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C W Turck

Publications and source records attributed to C W Turck.

At least 73 records · Page 4Linked to original sources
PubMed1994

Crystallographic structures of thrombin complexed with thrombin receptor peptides: existence of expected and novel binding modes.

Many of the vital actions of thrombin on platelets and other cells appear to be mediated by the recently cloned seven-transmembrane-domain thrombin receptor. Thrombin activates this receptor by a novel proteolytic mechanism. The amino-terminal exodomain of the receptor contains the sequence LDPRSFLLRNPNDKYEPF. Structure-activity studies with mutant receptors and receptor peptides suggest that this sequence binds to thrombin at two sites: LDPR with the active center of thrombin and KYEPF with the fibrinogen recognition exosite of thrombin. Thrombin then cleaves the Arg41-Ser42 bond to unmask a new amino terminus, which functions as a tethered peptide ligand binding to as yet undefined sites within the body of the receptor to effect receptor activation. We have determined eight crystal structures of thrombin complexed with receptor-based peptides. Each of the two components of the bidentate docking model was captured in individual cocrystals. In one crystal type, the LDPR sequence docked in the active center of thrombin in a manner analogous to d-PheProArg chloromethyl ketone. In other crystals, the KYEPF sequence bound in the fibrinogen anion binding exosite of thrombin in a manner analogous to the DFEEI sequence of the carboxylate-terminal peptide of hirudin. Strikingly, however, generation of a single crystal that includes both components of the anticipated bidentate binding mode was not achieved, apparently because the peptides have a dominant solution S-like conformation that does not bind in a productive way at the active center. This peptide structure apparently favored a novel alternative mode of receptor peptide-thrombin interaction in which the receptor peptides formed an intermolecular bridge between neighboring thrombin molecules, resulting in an infinite peptide thrombin chain in crystals. In this structure, the KYEPF sequence docked in the expected manner at the exosite of one thrombin molecule, but the LDPR sequence docked in an unusual nonproductive mode with the active center of a neighboring molecule. Mutations that removed important determinants of the S-like receptor peptide structure underlying the bridging mode in the receptor itself did not significantly alter thrombin signaling. Additionally, a comparison of receptor density to the responsiveness of a cell did not support a role for receptor oligomerization in signaling. The physiological role for this unexpected intermolecular binding mode, if any, remains to be identified.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

SH2-dependent association of phosphatidylinositol 3'-kinase 85-kDa regulatory subunit with the interleukin-2 receptor beta chain.

Interleukin-2 (IL-2) signaling results in tyrosine phosphorylation of the 75-kDa IL-2 receptor (IL-2R) beta chain and the activation of phosphatidylinositol 3'-kinase (PI3-K). Herein, we demonstrate that the 85-kDa (p85) regulatory subunit of PI3-K physically associates with the tyrosine-phosphorylated IL-2R beta chain. A fusion protein containing both the amino- and the carboxyl-terminal src homology 2 domains of p85 precipitates an 80-kDa tyrosine-phosphorylated protein (pp80) from the lysates of IL-2-stimulated, but not unstimulated, human T lymphoblasts. Preclearing studies and immunoblotting with an antiserum to the IL-2R beta chain demonstrates that pp80 represents a portion of the IL-2R beta chain pool. A tyrosine-phosphorylated oligopeptide corresponding to tyrosine 392 of the IL-2R beta chain partially inhibits binding of the IL-2R beta chain by p85 fusion protein, raising the possibility that this residue plays a role in the interaction of PI3-K with the receptor.

Amino Acid Sequence↗
PubMed1994

Inhibition of T cell activation by protein kinase C pseudosubstrates.

PKC pseudosubstrate consensus sequences can be found in the amino terminal region of all the different PKC isoenzymes characterized to date. Here we have used four peptides corresponding to the putative pseudosubstrate sequences from the PKC isoenzymes alpha, gamma, delta, and epsilon. These peptides showed PKC inhibitory activity when tested in a PKC-specific enzyme assay at concentrations of 25 to 100 microM, similar to what has been reported for the myristylated peptide KRTLR. Although the presence of a myristyl group at the amino terminal end of any of these peptides is not essential for their inhibitory activity, myristylation increased the inhibitory activity significantly. By contrast, the myristylated control peptide (GALRQQKNVHEVKN) was not active even at a 100 microM concentration. All of the PKC inhibitory peptides were also able to block PKC activity in a cell assay as demonstrated by their ability to inhibit the induction of IL-2R and TNF-beta expression in Jurkat cells. Finally, we confirmed a previous report of the inhibitory activity of the myristylated peptide KRTLR and showed that other related peptides (N-m-RLTRK, N-m-RRLKT) are also active in these assays.

Amino Acid Sequence↗
PubMed1994

Specific recognition of the human neuroendocrine receptor for vasoactive intestinal peptide by anti-peptide antibodies.

Rabbit polyclonal IgG antibodies were generated to three distinct synthetic peptide substituents of the human neuroendocrine-type 7 transmembrane-domain receptor for vasoactive intestinal peptide (VIP), including a portion of the amino-terminus, first extracellular loop, and carboxyl-terminus. Immunofluorescent staining of both human K293 cell transfectants, expressing recombinant VIP receptors, and HT-29 human intestinal epithelial cells, bearing native VIP receptors, was observed with each of the antibodies and was eliminated specifically after absorption of antibodies with the respective peptide immunogen. Each of the antibodies recognized the same approximately 70-kDa membrane proteins, extracted from both K293 cell transfectants and HT-29 cells, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis blots. Neither IgG nor Fab preparations of the antibodies inhibited VIP binding to cellular receptors at a concentration of 1 microgram/ml, that yielded optimal immunofluorescence, or at 5-300 micrograms/ml. In contrast, 5-200 micrograms/ml of anti-peptide antibodies as IgG, but not Fab, significantly inhibited the increase in concentration of cyclic AMP in HT-29 cells elicited by 1 nM VIP, without affecting the greater increase evoked by 100 nM VIP or alone altering the level of cyclic AMP. Antibodies to several peptide substituents thus bind specifically to VIP receptors in immunoblots and permeabilized cells, and may affect the cellular functions of VIP receptors with sufficient selectivity to reduce transduction of signals, without altering the binding of VIP.

Amino Acid Sequence↗
PubMed1994

Substrate specificities and identification of a putative binding site for PI3K in the carboxy tail of the murine Flt3 receptor tyrosine kinase.

Flt3 is a receptor tyrosine kinase (RTK) structurally related to the CSF-1R encoded by the c-fms locus, Kit and the PDGFR which is restricted in its expression to hematopoietic precursor populations and several distinct cell types within the central nervous system. Although the ligand for Flt3 has recently been identified, the developmental function of Flt3 within these tissues has not yet been described. In order to examine the signalling properties of this receptor, we previously constructed a chimeric molecule containing the extracellular domain of CSF-1R fused to the transmembrane and cytoplasmic domain of mouse Flt3 (FF3). The ability of the FF3 to directly associate with or tyrosine phosphorylate specific cytoplasmic signalling molecules in vivo was examined. GAP, Vav, Shc, and to a lesser extent PLC gamma become tyrosine-phosphorylated but no in vivo association with the receptor was detectable. FF3 associates with PI3K activity and the SH2 domains of p85 and Grb-2. Phosphopeptide competition experiments suggest that the PI3K binding site is located outside of the kinase insert in the carboxy tail of the receptor.

Amino Acid Sequence↗
PubMed1993

Inhibition of CDK2 activity in vivo by an associated 20K regulatory subunit.

The major events of the cell division cycle are triggered by periodic changes in the activity of cyclin-dependent protein kinases (CDKs). In mammals, the members of the CDK family include CDK2 and CDC2, which are thought to be involved in the control of DNA replication and mitosis, respectively. The protein kinase activity of these enzymes is controlled by a complex array of mechanisms. Activation of the CDK catalytic subunit requires association with a positive regulatory subunit (cyclin) and phosphorylation (at Thr 160 in CDK2). This activated complex can be inhibited by additional phosphorylation at Thr 14 and Tyr 15. Here we report the identification of a new mechanism for the regulation of CDK2 activity. We find that CDK2/cyclin complexes in mouse fibroblasts associate tightly with a 20K protein (CAP20). Complexes containing CAP20 were isolated from cell lysates and found to have negligible kinase activity, indicating that CAP20 association in vivo may inhibit CDK2 activity. We purified CAP20 from 3T3 cells and found that low concentrations of the protein completely inhibit the kinase activity of CDK2 in vitro. Thus CAP20 represents a new negative regulatory subunit that inhibits the activity of CDK2/cyclin complexes in mammalian cells.

3T3 Cells↗
PubMed1993

Primary structure of the soluble lactose binding lectin L-29 from rat and dog and interaction of its non-collagenous proline-, glycine-, tyrosine-rich sequence with bacterial and tissue collagenase.

A lactose-binding lectin from rat lung (RL-29) and a related lectin from Madin-Darby canine kidney (MDCK) cells have been analyzed with the primary goal of identifying post-translational modifications. The sequences show that RL-29 and the dog lectin are homologues of a lectin designated here as L-29 and elsewhere as CBP-35, epsilon BP, Mac-2, or L-34. RL-29 has a 140-amino-acid COOH-terminal carbohydrate-binding domain, a 20-amino-acid NH2-terminal domain, and an intervening domain consisting of 11 repeating elements rich in Pro, Gly, and Tyr (R-domain). The dog homologue has 14 repeating elements in its R-domain explaining its larger size. The sensitivity of the R-domain to bacterial collagenase allowed us to isolate the NH2-terminal domain and show that the NH2 terminus was blocked by acetylation and, in the accompanying paper (Huflejt, M. E., Turck, C. W., Lindstedt, R., Barondes, S. H., and Leffler, H. (1993) J. Biol. Chem. 268, 26712-26718), that the NH2-terminal domain is phosphorylated. In addition, we unexpectedly found an endogenous component, resembling 92-kDa type IV collagenase, that co-purified with L-29 and slowly digested the R-domain. Hence, L-29 is a substrate for bacterial and tissue collagenases even though the R-domain is non-collagenous. Moreover, the co-purification suggests a non-enzymatic interaction between 92-kDa collagenase and L-29.

Amino Acid Sequence↗
PubMed1993

L-29, a soluble lactose-binding lectin, is phosphorylated on serine 6 and serine 12 in vivo and by casein kinase I.

L-29, a mammalian soluble lactose-binding lectin, was previously shown to be phosphorylated in confluent 3T3 fibroblasts (Cowles, E. A., Agrwal, N., Anderson, R. L., and Wang, J. L. (1990) J. Biol. Chem. 265, 17706-17712), which contain a small amount of this protein. We have determined the site of phosphorylation on L-29, taking advantage of the abundance of L-29 (about 1% of total soluble cell protein) in confluent polarized Madin-Darby canine kidney (MDCK) cells. Approximately 15-20% of the L-29 is phosphorylated in these cells. Phosphoamino acid analysis showed phosphate incorporation only at serine. Analysis of chymotryptic and endoproteinase Asp-N-generated NH2-terminal fragments by Edman degradation showed that 90% of the phosphate was at Ser6 and 10% at Ser12. The sequence surrounding Ser6, which is conserved in all known L-29 sequences, indicated that this serine might be phosphorylated by casein kinase I or casein kinase II. Reaction of human recombinant L-29 with [gamma-32P]ATP and each of these casein kinases showed that only casein kinase I catalyzed significant incorporation of 32P into L-29; and, as with the L-29 from the MDCK cell extracts, most of the phosphate was incorporated at Ser6 and a small amount was incorporated at Ser12.

Amino Acid Sequence↗
PubMed1993

Variants of vasoactive intestinal peptide in mouse mast cells and rat basophilic leukemia cells.

Radioimmunoassays for neuroendocrine vasoactive intestinal peptide (VIP1-28) detected 30-120 fmol of structurally related peptides in extracts of 10(7) mouse peritoneal mast cells, bone marrow-derived mast cells, cultured PT-18 and C1.MC/C57.1 lines of mast cells, and rat basophilic leukemia (RBL) cells. No VIP was found in peritoneal cells of mast cell-deficient WBB6F1-W/Wv mice, whereas the amounts extracted from peritoneal cells of the congenic normal (WBB6F1-+/+) mice were similar to those from cultured mouse mast cells. Sephadex G-25 gel filtration resolved two different-sized variants of VIP from mouse mast cells and RBL cells. Amino acid sequence analyses showed that the smaller variant is VIP10-28. The principal amino-terminally larger variant of VIP from C1.MC/C57.1 mouse mast cells and RBL cells exhibited amino acid sequence homology with VIP(-6)-28, and this sequence was established for the corresponding larger VIP from PT-18 mast cells. Polymerase chain reaction amplification of two different substituent sequences of prepro VIP in RBL cell RNA identified the VIP message. VIP10-28 was released from mouse mast cells concurrently with histamine by IgE-dependent stimulation. Rodent mast cell-derived VIP thus consists of both the truncated VIP10-28 and amino-terminally larger forms that appear to be generated by peptidolysis of a preproVIP similar to that found in neural cells.

Amino Acid Sequence↗
PubMed1993

Cloning and gene defects in microsomal triglyceride transfer protein associated with abetalipoproteinaemia.

The microsomal triglyceride transfer protein (MTP), which catalyses the transport of triglyceride, cholesteryl ester and phospholipid between phospholipid surfaces, is a heterodimer composed of the multifunctional protein, protein disulphide isomerase, and a unique large subunit with an apparent M(r) of 88K (refs 1-3). It is isolated as a soluble protein from the lumen of the microsomal fraction of liver and intestine. The large subunit of MTP was not detectable in four unrelated subjects with abetalipoproteinaemia, a rare autosomal recessive disease characterized by a defect in the assembly or secretion of plasma lipoproteins that contain apolipoprotein B (ref. 6). We report here the isolation and sequencing of complementary DNA encoding the large subunit of MTP. A comparison of this sequence to corresponding genomic sequences from two abetalipoproteinaemic subjects revealed a homozygous frameshift mutation in one subject and a homozygous nonsense mutation in the other. The results indicate that a defect in the gene for the large subunit of MTP is the proximal cause of abetalipoproteinaemia in these two subjects, and that MTP is required for the secretion of plasma lipoproteins that contain apolipoprotein B.

Abetalipoproteinemia↗
PubMed1993

Targeted activation of CD8 cells and infection of beta 2-microglobulin-deficient mice fail to confirm a primary protective role for CD8 cells in experimental leishmaniasis.

CD8+ T cells play an important role in the immunologic control of intracellular pathogens, particularly viruses. Leishmania are obligate intracellular parasites of macrophages in the mammalian host, and previous studies using deletion of CD8+ cells by administration of mAb to infected animals have suggested a protective role for these cells. Two complementary approaches were used to define more carefully the role of CD8+ cells in leishmaniasis. In BALB/c mice susceptible to Leishmania major (L. major) infection, targeted activation of CD8+ T cells was attempted by immunization with nonapeptides derived from the conserved major outer surface protein of the organism, gp63, that contained the consensus binding motif for MHC class I H-2Kd molecules. Two of the nonapeptides induced CTL activity in subsequently infected BALB/c mice that could be elicited against P815 cells pulsed either with peptide or lysates of L. major. Purified CD8+ T cells from immunized mice had elevated levels of IFN-gamma mRNA transcripts as compared to unimmunized mice. Despite evidence for activation of CD8+ cells, none of the mice immunized with nine different peptides alone or in combination were protected from progressive disease. In a second series of experiments, beta 2-microglobulin deficient mice that lack CD8+ cells were infected with L. major and the course of infection monitored. These mice cured disease as rapidly as beta 2-m +/- and +/+ littermates, and cure was associated with comparable levels of IFN-gamma mRNA in the draining lymph node population. Neither of these approaches was able to confirm a substantive role for CD8+ T cells in the primary protective response to L. major.

Amino Acid Sequence↗
PubMed1993

Multiple active conformers of mouse ornithine decarboxylase.

Purified recombinant mouse ornithine decarboxylase (ODC) was denatured with urea or with guanidinium chloride. Enzymic activity was efficiently recovered upon dilution of the denaturing agent. ODC renatured after urea treatment was further characterized. Kinetics of decarboxylation of the natural substrate ornithine or of the suicide substrate alpha-difluoromethylornithine (DFMO) were not significantly changed by denaturation/renaturation. Surprisingly, the renatured enzyme was not stably labelled with radioactive DFMO, in contrast with the native enzyme not subjected to denaturation. Native and renatured ODC did not differ in their c.d. spectra, but the former contained four reactive cysteine residues and the latter seven. These data indicate that a conformational change results from denaturation/renaturation that does not alter decarboxylation of substrates, but does change the accessibility or stability of the cysteine-360 residue modified by decarboxylated DFMO.

Animals↗
PubMed1993

Mapping the S' subsites of serine proteases using acyl transfer to mixtures of peptide nucleophiles.

We have developed a rapid and convenient procedure for the characterization of the S' subsite specificity of serine proteases. A mixture of peptide nucleophiles is incubated with the enzyme in the presence of excess of a specific ester substrate. The decrease in each nucleophile concentration is monitored by high-performance liquid chromatography analysis of the dansylated mixture. Relative kinetic parameters for each nucleophile in the mixture are then calculated using a new statistical algorithm that relates all pairs of nucleophiles. As a first application, we investigated the S'1 subsite specificity of chymotrypsin, trypsin, and a recently described trypsin mutant, Tr-->Ch[S1 + L1 + L2] with chymotrypsin-like primary specificity [Hedstrom, L., Szilagyi, L., & Rutter, W. J. (1992) Science 255, 1249-1253]. For this purpose 21 peptide nucleophiles of the general structure H-Xaa-Ala-Ala-Ala-Ala-NH2 were prepared by multiple solid-phase synthesis, where Xaa represents D-alanine, citrulline, and all natural amino acids except cysteine. Relative second-order rate constants for the enzyme-catalyzed acyl transfer to these nucleophiles were determined over a range of 10(2). Chymotrypsin and trypsin have markedly different S'1 specificities. The order of preference in chymotrypsin-catalyzed acyl transfer reactions is positively charged > aliphatic > aromatic >> negatively charged, D-Ala, Pro P'1 side chain. Trypsin prefers hydrophobic residues, but like chymotrypsin aliphatic residues are better than aromatic residues in P'1 position. The S'1 specificity of the mutant Tr-->Ch[S1 + L1 + L2] is similar to the specificity of trypsin; however, P'1 aromatic residues have low reactivity characteristic of chymotrypsin.

Acyltransferases↗
PubMed1993

Soluble lactose-binding lectin from rat intestine with two different carbohydrate-binding domains in the same peptide chain.

Of the multiple soluble lactose-binding (S-Lac) lectins in rat intestine, the major one, tentatively designated RI-H, was previously isolated as a polypeptide of molecular weight approximately 17,000. We here report the sequence of RI-H, as determined both at the peptide level and at the nucleotide level. Surprisingly the cDNA encodes a protein of molecular weight approximately 36,000, and this protein contains two homologous but distinct domains each with sequence elements that are conserved among all S-Lac lectins. The C-terminal domain, designated domain II, corresponds to the lectin with M(r) of 17,000 previously isolated from intestinal extracts and shown to have lactose binding activity. By preparing recombinant protein containing only the N-terminal domain, designated domain I, we here directly demonstrate that it too binds lactose and a related range of sugars that are roughly similar to domain II, but clearly distinct. The new lectin, which we designate L-36, is highly expressed in full-length form in rat small and large intestine and stomach but was not detected in eight other tissues including lung, liver, kidney, and spleen. Each domain has approximately 35% sequence identity with the other domain and with the carbohydrate-binding domain of L-29, another S-Lac lectin, but only about 15% identity with other known S-Lac lectins.

Amino Acid Sequence↗
PubMed1993

Regulation of protein kinase C isoform proteins in phorbol ester-stimulated Jurkat T lymphoma cells.

Activation of protein kinase C (PKC) in T cells leads to a variety of responses including IL-2 production and IL-2 receptor expression. PKC consists of several isoforms that exhibit some different in vitro properties. We have set up a Western blotting system to explore the regulation of PKC isoforms during T cell activation. In Jurkat T lymphoma cells, PKC alpha, beta, delta, epsilon, and zeta were detected. PKC alpha and beta existed primarily in the cytosol, translocated to the membrane fraction after 10 minutes of treatment with PMA, and almost completely disappeared within 16 h. A larger fraction of PKC delta and epsilon existed in the membrane fraction compared to PKC alpha or beta, and PKC epsilon translocated to the membrane fraction rapidly. Translocation of PKC delta was not apparent after 1 h treatment with PMA, but total PKC delta protein was reduced within 4 to 6 h of treatment. Consistent with this, overnight treatment with PMA caused down-regulation of both PKC delta and epsilon, but to a lesser degree than was observed with PKC beta. Anti-PKC zeta antibody detected two bands at 82 and 75 kDa. The 75-kDa band existed mostly in the cytosol fraction and showed no translocation or down regulation after PMA. We present evidence that this 75-kDa band represents PKC zeta. Similar PMA-induced translocation responses were observed in murine thymocytes showing that the responses are not unique to PKC isoforms in Jurkat. These results demonstrate that it is possible for the PKC isoforms to be differentially regulated during T cell activation.

Animals↗
PubMed1993

Cloning and functional characterization of early B-cell factor, a regulator of lymphocyte-specific gene expression.

Early B-cell factor (EBF) was identified previously as a tissue-specific and differentiation stage-specific DNA-binding protein that participates in the regulation of the pre-B and B lymphocyte-specific mb-1 gene. Partial amino acid sequences obtained from purified EBF were used to isolate cDNA clones, which by multiple criteria encode EBF. The recombinant polypeptide formed sequence-specific complexes with the EBF-binding site in the mb-1 promoter. The cDNA hybridized to multiple transcripts in pre-B and B-cell lines, but transcripts were not detected at significant levels in plasmacytoma, T-cell, and nonlymphoid cell lines. Expression of recombinant EBF in transfected nonlymphoid cells strongly activated transcription from reporter plasmids containing functional EBF-binding sites. Analysis of DNA binding by deletion mutants of EBF identified an amino-terminal cysteine-rich DNA-binding domain lacking obvious sequence similarity to known transcription factors. DNA-binding assays with cotranslated wild-type and truncated forms of EBF indicated that the protein interacts with its site as a homodimer. Deletions delineated a carboxy-terminal dimerization region containing two repeats of 15 amino acids that show similarity with the dimerization domains of basic-helix-loop-helix proteins. Together, these data suggest that EBF represents a novel regulator of B lymphocyte-specific gene expression.

Amino Acid Sequence↗
PubMed1992

ZAP-70: a 70 kd protein-tyrosine kinase that associates with the TCR zeta chain.

Protein-tyrosine kinases (PTKs) play an integral role in T cell activation. Stimulation of the T cell antigen receptor (TCR) results in tyrosine phosphorylation of a number of cellular substrates. One of these is the TCR zeta chain, which can mediate the transduction of extracellular stimuli into cellular effector functions. We have recently identified a 70 kd tyrosine phosphoprotein (ZAP-70) that associates with zeta and undergoes tyrosine phosphorylation following TCR stimulation. Here we report the isolation of a cDNA clone encoding ZAP-70. ZAP-70 represents a novel PTK and is expressed in T and natural killer cells. Moreover, tyrosine phosphorylation and association of ZAP-70 with zeta require the presence of src family PTKs and provide a potential mechanism by which the src family PTKs and ZAP-70 may interact to mediate TCR signal transduction.

Amino Acid Sequence↗