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M J Crumpton

Publications and source records attributed to M J Crumpton.

At least 37 records · Page 2Linked to original sources
PubMed1991

Human CD2 is functional in CD2 transgenic mice.

We aimed to study the biochemical consequences of T-cell activation via the CD2 antigen in mouse T cells. The lack of stimulatory monoclonal antibodies against the mouse CD2 antigen led us to analyse this problem in transgenic mice carrying and expressing the human CD2 gene. Monoclonal antibodies to the human CD2 antigen that were mitogenic for human T cells induced proliferation of mouse T cells from the CD2 transgenic mice. Stimulation was accompanied by rapid phosphorylation of the murine CD3 gamma chain and T-cell receptor zeta chain. These results demonstrate that the human CD2 antigen is functional in the CD2 transgenic mice and indicate a considerable conservation of the signal transducing processes and also the activation mechanisms between mouse and man.

Animals↗
PubMed1990

Crystallization and preliminary X-ray crystallographic studies of human placental annexin IV.

Human placental annexin IV, a member of the annexin family of calcium and phospholipid-binding proteins, has been crystallized by the vapour diffusion method in the presence of calcium, using polyethylene glycol 8000. The crystals are orthorhombic, space C222(1), cell dimensions a = 105.4 A, b = 115.7 A, c = 80.7 A and diffract to at least 2.5 A resolution on a synchrotron source.

Binding Sites↗
PubMed1990

A method for measuring protein kinase C activity in permeabilized T lymphocytes by using peptide substrates. Evidence for multiple pathways of kinase activation.

Activation of protein kinase C (PKC) in human T lymphocytes is an immediate consequence of mitogenic signalling via the antigen-receptor complex and CD2 antigen. In order to investigate further the signal-transduction pathways which result in PKC activation, we have established a novel PKC assay system using streptolysin-O-permeabilized T cells. Known peptide substrates of PKC were introduced into permeabilized cells in the presence of [gamma-32P]ATP, 3 mM-Mg2+ and 150 nM free Ca2+. The peptide found to have the lowest background phosphorylation had the sequence Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys (peptide GS), and the phosphorylation of the peptide was increased up to 6-fold by direct activation of PKC with phorbol 12,13-dibutyrate. Induction of PKC activation with the UCHT1 antibody against the CD3 antigen, or with phytohaemagglutinin (PHA) or guanosine 5'-[gamma-thio]triphosphate (GTP[S]), increased peptide-GS phosphorylation by 2-3 fold. The specificity of PKC action on peptide GS was demonstrated by blocking increases in phosphorylation with a pseudosubstrate peptide PKC inhibitor. PKC activation by this technique could be detected within 1 min of adding external ligand. Dose-response curves revealed that PHA-induced production of inositol phosphates correlated closely with PKC activities, whereas only a partial correlation between these parameters was observed with GTP[S]. Our data are consistent with the presence of more than one G-protein-mediated pathway of PKC regulation in T cells. The quantitative PKC assay system described is both simple and reproducible, and its potential application to a wide range of cell types should prove useful in further investigations of PKC activation mechanisms.

Antibodies, Monoclonal↗
PubMed1990

Alternative splicing gives rise to two forms of the p68 Ca2(+)-binding protein.

The p68 Ca2+ and phospholipid binding protein of the lipocortin/calpactin family appears to exist as two forms. These may be resolved into a closely-spaced polypeptide doublet by SDS-PAGE. The cloning and sequencing of p68 revealed an apparent 18 nucleotide alternative splice sequence, which could account for this observation. We show here that an antiserum directed against a synthetic peptide corresponding to the region containing the splice sequence, recognises only the upper band of the p68 doublet by both immunoprecipitation and Western blotting. These results are consistent with alternative splicing being responsible for the generation of the two forms of p68.

Amino Acid Sequence↗
PubMed1990

Activation of human T lymphocytes via the CD2 antigen results in tyrosine phosphorylation of T cell antigen receptor zeta-chains.

The phosphorylation of the invariant chains associated with the human TCR has been investigated after the stimulation of T lymphocytes with CD2 mAb T11(2) and T11(3), PHA, or phorbol 12,13-dibutyrate. As described previously, stimulation of T cells with either CD2 mAb or phorbol 12,13-dibutyrate resulted in the phosphorylation of the CD3 gamma-chain. The combination of T11(2) and T11(3) mAb also induced phosphorylation of the TCR zeta-chain. The phosphorylated zeta-polypeptide of CD2-activated cells was immunoprecipitated with antiphosphotyrosine antibodies and migrated to a 21- to 23-kDa position during SDS/PAGE. These results indicate that stimulation of human T cells via the CD2 Ag with the T11(2) and T11(3) mAb activates not only protein kinase C but also tyrosine kinase(s), resulting in the phosphorylation of the CD3 gamma-chain and the tyrosine phosphorylation of the zeta-chain, respectively.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

The CD2 antigen associates with the T-cell antigen receptor CD3 antigen complex on the surface of human T lymphocytes.

T lymphocytes can be activated by various stimuli directed either against the T-cell antigen receptor-CD3 antigen complex (Ti-CD3) or the CD2 molecule; see ref. 1 for a review. Activation signals generated by antigen binding to the antigen-specific alpha/beta heterodimer (Ti) are thought to be transduced via the invariant CD3 gamma, epsilon and delta chains, and the associated zeta and eta subunits. The physiological role of the interaction of CD2 with its homologous cell-surface associated ligand LFA-3 remains to be fully elucidated. It has been suggested that CD2 regulates an antigen-independent pathway of activation or that signals delivered via CD2 are an integral part of the antigen-specific pathway. Several recent studies have indicated a requirement for the Ti-CD3 complex in CD2 signalling. Thus, mutant T-cell lines expressing CD2, but not Ti-CD3, on the cell surface cannot be activated via the CD2 molecules. Functional interaction between the Ti-CD3 complex and the CD2 antigen suggests that these T-lymphocyte cell-surface structures are physically associated. Here we use a digitonin-based solubilization procedure to explore this possibility and show that 40% of the cell-surface CD2 molecules can be specifically co-precipitated in association with the Ti-CD3 complex.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

A protein kinase C pseudosubstrate peptide inhibits phosphorylation of the CD3 antigen in streptolysin-O-permeabilized human T lymphocytes.

Activation of human T lymphocytes leads to the phosphorylation of the CD3-antigen gamma polypeptide. We have investigated a possible role for protein kinase C (PKC) in mediating this phosphorylation event by using T cells permeabilized with streptolysin-O in the presence of 120 mM-K+ buffers containing Ca2+-EGTA. The gamma-chain was phosphorylated by [gamma-32P]ATP in permeabilized T lymphoblasts in the presence of phorbol 12,13-dibutyrate (Pdbu) or phytohaemagglutinin (PHA). Ca2+ alone in the range 0.5-1.0 microM also induced gamma-chain phosphorylation in some T-lymphoblast preparations; that in Jurkat-6 cells occurred at lower concentrations (50-500 nM). Two experimental approaches were used to investigate the possible involvement of PKC. Firstly, when permeabilization was carried out in buffer lacking free Ca2+, PKC was lost from the cells, and gamma-chain phosphorylation could then no longer be induced on subsequent addition of Pdbu or PHA in 400 nM-Ca2+, or 800 nM-Ca2+ alone, to permeabilized cells. However, when permeabilization was carried out in the presence of these three agents, PKC was translocated to intracellular membranes, and subsequent addition of [gamma-32P]ATP to these cells then resulted in gamma-chain phosphorylation. In the second approach, induction of gamma-chain phosphorylation by Pdbu, 1-oleoyl-2-acetylglycerol, 1,2-diolein, PHA or Ca2+ alone was effectively blocked by permeabilizing T cells in the presence of a PKC pseudosubstrate peptide (50 microM). Pseudosubstrate concentrations in the range 7-20 microM inhibited gamma-chain phosphorylation by 50%. In contrast, addition of four other 'irrelevant' basic peptides (50 microM) did not result in detectable inhibition, and 50 microM-pseudosubstrate did not inhibit the phosphorylation of 17 other polypeptides isolated from permeabilized T cells. These data suggest that Pdbu-, 1,2-diacylglycerol-, PHA- and Ca2+-induced phosphorylation of the CD3-antigen gamma chain in permeabilized T cells is mediated by PKC.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

Dephosphorylation of the human T lymphocyte CD3 antigen.

Previous studies demonstrated that activation of T lymphocytes by phorbol ester or mitogenic lectin leads to phosphorylation of Ser 126 of the CD3 antigen gamma chain, whereas treatment with ionomycin results in phosphorylation of both Ser 123 and 126 [Davies, A. A. et al. (1987) J. Biol. Chem. 262, 10918-10921]. In the present study, the dephosphorylation of Ser 123 and Ser 126 of the gamma chain was investigated. Phorbol-ester-induced phosphorylation of the gamma-chain Ser 126 in vivo was reversed following removal of phorbol ester. Dephosphorylation of both Ser 123 and 126 was also observed in vitro using the microsome fraction of T lymphocytes. In order to identify the phosphatases acting at these two sites, the immunoprecipitated gamma chain was used as substrate either following treatment with protein kinase C in vitro, in which case phosphorylation occurs mainly at Ser 123, or following in vivo phosphorylation of Ser 126. Purified oligomeric forms of the polycation-stimulated phosphatases were more effective in dephosphorylating both phosphorylated forms of the gamma chain compared with equivalent amounts of ATP,Mg2+-dependent phosphatases or calcineurin. By using phosphopeptide analogues of the CD3 gamma chain containing Ser 123 or Ser 126 as substrates (A3 and A6), it was shown that polycation-stimulated phosphatases selectively dephosphorylated Ser 123 compared to Ser 126. In order to determine which phosphatases dephosphorylate the gamma chain in microsomes, A3 and A6 were used as substrates for characterising phosphatases in microsomes from human T leukaemia Jurkat 6 cells. Three phosphopeptide phosphatases (250-400 kDa) co-eluted through five purification steps with three forms of polycation-stimulated phosphorylase phosphatase. The partially purified A3/A6 phosphopeptide phosphatases were insensitive to Ca2+, calmodulin and inhibitor-1, and dephosphorylated A3 preferentially compared with A6. A latent form of microsomal ATP,Mg2+-dependent phosphorylase phosphatase was stimulated 10-fold by trypsinisation, but did not dephosphorylate phosphopeptides A3 and A6. The results show that high-Mr forms of polycation-stimulated phosphatases are the only enzymes in human T leukaemia cell microsomes which dephosphorylate gamma chain phosphopeptides. The data point to an important role for polycation-stimulated phosphatases in regulating the phosphorylation state, and so function(s), of the CD3 antigen.

Amino Acid Sequence↗
PubMed1989

Crystallization of p68 on lipid monolayers and as three-dimensional single crystals.

Two-dimensional crystals of p68, a Ca2+ -binding protein that has homology with members of the lipocortin/calpactin family, were obtained by interaction with a phospholipid monolayer. By measuring surface pressure at constant surface area, p68 was found to interact in a Ca2+ -dependent manner specifically with phosphatidylethanolamine, less so with phosphatidylserine and not at all with phosphatidylcholine. With dimyristoyl-phosphatidylethanolamine, two-dimensional crystalline arrays were formed. Image analysis of electron micrographs of these crystals, which diffracted to about 50 A, revealed p3 symmetry with a unit cell of about 178 A by 178 A; the protein densities showed a two-domain structure giving a cylindrical molecule of about 100 A by 35 A diameter packed as trimers. Three-dimensional microcrystals obtained without lipid or Ca2+ were suitable for electron microscopy and gave a tetragonal unit cell of about 256 A by 68 A. The implications of these observations on the structure and lipid specificity of p68 binding are discussed.

Annexin A6↗
PubMed1989

Evidence that a kinase distinct from protein kinase C induces CD3 gamma-subunit phosphorylation without a concomitant down-regulation in CD3 antigen expression.

An immediate consequence of Ag-specific activation of T cells is phosphorylation of the gamma-subunit of the CD3 gamma-chain. There is good evidence that the kinase that mediates CD3 gamma-chain phosphorylation is protein kinase C (pkC). It has also been proposed that the interaction between pkC and CD3 gamma-chains controls the cell surface expression of the antigen receptor/CD3 Ag complex. In the present study we present data relevant to these two points. Thus we show that CD3 gamma-subunit phosphorylation can be triggered by the calcium ionophore ionomycin. However, as judged by several criteria, ionomycin does not stimulate cellular pkC. Accordingly, ionomycin must regulate phosphorylation of the CD3 Ag by a kinase distinct from pkC. The phosphorylation of CD3 Ag induced by ionomycin is not accompanied by a modulation of the cell surface expression of CD3 molecules which implies that CD3 gamma-chain phosphorylation is not a sufficient signal for the endocytosis of the CD3/Ag receptor complex.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

The gene coding for the p68 calcium-binding protein is localised to bands q32-q34 of human chromosome 5, and to mouse chromosome 11.

The gene coding for human p68, a membrane-associated calcium-binding protein, has been assigned to chromosome 5, using a cDNA clone to probe genomic DNA from rodent-human somatic cell hybrids by Southern hybridisation. The gene was localised, by in situ hybridisation, to 5q32-34. The murine gene was assigned to chromosome 11, using a murine cDNA clone to probe genomic DNA from rodent-rodent somatic cell hybrids.

Animals↗