Search PubMed⌕ Search

Biomedical subjects

C J Pallen

Publications and source records attributed to C J Pallen.

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

Expression of receptor-like protein tyrosine phosphatase alpha in rat embryo fibroblasts activates mitogen-activated protein kinase and c-Jun.

Anti-c-Src and anti-phosphotyrosine immunoprecipitates from receptor-like protein tyrosine phosphatase alpha (PTP alpha)-transfected and control rat embryo fibroblasts contain a 39-kDa phosphoprotein (p39) whose phosphorylation is enhanced by PTP alpha expression. The p39 that co-immunoprecipitates with c-Src has been identified as c-Jun by immunological and functional criteria; it is recognized by several different anti-c-Jun antibodies and binds to a c-Jun recognition element-containing oligonucleotide. Whereas the association of c-Src and c-Jun is unexpected, it may be of significance in PTP alpha signaling since we have previously demonstrated that c-Src is activated by PTP alpha (Zheng, X. M., Wang, Y., and Pallen, C. J. (1992) Nature 359, 336-339. Examination of c-Jun activity in these fibroblasts demonstrates that c-Jun DNA binding activity and c-Jun-mediated transcription of a chloramphenicol acetyltransferase reporter gene are elevated in PTP alpha-expressing cells. In addition to c-Jun activation, mitogen-activated protein kinase is activated in PTP alpha-expressing cells and translocated to the nuclei of these cells. The nuclear localization of activated mitogen-activated protein kinase and c-Jun suggests that their activation represents downstream events in the receptor-like PTP alpha-initiated signaling pathway(s).

Animals↗
PubMed1993

The receptor-like protein tyrosine phosphatase alpha: a role in cell proliferation and oncogenesis.

The transmembrane nature of the receptor-like protein tyrosine phosphatases (PTPases) suggests that they transduce as yet unidentified extracellular signals to intracellular events via a phosphotyrosyl-protein dephosphorylation step, although little is known of their regulation and cellular activities. Structure/function studies of PTP alpha demonstrate that both catalytic domains are required for full enzymatic efficiency and that interdomain interactions may modulate PTP alpha activity and specificity. Overexpression of PTP alpha results in cell transformation and tumorigenesis, likely as a consequence of the ability of PTP alpha to dephosphorylate and activate the c-src tyrosine kinase. This suggests a role for PTP alpha in normal cell proliferation. PTP alpha is so far unique among the PTPases in terms of its oncogenic potential, and overexpression or deregulation of PTP alpha may be involved in the genesis, progression or maintenance of certain tumor states.

Cell Division↗
PubMed1992

Cell transformation and activation of pp60c-src by overexpression of a protein tyrosine phosphatase.

The kinase activity of pp60c-src is specifically and transiently increased during mitosis and repressed during interphase. Loss of cell-cycle control of pp60c-src occurs on mutation of Tyr527 to Phe or when pp60c-src is associated with polyoma middle-T-antigen, and these conditions result in cell transformation or tumorigenesis. In both cases, pp60c-src has elevated kinase activity which is maintained throughout the cell cycle and accompanied by dephosphorylation of the carboxy-terminal negative regulatory Tyr527 site, or mimicry of Tyr527 dephosphorylation in the case of the mutant. Here we report that overexpression of the receptor-like protein tyrosine phosphatase PTP alpha results in persistent activation of pp60c-src kinase, with concomitant cell transformation and tumorigenesis. In PTP alpha-overexpressing cells, the pp60c-src kinase activation is accompanied by dephosphorylation at Tyr527, and direct dephosphorylation of this site by purified PTP alpha occurs in vitro. Our results suggest that PTP alpha is involved in the regulation of cell proliferation, exerting at least some of its effects through pp60c-src kinase, and has oncogenic capability when overexpressed.

Amino Acid Sequence↗
PubMed1992

Expression and characterization of wild type, truncated, and mutant forms of the intracellular region of the receptor-like protein tyrosine phosphatase HPTP beta.

Human HPTP beta is unique among mammalian receptor-like protein tyrosine phosphatases in that it has only a single catalytic domain. The intracellular region of HPTP beta was expressed in bacteria, purified, and characterized. It exhibits high activity toward all substrates tested and is potently inhibited by zinc. Vanadate and polyanions also inhibited activity. The juxta-membrane segment of HPTP beta (residues 1622-1639) potentially functions as a negative regulatory sequence since its deletion can increase HPTP beta activity 5-fold. This segment contains up to two sites for protein kinase C phosphorylation, although in vitro phosphorylation by this kinase did not affect HPTP beta activity. The boundaries of the catalytic domain were delineated by truncation analyses. Successive deletion of N-terminal sequence prior to residue 1684 had little effect on substrate affinity and at most reduced activity about 6-fold. Further removal of residues 1684-1686 resulted in a marked 50-500-fold drop in activity, and loss of N-terminal sequence prior to residue 1690 abolished activity. Based on these analyses a highly conserved motif was identified in all mammalian tyrosine phosphatases (E/q) (F/y)XX(L/i), corresponding to positions 1684-1688 of HPTP beta. Mutation of residue 1684 or 1685 generally gave rise to proteins with marked temperature sensitivity. These mutant HPTP beta were active but had reduced activity compared to the wild type enzyme. In conjunction, these results suggest that this region represents the N-terminal border of the catalytic domain and is essential for correct phosphatase folding although not directly involved in catalysis. Parallel truncation studies have defined residues 1930-1939/40 as the C-terminal border of the catalytic domain.

Amino Acid Sequence↗
PubMed1992

Protein phosphatases in cell signalling.

Protein phosphorylation is the most common post-translational modification and plays a role in all known pathways of signal transduction. Net phosphorylation is a result of the balance of activities of protein kinases and phosphatases. There is increasing evidence that the regulated removal of phosphate groups from proteins, catalyzed by protein phosphatases, is required for the downstream activation of other signalling proteins.

Animals↗
PubMed1992

Pituitary-specific transcriptional initiation sites of the rat carboxypeptidase-H gene and the influence of thyroid hormone status.

Carboxypeptidase-H (CPH) is a metallocarboxypeptidase implicated in the processing of peptide hormones. Consistent with such a role, the gene for CPH is expressed in cells that secrete regulatory peptides, such as those of the brain and endocrine tissues. In the rat brain, CPH is transcribed from a single transcriptional start site associated with the initiator-type element first described in the gene encoding lymphocyte-specific terminal deoxynucleotidyltransferase. We have used a combination of Northern blot analysis and S1 nuclease protection mapping to describe the expression and transcription initiation pattern of the gene for CPH in the peripheral tissues and brain regions of the normal rat. The single transcriptional start site is used in all tissues examined, except the pituitary, where two additional specific initiation sites are found. The expression of the CPH gene is up-regulated in the anterior pituitary gland as a consequence of systemic thyroid hormone depletion, and this increase is associated with a preferential utilization of the novel upstream transcriptional initiation sites. Thus, the use of different major transcriptional initiation sites of the CPH gene in the pituitary gland is subject to differential direct or indirect thyroid hormone regulation.

Amino Acid Sequence↗
PubMed1991

Elevation of membrane tyrosine phosphatase activity in density-dependent growth-arrested fibroblasts.

Swiss 3T3 cells harvested at high density contain a membrane protein-tyrosine-phosphatase (EC 3.1.3.48) whose specific activity is on average 8-fold higher than that of cells harvested at low or medium densities. Investigation of the conditions affecting this elevation of specific activity suggests that it is associated with density-dependent growth arrest. Fibroblasts in the exponentially doubling phase have a relatively low level of membrane phosphatase specific activity, which rises only as the rate of cell proliferation decreases and is maximal when cell growth is contact inhibited. These observations have been extended to BALB/c 3T3 fibroblasts and normal human diploid fibroblasts. The increase in membrane tyrosine phosphatase activity is coupled to density arrest and not to cellular quiescence in general, as no increase in phosphatase specific activity is detected when non-contact-inhibited cells are induced to arrest their growth through serum deprivation. The observed alterations in specific activity are attributable to a tyrosine phosphatase of Mr 37,000 that was purified and characterized from solubilized membrane fractions of Swiss 3T3 cells. A regulatory mechanism controlling tyrosine phosphatase activity may play a role in cell proliferation and growth arrest caused by cell contact.

Amino Acid Sequence↗
PubMed1991

Purification and characterization of a higher-molecular-mass form of protein phosphotyrosine phosphatase (PTP 1B) from placental membranes.

Purification of a major placental membrane protein phosphotyrosine phosphatase (PTP-I) through the use of a nonhydrolysable phosphotyrosine analogue affinity ligand has enabled identification of the enzyme as a single polypeptide of at least 46 kDa. This phosphatase specifically dephosphorylates phosphotyrosine-containing substrates, including the src peptide, the epidermal-growth-factor receptor tyrosine kinase and the non-receptor tyrosine kinase p56lck. The p56lck can be dephosphorylated by PTP-I at two tyrosine residues (Tyr-394 and Tyr-505), which are differentially phosphorylated in vitro and in vivo and have been suggested to modulate kinase activity. The activity of PTP-I towards these substrates indicates a possible function of regulation of cellular tyrosine phosphorylation pathways at the level of growth-factor receptor and/or oncogene/proto-oncogene tyrosine kinases. Kinetic analyses show that PTP-I exhibits a Km value of about 2 microM with either src peptide or reduced, carboxyamidomethylated and maleylated (RCM)-lysozyme as substrate, and is inhibited in a mixed competitive manner by the polyanions heparin and poly(Glu4,Tyr1). Sequencing of PTP-I peptides reveals almost complete identity with sequences within the N-terminal half of the 37 kDa non-receptor tyrosine phosphatase 1B. However, the size and amino acid composition of PTP-I are similar to that of a higher-molecular-mass form of PTP 1B predicted from cDNA cloning. These results suggest that the 37 kDa PTP 1B is a proteolysed form of PTP-I, and provide evidence that a larger form of PTP 1B exists in vivo, at least in association with placental membranes.

Amino Acid Sequence↗
PubMed1991

The receptor-like protein tyrosine phosphatase HPTP alpha has two active catalytic domains with distinct substrate specificities.

Cloning and expression of the homologous domains of the receptor-like tyrosine phosphatase HPTP alpha shows that both domain 1 (D1) and domain 2 (D2) are enzymatically active. The two domains display different substrate specificities with D1 preferentially dephosphorylating MBP approximately RR-src greater than PNPP while D2 favours PNPP much much greater than RR-src and is inactive towards MBP. Each domain has lower activity than an expressed protein containing both domains. Analysis of chimaeric D1/2 proteins suggests that no particular region of D2 is responsible for the low activity of D2 on RR-src and that the specificity differences of D1 and D2 reflect overall sequence dissimilarities. Activities of D1 and D2 are inhibited by zinc, vanadate and EDTA and differentially susceptible to inhibition by heparin and poly(Glu4:Tyr1). Unusually, the activity of the protein containing both domains is stimulated by these polyanions. Regions amino-terminal to each domain are important for catalysis since deletion of these sequences abolishes phosphatase activity. Activity of the double domain polypeptide was also lost upon deletion of the sequence amino-terminal to D1, indicating that inactivation of D1 may suppress D2 activity. Differences in substrate specificity and responses to effectors and the interdependence between the two domains are likely important properties in the function of this PTPase in signal transduction.

Amino Acid Sequence↗
PubMed1989

Calcium-dependent and phosphorylation-stimulated proteolysis of lipocortin I by an endogenous A431 cell membrane protease.

Purified placental lipocortin I but not lipocortin II was proteolyzed during A431 cell membrane-catalyzed phosphorylation reactions. Proteolysis was Ca2+-dependent but was not prevented in the presence of a variety of inhibitors of Ca2+-dependent proteases, suggesting that the Ca2+ effect is a property of lipocortin I itself. Proteolysis was inhibited by Triton X-100 or dithiothreitol and was temperature-dependent, occurring at 30 degrees C but not at 0 degrees C. Tyrosine phosphorylation and proteolysis are distinct events as both phosphorylated and nonphosphorylated lipocortins could be cleaved by the membrane protease, but prephosphorylation enhanced the rate of proteolysis 2-fold during the initial reaction and by 60 min almost half of the phosphorylated lipocortin was proteolyzed. Cleavage of the 38-kDa phosphotyrosyl lipocortin I generated a truncated 37-kDa form of lipocortin which retained the phosphate label, indicating that proteolysis occurred at a site N-terminal to the site of tyrosine phosphorylation, possibly at tryptophan 12. Ando, Y., Imamura, S., Hong, Y.-M., Owada, M.K., Kakunaga, T., and Kannagi, R. [1989) J. Biol. Chem. 264, 6948-6955) have recently reported that in vitro cleavage at sites in the N-terminal tail region of lipocortin I by exogenously added proteases dramatically enhanced the Ca2+ sensitivity of phospholipid binding by lipocortin. The demonstrated ability of an endogenous membrane protease to catalyze a similar and specific cleavage in a Ca2+-dependent manner indicates that this event may occur in the cell where it would have important effects on the functional properties of lipocortin I.

Actins↗
PubMed1988

Conversion of a phosphoseryl/threonyl phosphatase into a phosphotyrosyl phosphatase.

By use of the autophosphorylated epidermal-growth-factor receptor and the synthetic peptide RRLIE-DAEY(P)AARG, representing an autophosphorylation site of the transforming protein of Rous-sarcoma virus, it is demonstrated that the phosphotyrosyl phosphatase activity of the polycation-stimulated phosphatases is substantially increased by an enzyme-directed effect of ATP or PPi. Concomitant with this increase in phosphotyrosyl phosphatase activity, the phosphorylase phosphatase activity is decreased, thus dramatically changing the substrate specificity of these enzymes. The dephosphorylation of four different phosphotyrosyl sites of the epidermal-growth-factor receptor is neither consecutive nor at random, but a preferred dephosphorylation of the P1 site over the P3 greater than P2 greater than P4 sites is observed. This phosphatase activity represents a substantial fraction of the total phosphotyrosyl phosphatase activity in the post-mitochondrial supernatant of Xenopus laevis oocytes.

Adenosine Triphosphate↗
PubMed1986

Stoichiometry and dynamic interaction of metal ion activators with calcineurin phosphatase.

Calcineurin, a calmodulin-regulated phosphatase, is composed of two distinct subunits (A and B) and requires certain metal ions for activity. The binding of the two most potent activators, Ni2+ and Mn2+, to calcineurin and its subunits has been studied. Incubation of the protein with 63Ni2+ (or 54Mn2+) followed by gel filtration to separate free and protein-bound ions indicated that calcineurin could maximally bind 2 mol/mol of Ni2+ or Mn2+. While isolated A subunit also bound 2 mol/mol of Ni2+, no Mn2+ binding was demonstrated for either isolated A or B subunit. When bindings were monitored by nitrocellulose filter assay, only 1 mol/mol bound Ni2+ or Mn2+ was detected, suggesting that the two Ni2+ (or Mn2+) binding sites had different relative affinities and that only metal ions bound at the higher affinity sites were detected by the filter assay. Preincubation of calcineurin with Mn2+ (or Ni2+) decreased the filter assay-measured Ni2+ (or Mn2+) binding by only 30%. Preincubation of the protein with Zn2+ decreased the filter assay-measured Ni2+ or Mn2+ binding by 90 or 17%, respectively. The results suggest that the higher affinity sites are a Ni2+-specific site and a distinct Mn2+-specific site. Preincubation of calcineurin with Mn2+ (or Ni2+) decreased the gel filtration-determined Ni2+ (or Mn2+) binding from 2 to 1 mol/mol suggesting that calcineurin also contains a site which binds either metal ion. The time course of Ni2+ (or Mn2+) binding was correlated with that of the enzyme activation, and the extent of deactivation of the Ni2+-activated calcineurin by EDTA or by incubation with Ca2+ and calmodulin (Pallen, C. J., and Wang, J. H. (1984) J. Biol. Chem. 259, 6134-6141) was correlated with the release of the bound ions, thus suggesting that the bound ion is directly responsible for enzyme activation.

Animals↗
PubMed1986

Characterization of the phosphotyrosyl protein phosphatase activity of calmodulin-dependent protein phosphatase.

Calmodulin-dependent protein phosphatase from bovine brain and heart was assayed for phosphotyrosine and phosphoserine phosphatase activity using several substrates: 1) smooth muscle myosin light chain (LC20) phosphorylated on tyrosine or serine residues, 2) angiotensin I phosphorylated on tyrosine, and 3) synthetic phosphotyrosine- or phosphoserine-containing peptides with amino acid sequences patterned after the autophosphorylation site in Type II regulatory subunit of the cAMP-dependent protein kinase. The phosphatase was activated by Ni2+ and Mn2+, and stimulated further by calmodulin. In the presence of Ni2+ and calmodulin, it exhibited similar kinetic constants for the dephosphorylation of phosphotyrosyl LC20 (Km = 0.9 microM, and Vmax = 350 nmol/min/mg) and phosphoseryl LC20 (Km = 2.6 microM, Vmax = 690 nmol/min/mg). Dephosphorylation of phosphotyrosyl LC20 was inhibited by phosphoseryl LC20 with an apparent Ki of 2 microM. Compared to the reactions with phosphotyrosyl LC20 as the substrate, reactions with phosphotyrosine-containing oligopeptides exhibited slightly higher Km and lower Vmax values. The reaction with the phosphoseryl peptide based on the Type II regulatory subunit sequence exhibited a slightly higher Km (23 microM), but a much higher Vmax (4400 nmol/min/mg) than that with its phosphotyrosine-containing counterpart. Micromolar concentrations of Zn2+ inhibited the phosphatase activity; vanadate was less potent, and 25 mM NaF was ineffective. The study provides quantitative data to serve as a basis for comparing the ability of the calmodulin-dependent protein phosphatase to act on phosphotyrosine- and phosphoserine-containing substrates.

Amino Acid Sequence↗
PubMed1985

Use of fluorinated tyrosine phosphates to probe the substrate specificity of the low molecular weight phosphatase activity of calcineurin.

Calcineurin, a calmodulin-activated protein phosphatase, is known to dephosphorylate certain low molecular weight phosphate esters. The low molecular weight phosphatase activity of calcineurin has been studied by utilizing tyrosine phosphate derivatives. Kinetic studies suggest that the substrate specificity is dependent upon the electronic nature of the substrate in contrast to results obtained with alkaline phosphatase from Escherichia coli. Comparison of calcineurin and acid-catalyzed hydrolyses indicates a 1:1 correlation between the rate constants for the two processes. This correlation and other model studies have been utilized to provide insight into the chemical mechanism of calcineurin. Possible chemical mechanisms for calcineurin are discussed.

Alkaline Phosphatase↗
PubMed1985

Survey of calcineurin activity towards nonprotein compounds and identification of phosphoenol pyruvate as a substrate.

Calcineurin, originally identified as a calmodulin-dependent phosphoprotein phosphatase (Stewart, A.A. et al. (1982) FEBS Lett. 137, 80-84) also uses p-nitrophenyl phosphate and phosphotyrosine as substrates (Pallen, C.J. and Wang, J.H. (1983) J. Biol. Chem. 258, 8550-8553). We have surveyed a wide range of nonprotein phosphocompounds and found that several synthetic aryl phosphocompounds serve as calcineurin substrates. Among more than 20 naturally occurring phosphocompounds tested, only phosphoenol pyruvate possesses significant calcineurin substrate activity. The phosphoenol pyruvate phosphatase activity is dependent on Ni2+ and Mn2+, is stimulated by calmodulin, and is inhibited by a monoclonal antibody to calcineurin, thus indicating that it is an intrinsic property of calcineurin. The results suggest that functional roles of calcineurin may include actions of the enzyme toward nonprotein phosphocompounds.

Amino Acids↗
PubMed1985

Calcineurin-mediated dephosphorylation of the human placental membrane receptor for epidermal growth factor urogastrone.

The findings of our work were 2-fold: (1) calcineurin (from bovine brain) can catalyze the complete dephosphorylation of the phosphotyrosine and phosphoserine residues in the human placental receptor for epidermal growth factor urogastrone (EGF-URO), and (2) the major calmodulin-binding protein of human placental membranes is a calcineurin-related protein. In terms of its metal ion dependence (Ni2+ greater than Mn2+ greater than Co2+), its calmodulin dependence, and its sensitivity to inhibitors (Zn2+, fluoride, orthovanadate), the phosphotyrosyl protein phosphatase activity of calcineurin, using the EGF-URO receptor as substrate, paralleled the enzyme activity measured with p-nitrophenyl phosphate (PNPP) as a substrate. These characteristics distinguish calcineurin from other classes of protein phosphotyrosyl phosphatases. Calcineurin purified from placental membranes was similar to, if not identical with, bovine brain calcineurin in terms of enzymatic specific activity toward PNPP, subunit electrophoretic mobilities, and immunological cross-reactivity. The enzymatic properties and comparative abundance of calcineurin in the placenta membranes suggest that this enzyme may play an important role in regulating the phosphorylation state of those receptors (e.g., for EGF-URO or insulin) also known to be present in the membranes.

Brain↗