosine phosphatases in intact cells.

We have employed transient co-overexpression of protein tyrosine phosphatases (PTPs) with a panel of receptor tyrosine kinases (RTKs) to investigate molecular parameters that regulate dephosphorylation activity and specificity in intact cells. Our results demonstrate clear differences in susceptibility of various forms of different RTKs to the action of PTP 1B, T-cell phosphatase (TC-PTP), and CD45, which suggests cellular compartmentalization as a major factor defining activity and overall function. TC-M PTP, a nonlocalized cytosolic mutant, is deregulated and is therefore able to efficiently suppress v-erbB- and v-fms-induced cell transformation, which is not observed with the intact TC-PTP or PTP 1B. The transmembrane PTP CD45 displays more selectivity but appears to be already active during transport to the cell surface. Dephosphorylation activity is also dependent on relative RTK/PTP expression levels and can be modulated by the SH2 domain-containing noncatalytic subunit of phosphatidylinositol 3'-kinase, p85. Overexpression of high affinity binding proteins could therefore contribute to RTK-induced cell transformation and cancer.

Base Sequence↗

PubMed1993

A general peptide substrate for protein tyrosine phosphatases.

The determination of protein tyrosine phosphatase (PTP) activity using different protein substrates such as modified lysozyme or myelin basic protein is greatly affected by the type of enzyme involved, the condition of the assay, and the presence of effectors. The purpose of this study was to develop a general substrate of wide applicability with which variations in enzymatic activity would be minimized. A nonapeptide ENDYINASL derived from a highly conserved region of the T-cell phosphatase TC.PTP (Cool et al. (1989) Proc. Natl. Acad. Sci. USA 86, 5257-5261) was phosphorylated with a recombinant tyrosine kinase domain of the EGF receptor and purified on a C18 cartridge. Phosphatase activities of intracellular and receptor-linked PTPs were as high as the highest values obtained with the protein substrates. The intracellular, low-molecular-weight PTPs exhibited Km values between 0.5 and 1.3 microM, whereas the receptor forms CD45 and RPTP alpha gave values of 14 and 35 microM, respectively. All PTPs displayed similar properties toward the peptide including a low pH optimum and inhibition by vanadate, divalent cations, and heparin. Following immunoprecipitation, 1 ng of TC.PTP could be detected with ENDY(P)INASL compared to 10 ng in presence of protein substrates.

Amino Acid Sequence↗

PubMed1993

Stimulation by phospholipids of a protein-tyrosine-phosphatase containing two src homology 2 domains.

PTP1C, a protein-tyrosine-phosphatase (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) containing two src homology 2 domains, is poorly active when assayed with various protein substrates in vitro. Its activity is stimulated > 1000-fold by anionic phospholipids when myelin basic protein or mitogen-activated protein kinase is used as substrate but reduced in the presence of several other substrates. Data are presented to indicate a direct interaction of the enzyme with phospholipids. Enzyme stimulation directed only toward certain specific substrates is interpreted by assuming that these compounds also bind to the phospholipid vesicles where they will be subjected to rapid enzymatic attack. A possible regulation of PTP1C by its translocation to the cell membrane is hypothesized.

Amino Acid Sequence↗