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Biomedical subjects

D G Campbell

Publications and source records attributed to D G Campbell.

At least 19 recordsLinked to original sources
PubMed1995

The cyanobacterial toxin microcystin binds covalently to cysteine-273 on protein phosphatase 1.

The interaction between protein phosphatase 1 (PP1) and microcystin (MC) was stable in 1% SDS or 70% formic acid indicative of a covalent interaction. Here we isolate the MC-binding peptide and demonstrate that Cys273 of PP1 binds covalently to the methyl-dehydroalanine (Mdha) residue of the toxin. Mutation of Cys273 to Ala, Ser or Leu abolished covalent binding to MC, as did reduction of the Mdha residue of the toxin with ethanethiol. The abolition of covalent binding increased the IC50 for toxin inhibition of PP1 by 5- to 20-fold. The covalent binding of MC to protein serine/threonine phosphatases explains the failure to detect this toxin post-mortem in suspected cases of MC poisoning.

Alanine↗
PubMed1995

Modulation of antigen processing by bound antibodies can boost or suppress class II major histocompatibility complex presentation of different T cell determinants.

Bound antibodies can modulate antigen processing but it is not clear to what extent this affects antigen presentation. Here we show that presentation of T cell determinants in tetanus toxin can be either enhanced or suppressed as a direct consequence of antibody modulation of antigen processing in human B lymphoblastoid cells. Remarkably, a single bound antibody or its Fab fragment can simultaneously enhance the presentation of one T cell determinant by more than 10-fold while strongly suppressing the presentation of a different T cell determinant. Biochemical analysis demonstrates that both the suppressed and boosted determinants fall within an extended domain of antigen stabilized or "footprinted" by this antibody during proteolysis. These results demonstrate that bound antibodies can modulate the capture of peptides by class II major histocompatibility complex (MHC), thus manipulating the T cell response towards or away from particular determinants. Altered processing of protein-protein complexes leading to enhanced loading of class II MHC and substantially lowered threshold for T cell activation suggests a novel mechanism that might reveal "cryptic" self determinants.

Amino Acid Sequence↗
PubMed1995

p53 is phosphorylated in vitro and in vivo by an ultraviolet radiation-induced protein kinase characteristic of the c-Jun kinase, JNK1.

The p53 tumor suppressor protein is thought to play a major role in the defense of the cell against agents that damage DNA. In this report, we describe the identification and characterization of a protein kinase that phosphorylates mouse p53 at a single site, serine 34, a major site of phosphorylation in the cell. The protein kinase is activated strikingly following treatment of cells with ultraviolet radiation, has a native molecular weight of approximately 45,000, and can be resolved from mitogen-activated protein (MAP) kinase by chromatography on Superose 6 and DEAE-cellulose. The p53 kinase activity co-purifies with UV-activated c-Jun kinase activity on heparin-Sepharose and on a c-Jun (but not a v-Jun-) affinity column. Treatment of the partially purified kinase with CL100, a protein phosphatase that specifically dephosphorylates MAP kinase homologues, inhibits its activity. Taken together, the data suggest that this p53 kinase is likely to be activated by phosphorylation and may be a member of the stress-activated protein kinase subfamily of MAP kinases. UV irradiation of SV3T3 cells leads to increased phosphorylation of p53 at serine 34, indicating that phosphorylation of p53 by this kinase is likely to be physiological. Phosphorylation of p53 by this protein kinase may be a key event in a signal transduction mechanism that coordinately controls key nuclear proteins in response to oxidative stress or DNA damaging agents.

3T3 Cells↗
PubMed1995

Early patellofemoral revision following total knee arthroplasty.

A consecutive series of 289 Miller-Galante (Zimmer, Warsaw, IN) total knee arthroplasties were studied, with particular reference to the patellofemoral joint. Sixteen knees were initially excluded; the remaining 273 arthroplasties were followed for 14 to 44 months. Thirty patients (11%) had patellofemoral pain. Twenty patients (7.3%) had revision patellofemoral surgery. Fourteen patients had revision surgery for patellar maltracking, of which 10 had resolution of their symptoms, 2 were improved, 1 had no change, and 1 developed a prosthetic infection. Six patients who had no evidence of patellar maltracking had revision surgery with a cemented metal-backed patellar component. Only two of these patients had symptom improvement from their revision surgery. A higher than usual incidence of patellar maltracking (5%) is reported. The first-generation Miller-Galante femoral component may contribute to the relative instability of patellofemoral tracking. Those patients with patellar maltracking were greatly improved by revision surgery; the results of revision surgery for anterior knee pain without associated patellar maltracking were disappointing.

Adult↗
PubMed1994

Molecular cloning of cDNA encoding the 110 kDa and 21 kDa regulatory subunits of smooth muscle protein phosphatase 1M.

The structures of the M110 and M21 regulatory subunits of protein phosphatase-1M, the major enzyme which dephosphorylates myosin in smooth muscle, have been deduced from cloned cDNAs. The N-terminus of the M110 subunit from rat aorta contains seven ankyrin repeats, while the C-terminus of the M21 subunit from chicken gizzard contains a leucine zipper motif. The M110 subunit is expressed in two different forms which differ in their C-terminal sequences. One of these is highly homologous to the whole of the M21 subunit.

Amino Acid Sequence↗
PubMed1994

O- and N-glycosylation of the Leishmania mexicana-secreted acid phosphatase. Characterization of a new class of phosphoserine-linked glycans.

The protozoan parasite Leishmania mexicana secretes a heavily glycosylated 100-kDa acid phosphatase (sAP) which is associated with one or more polydisperse proteophosphoglycans. Most of the glycans in this complex were released using mild acid hydrolysis conditions that preferentially cleave phosphodiester linkages. The released saccharides were shown to consist of monomeric mannose and a series of neutral and phosphorylated glycans by Dionex high performance liquid chromatography, methylation analysis, exoglycosidase digestions, and one-dimensional 1H NMR spectroscopy. The neutral species comprised a linear series of oligosaccharides with the structures [Man alpha 1-2]1-5Man. The phosphorylated oligosaccharides were characterized as PO4-6Gal beta 1-4Man and PO4-6[Glc beta 1-3]Gal beta 1-4Man. The attachment of these glycans to the polypeptide backbone via the linkage, Man alpha 1-PO4-Ser, is suggested by: 1) the finding that more than 60% of the serine residues in the polypeptide are phosphorylated and 2) the resistance of the phosphoserine residues to alkaline phosphatase digestion unless the sAP was first treated with either mild acid (to release all glycans) or jack bean alpha-mannosidase (to release neutral mannose glycans). Analysis of the partially resolved components of the complex indicated that the most of the O-linked glycans on the 100-kDa phosphoglycoprotein comprised mannose and the mannose-oligosaccharides. In contrast the major O-linked glycans on the proteophosphoglycan were short phosphoglycan chains, containing on average two repeat units per chain. In addition to the O-linked glycans, both components in the sAP complex contained N-linked glycans. The N-glycanase F-released glycans were characterized by Bio-Gel P4 chromatography and exoglycosidase digestions to be the biantennary oligomannose type with the structures Glc1Man6GlcNAc2 and Man6GlcNAc2. The O-linked glycans of the sAP complex are similar to those found in the phosphoglycan chains of the abundant surface lipophosphoglycan, but differ in having much shorter phosphoglycan chains and a more diverse series of mannose cap oligosaccharides. These data suggest that there are marked differences in the ability of different glycosyltransferases to utilize peptide-linked versus glycolipid-linked acceptors.

Acid Phosphatase↗
PubMed1994

Phosphorylation of the tumor suppressor protein p53 by mitogen-activated protein kinases.

The p53 tumor suppressor protein is tightly regulated in the cell and is phosphorylated at multiple sites by several different protein kinases. We have investigated the phosphorylation of p53 by mitogen-activated protein (MAP) kinase, a protein kinase that plays a central role in mediating many mitogenic and differentiation signals. Recombinant wild-type mouse p53 was phosphorylated in vitro by activated recombinant p42-MAP kinase but not by inactive MAP kinase or by the activating protein, MAP kinase kinase. Phosphorylation of p53 by MAP kinase occurred at two N-terminal sites, threonine residues 73 and 83. Tryptic phosphopeptides of recombinant p53 phosphorylated in vitro by MAP kinase comigrated on two-dimensional maps with p53 from SV3T3 cells labeled in vivo with [32P]orthophosphate, suggesting that MAP kinase targets a site in p53 that is phosphorylated in the cell. Following serum stimulation of quiescent C57MG cells, two p53 kinases, which were resolved by chromatography on Mono Q, were stimulated 15-20-fold within 5 min. Each of these kinase activities co-eluted with myelin basic protein kinase activity and could be inactivated following treatment with protein phosphatase 2A, a serine/threonine phosphatase, or leukocyte antigen receptor, a protein tyrosine phosphatase, suggesting that these activities were members of the MAP kinase family. The two kinase activities from the lysates targeted the same phosphorylation sites on p53 as the purified recombinant MAP kinase. These protein kinase activities were also stimulated following exposure of the cells to ultraviolet radiation, but with slightly delayed kinetics. Phorbol ester treatment of SV3T3 cells led to increased phosphorylation of the peptide containing the residues targeted by MAP kinase. The data suggest that p53 may be phosphorylated by MAP kinase physiologically and that this interaction may be involved in the cell's response to UV exposure, growth factor stimulation, or transformation by oncogenes.

Amino Acid Sequence↗
PubMed1994

The threonine residues in MAP kinase kinase 1 phosphorylated by MAP kinase in vitro are also phosphorylated in nerve growth factor-stimulated rat phaeochromocytoma (PC12) cells.

The residues on MAP kinase kinase-1 (MAPKK1) phosphorylated by MAP kinase in vitro have been identified as Thr-291 and Thr-385. Both threonines are phosphorylated in PC12 cells and the 32P-labelling of each residue increases after stimulation with nerve growth factor (NGF). The results establish that MAPKK1 is a physiological substrate for MAP kinase. The two active forms of MAPKK that are resolved by Mono Q chromatography of PC12 cell extracts are both phosphorylated at Thr-291 and Thr-385, demonstrating that neither species is the MAPKK2 isoform which lacks Thr-291.

Amino Acid Sequence↗
PubMed1994

Tandem overproduction and characterisation of the nuclease domain of colicin E9 and its cognate inhibitor protein Im9.

We report the overproduction of the non-specific endonuclease domain of the bacterial toxin colicin E9 and its preliminary characterisation in vitro. The enzymatic colicins (61 kDa) are normally released from producing cells in a complex with their cognate inhibitors, known as the immunity proteins (9.5 kDa). Tryptic digestion of the purified ColE9 complex was found to generate two major components, a monomer derived from the N-terminal and central regions of the toxin and a heterodimer comprising the catalytically active C-terminal domain of the colicin bound to its intact immunity protein, Im9. N-terminal amino acid sequencing, in conjunction with electrospray mass spectrometry, shows that preparations of the DNase domain isolated by this method are heterogeneous, thus making subsequent mechanistic and structural analysis difficult. This problem was circumvented by selectively overexpressing the C-terminal 15-kDa nuclease domain of colicin E9 in tandem with its cognate inhibitor in Escherichia coli. This tandem overexpression strategy allowed high-level production of a 25-kDa protein complex comprising the C-terminal DNase domain of colicin E9 tightly bound to its specific inhibitor Im9, thus masking the anticipated toxicity of the nuclease. The DNase domain was then separated from Im9 under denaturing conditions, refolded by removal of the denaturant and the renatured protein shown to possess both endonuclease and Im9 binding activity. These results describe a novel method for the overproduction of a nuclease in bacteria by co-expressing its specific inhibitor and lay the foundations for a full mechanistic, biophysical and structural characterization of the isolated DNase domain of the colicin E9 toxin.

Amino Acid Sequence↗
PubMed1994

Sterilization of HIV by gamma irradiation. A bone allograft model.

A human immuno-deficiency virus (HIV) infected bone allograft model has been created using HTLV-IIIB virus in a concentration simulating a massively HIV infected bone allograft donor [HTLV-III is the denomination initially given to the human immuno-deficiency by the american team of Prof. Gallo. It represent the virus HIV 1 of the present international nomenclature]. 5 x 10(4) tissue culture infective doses per ml. of virus were placed within the medullary cavity of bovine femora and tibiae with a radiation dosimeter, and the ends sealed with lead. The bone/virus model was maintained at -70 degrees C while being irradiated with 1 to 4 megarads of gamma irradiation in increments of 0.5 megarads. The study showed that the HTLV-IIIB virus is a relatively radio-resistant organism, a property common to most viruses. The results suggest that HTLV-IIIB can be inactivated in bone infected with a clinically significant viral load, as may be found in donors who are initially negative when screened for HIV. It is recommended that bone allografts which are secondarily sterilized by gamma irradiation receive at least 2.5 megarads. The amount of radiation absorbed by the bone cortex was minimal.

Animals↗
PubMed1993

Phosphorylation and activation of human tyrosine hydroxylase in vitro by mitogen-activated protein (MAP) kinase and MAP-kinase-activated kinases 1 and 2.

Mitogen-activated protein-kinase (MAP) kinase-activated protein kinases 1 and 2 (MAPKAP kinase-1, MAPKAP kinase-2), were found to phosphorylate bacterially expressed human tyrosine hydroxylase in vitro at comparable rates to other proteins thought to be physiological substrates of these protein kinases. The phosphorylation of all four alternatively spliced forms of human tyrosine hydroxylase by MAPKAP kinases-1 and -2 reached plateau values at 1 mol/mol subunit and 2 mol/mol subunit, respectively; the sites of phosphorylation were identified as Ser40 (MAPKAP kinase-1) and Ser19 and Ser40 (MAPKAP kinase-2). In contrast to calmodulin-dependent protein kinase-II, which phosphorylates Ser19 faster than Ser40, MAPKAP kinase-2 phosphorylated Ser40 about twice as fast as Ser19. The maximal activation of tyrosine hydroxylase by MAPKAP kinase-1 or-2 was about 3-fold, and activation by MAPKAP kinases-1 and -2 or calmodulin-dependent protein kinase-II correlated with the extent of phosphorylation of Ser40. The four alternatively spliced forms of human tyrosine hydroxylase were phosphorylated at Ser31 by MAP kinase, but at markedly different rates (3 = 4 > 1 >> 2). Forms 3 and 4 were phosphorylated rapidly and stoichiometrically by MAP kinase doubling the activity, while phosphorylation of form 1 by MAP kinase to 0.4 mol/mol subunit increased activity by 40%. The effect on activity of phosphorylating both Ser31 and Ser40 was not additive. The possible roles of MAPKAP kinase-1, MAPKAP kinase-2 and MAP kinase in the regulation of tyrosine hydroxylase in vivo are discussed.

Alternative Splicing↗
PubMed1993

The phosphorylation of stathmin by MAP kinase.

Stathmin, a ubiquitous cytosolic phosphoprotein which may play a role in integrating the effects of diverse signals regulating proliferation, differentiation and other cell functions, was found to be phosphorylated rapidly and stoichiometrically by mitogen-activated protein (MAP) kinase in vitro. Ser-25 was identified as the major site and Ser-38 as a minor site of phosphorylation, while the p42 and p44 isoforms of MAP kinase were the only significant stathmin kinases detected in PC12 cells after stimulation by nerve growth factor (NGF). The results suggest that MAP kinases are the enzymes responsible for increasing the level of phosphorylation of Ser-25, which has been observed previously in PC12 cells following stimulation by NGF.

Amino Acid Sequence↗
PubMed1992

A myofibrillar protein phosphatase from rabbit skeletal muscle contains the beta isoform of protein phosphatase-1 complexed to a regulatory subunit which greatly enhances the dephosphorylation of myosin.

A form of protein phosphatase-1 (PP1M), which possesses 25-fold higher activity towards the P light chain of myosin (in heavy meromyosin) than other forms of protein phosphatase-1, was purified over 200,000-fold from the myofibrillar fraction of rabbit skeletal muscle. PP1M, which eluted from Superose 12 with an apparent molecular mass of 60 kDa, was dissociated by LiBr into two subunits. One of these displayed enzymic properties identical to those of the catalytic subunit of protein phosphatase-1 (PP1C) and was identified as the beta isoform of PP1C by amino acid sequencing. The second subunit had no intrinsic protein phosphatase activity, but greatly increased the rate at which PP1C dephosphorylated skeletal-muscle heavy meromyosin and decreased the rate at which it dephosphorylated glycogen phosphorylase. The properties of PP1M, together with those of smooth muscle PP1M [Alessi, D., MacDougall, L. K., Sola, M. M., Ikebe, M. & Cohen, P. (1992) Eur. J. Biochem. 210, 1023-1035] and the previously characterised glycogen-associated form of protein phosphatase-1 (PP1G), indicate that the subcellular localisation and substrate specificity of PP1 is determined by its interaction with specific targetting subunits.

Amino Acid Sequence↗
PubMed1992

Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins.

MAP kinase-activated protein kinase-2 (MAPKAP kinase-2) phosphorylates the serine residues in murine heat shock protein 25 (hsp25) and human heat shock protein 27 (hsp27) which are phosphorylated in vivo in response to growth factors and heat shock, namely Ser15 and Ser86 (hsp25) and Ser15, Ser78 and Ser82 (hsp27). Ser86 of hsp25 and the equivalent residue in hsp27 (Ser82) are phosphorylated preferentially in vitro. The small heat shock protein is present in rabbit skeletal muscle and hsp25 kinase activity in skeletal muscle extracts co-purifies with MAPKAP kinase-2 activity throughout the purification of the latter enzyme. These results suggest that MAPKAP kinase-2 is the enzyme responsible for the phosphorylation of these small heat shock proteins in mammalian cells.

Amino Acid Sequence↗
PubMed1992

MAP kinase kinase from rabbit skeletal muscle. A novel dual specificity enzyme showing homology to yeast protein kinases involved in pheromone-dependent signal transduction.

MAP kinase kinase (MAPKK) was purified 30,000-fold to homogeneity from extracts of rabbit skeletal muscle and shown to be a monomeric protein of apparent molecular mass 44 kDa. MAPKK activated the 42 kDa isoform of MAP kinase by phosphorylation of Thr-183 and Tyr-185, and phosphorylated itself slowly on tyrosine, threonine and serine residues, establishing that it is a 'dual specificity' protein kinase. Peptide sequences from MAPKK were homologous to other protein serine/threonine kinases, especially to the subfamily that includes yeast protein kinases that lie upstream of yeast MAP kinase homologues in the pheromone-dependent mating pathways.

Amino Acid Sequence↗
PubMed1992

Mammalian protein serine/threonine phosphatase 2C: cDNA cloning and comparative analysis of amino acid sequences.

Complementary DNA encoding rat protein phosphatase 2C alpha was obtained from a liver library and used to isolate the homologous cDNAs from rabbit liver and human teratocarcinoma libraries. The amino acid sequences of the three enzymes deduced from the cDNA (382 amino acids) were extremely similar (greater than 99% identity), the maximum number of differences (between rat and human) being four. Amino acid sequences of peptides corresponding to 238 residues (61%) of the protein phosphatase 2C beta isoform from rabbit skeletal muscle were determined and showed 12 differences from the recently published sequence of the rat liver enzyme deduced from the cDNA (95% identity).

Amino Acid Sequence↗
PubMed1992

MAPKAP kinase-2; a novel protein kinase activated by mitogen-activated protein kinase.

A novel protein kinase, which was only active when phosphorylated by the mitogen-activated protein kinase (MAP kinase), has been purified 85,000-fold to homogeneity from rabbit skeletal muscle. This MAP kinase activated protein kinase, termed MAPKAP kinase-2, was distinguished from S6 kinase-II (MAPKAP kinase-1) by its response to inhibitors, lack of phosphorylation of S6 peptides and amino acid sequence. MAPKAP kinase-2 phosphorylated glycogen synthase at Ser7 and the equivalent serine (*) in the peptide KKPLNRTLS*VASLPGLamide whose sequence is similar to the N terminus of glycogen synthase. MAPKAP kinase-2 was resolved into two monomeric species of apparent molecular mass 60 and 53 kDa that had similar specific activities and substrate specificities. Peptide sequences of the 60 and 53 kDa species were identical, indicating that they are either closely related isoforms or derived from the same gene. MAP kinase activated the 60 and 53 kDa forms of MAPKAP kinase-2 by phosphorylating the first threonine residue in the sequence VPQTPLHTSR. Furthermore, Mono Q chromatography of extracts from rat phaeochromocytoma and skeletal muscle demonstrated that two MAP kinase isoforms (p42mapk and p44mapk) were the only enzymes in these cells that were capable of reactivating MAPKAP kinase-2. These results indicate that MAP kinase activates at least two distinct protein kinases, suggesting that it represents a point at which the growth factor-stimulated protein kinase cascade bifurcates.

3T3 Cells↗