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D P Leader

Publications and source records attributed to D P Leader.

At least 19 recordsLinked to original sources
PubMed2000

Identification of sites phosphorylated by the vaccinia virus B1R kinase in viral protein H5R.

BACKGROUND: Vaccinia virus gene B1R encodes a serine/threonine protein kinase. In vitro this protein kinase phosphorylates ribosomal proteins Sa and S2 and vaccinia virus protein H5R, proteins that become phosphorylated during infection. Nothing is known about the sites phosphorylated on these proteins or the general substrate specificity of the kinase. The work described is the first to address these questions. RESULTS: Vaccinia virus protein H5R was phosphorylated by the B1R protein kinase in vitro, digested with V8 protease, and phosphopeptides separated by HPLC. The N-terminal sequence of one radioactively labelled phosphopeptide was determined and found to correspond to residues 81-87 of the protein, with Thr-84 and Thr-85 being phosphorylated. A synthetic peptide based on this region of the protein was shown to be a substrate for the B1R protein kinase, and the extent of phosphorylation was substantially decreased if either Thr residue was replaced by an Ala. CONCLUSIONS: We have identified the first phosphorylation site for the vaccinia virus B1R protein kinase. This gives important information about the substrate-specificity of the enzyme, which differs from that of other known protein kinases. It remains to be seen whether the same site is phosphorylated in vivo.

Binding Sites↗
PubMed1999

Trafficking of Glut4-green fluorescent protein chimaeras in 3T3-L1 adipocytes suggests distinct internalization mechanisms regulating cell surface glut4 levels.

Insulin stimulates glucose transport in adipose and muscle tissue by stimulating the movement ('translocation') of an intracellular pool of glucose transporters (the Glut4 isoform) to the plasma membrane. We have engineered a series of chimaeras between Glut4 and green fluorescent protein (GFP) from Aequoria victoria and expressed these proteins in 3T3-L1 adipocytes by microinjection of plasmid cDNA. In the absence of insulin, GFP-Glut4 is localized intracellularly within a perinuclear compartment and multiple intracellular punctate structures. In response to insulin, chimaeric GFP-Glut4 species exhibit a profound redistribution to the cell surface with kinetics comparable with the endogenous protein. The intracellular localization of GFP-Glut4 overlaps partially with compartments labelled with Texas Red transferrin, but is largely distinct from intracellular structures identified using Lysotracker-Red(R). K(+)-depletion resulted in the accumulation of GFP-Glut4 at the cell surface, but to an lesser extent than that observed in response to insulin. In contrast with native Glut4, removal of the insulin stimulus or treatment of insulin-stimulated cells with phosphatidylinositol 3'-kinase inhibitors did not result in re-internalization of the chimaeric GFP-Glut4 from the plasma membrane, suggesting that the recycling properties of this species differ from the native Glut4 molecule. We suggest that the recycling pathway utilized by GFP-Glut4 in the absence of insulin is distinct from that used to internalize GFP-Glut4 from the plasma membrane after withdrawal of the insulin stimulus, which may reflect distinct pathways for internalization of endogenous Glut4 in the presence or absence of insulin.

3T3 Cells↗
PubMed1997

Tissue-specific distribution of mouse casein kinase I alpha mRNA.

A clone isolated from a mouse skeletal muscle cDNA library by differentially screening for sequences expressed in skeletal but not cardiac muscle was found to correspond to the a-isoform of casein kinase I. The bulk of the clone corresponded to the 3'-untranslated region of the mRNA, which is isoform-specific. Hybridization to mRNA indicated a tissue-specific distribution for this isoform of casein kinase I.

Animals↗
PubMed1995

Analysis of CpG dinucleotide frequency in relationship to translational reading frame suggests a class of genes in which mutation of this dinucleotide is asymmetric with respect to DNA strand.

Results are described from application of a computer program that compares the expected and actual incidence of CpG dinucleotides in relation to the codon reading frame of genes, assuming a conserved amino acid sequence and normalizing for the third-position incidences of C and G in the remainder of the sequence. Sequences encoding certain proteins showed a pronounced bias in favour of CpG in the (3,1) compared with the (2,3) codon position; whereas sequences encoding related proteins expressed to a similar extent or in the same tissue did not. We propose that the cases exhibiting this bias reflect a difference between the two strands of the DNA duplex in their susceptibility to loss of CpG dinucleotides by mutation. Although in vertebrates this loss of CpG dinucleotides from the sense strand might reflect strand-asymmetry in deamination of 5-methylcytosine residues, the fact that a similar CpG codon bias is found in some invertebrates indicates that other factor(s) must also be involved.

Actins↗
PubMed1995

Vaccinia virus gene H5R encodes a protein that is phosphorylated by the multisubstrate vaccinia virus B1R protein kinase.

Vaccinia virus gene B1R encodes a protein kinase, the previously identified substrates of which include the proteins S2 and Sa of 40S ribosomal subunits. This work characterizes another substrate of the B1R kinase: a 36-kDa protein induced at the early stage of infection. Partially purified 36-kDa protein, eluted from a single-stranded DNA-cellulose column by 0.5 M NaCl, was separated by two-dimensional gel electrophoresis. Phosphorylation in vitro yielded multiple forms of the 36-kDa protein with approximate isoelectric points (pI) of 5.5, 5.7, 5.9, and 6.3, in addition to the apparently unphosphorylated form with a pI of approximately 6.8. The tryptic peptides derived from 36-kDa proteins with pI values of 5.7, 5.9, and 6.3 yielded almost identical high-pressure liquid chromatography profiles, strongly suggesting that the 36-kDa protein was modified by the phosphorylation of at least four sites, which were characterized as threonine residues. The amino acid sequence of several tryptic peptides derived from the 36-kDa protein showed that the 36-kDa protein was encoded by gene H5R of vaccinia virus. Consistent with this, the B1R kinase--either expressed in Escherichia coli or highly purified from HeLa cells--phosphorylated a recombinant trpE-H5R fusion protein in vitro. Fingerprints of the trpE-H5R and 36-kDa proteins phosphorylated by recombinant B1R kinase revealed common sites of phosphorylation, although some tryptic peptides were specific to either protein. Comparison was made of fingerprints of tryptic phosphopeptides derived from 36-kDa single-stranded DNA-binding protein labelled in vivo or in vitro. A common subset of peptides was observed, suggesting that some sites on H5R protein are phosphorylated by the B1R kinase in infected cells. These results suggest that some of the multiple threonine sites in the H5R protein are phosphorylated in vivo by the B1R protein kinase.

Amino Acid Sequence↗
PubMed1994

Ribosomal protein S2/Sa kinase purified from HeLa cells infected with vaccinia virus corresponds to the B1R protein kinase and phosphorylates in vitro the viral ssDNA-binding protein.

A ribosomal protein S2 kinase was purified 6000-fold from cytoplasmic extracts of HeLa cells infected with vaccinia virus, using 80S ribosomes or 40S ribosomal subunits as a substrate. Although the preparation was not homogeneous, a 34K component was identified, the chromatographic behaviour of which correlated with enzyme activity. During its purification the ribosomal protein S2 kinase was resolved from a less abundant ribosomal protein S13 kinase, demonstrating the two to be different entities. A second protein kinase activity against a 43K ribosomal protein comigrated with the ribosomal protein S2 kinase activity during all five chromatographic procedures employed, and we conclude that the two activities are properties of a single species. Two-dimensional gel electrophoresis demonstrated that this second substrate was the acidic ribosomal protein Sa, of isoelectric point approximately 5.2, previously shown to be phosphorylated during infection with vaccinia virus. Another substrate for the ribosomal protein S2/Sa kinase in vitro was the 36K viral ssDNA-binding protein, of isoelectric point approximately 5.0, which is also known to be phosphorylated in vivo. The 34K protein correlating with the catalytic activity in the most purified preparations of the ribosomal protein S2/Sa kinase was recognized by an antibody specific for a protein expressed in Escherichia coli from vaccinia virus gene B1R. This and other evidence suggest strongly that the ribosomal protein S2/Sa kinase is the product of this gene.

DNA, Single-Stranded↗
PubMed1993

Phosphorylation of ribosomal proteins by the vaccinia virus B1R protein kinase.

Two proteins of the 40S ribosomal subunit were shown to be phosphorylated in vitro by a vaccinia virus-encoded serine/threonine protein kinase. These were identified by two-dimensional gel electrophoresis as ribosomal proteins Sa and S2 and were shown by phosphoamino acid analysis to both be phosphorylated on serine and threonine residues. The three phosphorylated forms of S2 produced by the B1R protein kinase in vitro matched the phosphorylated forms of S2 previously observed in cells infected with vaccinia virus. These data strongly suggest that this enzyme is responsible for the phosphorylation of S2 and Sa which occurs early during vaccinia virus infection.

Animals↗
PubMed1993

Viral protein kinases and protein phosphatases.

Certain large DNA viruses (e.g. herpesviruses and poxviruses) encode proteins related to cellular protein-serine/threonine kinases, and Hepatitis B virus and vesicular stomatitis virus may encode structurally different protein kinases. Other viruses activate cellular protein kinases, e.g. interferon-induced eukaryotic initiation factor-2 kinase, growth factor-induced kinases and protein kinases that regulate mitosis. Protein phosphatases are encoded by vaccinia virus and bacteriophage lambda and must also play a role in viral infection--as do cellular protein phosphatases. The functions of many of these viral enzymes remain to be determined, but they represent possible new targets for anti-viral therapy.

Amino Acid Sequence↗
PubMed1991

Further definition of the substrate specificity of the alpha-herpesvirus protein kinase and comparison with protein kinases A and C.

The pseudorabies virus protein kinase prefers model substrates containing arginyl residues on the amino-terminal side of a target seryl or threonyl residue. We have defined this substrate specificity more precisely in experiments using a new series of synthetic model peptides. When the number of arginyl residues was varied from two to four in substrates of the type RnASVA it was found that peptides with four arginyl residues constituted the best substrates, although the most marked decrease in Km was seen on increasing the number of arginyl residues from two to three. The effect of varying the number of 'spacer' alanyl residues from zero to three was investigated in peptides of the type R4AmSVA, and the peptide with one alanyl residue was found to be the best substrate, making R4X the optimal amino-terminal environment for this enzyme. A similar substrate specificity was observed with the herpes simplex type 1 protein kinase. Protein kinase C was found to have a quite similar substrate preference to the viral enzyme as far as the number and position of the amino-terminal basic residues was concerned; but, unlike the viral protein kinase, it also requires carboxy-terminal basic residues in optimal peptide substrates, and can tolerate the substitution of lysyl for arginyl residues. The cyclic AMP-dependent protein kinase, like the viral enzyme, had favourable kinetic constants for this series of peptides, but differed from the latter in being able to catalyze the phosphorylation of the peptides with two to four arginyl residues with similar efficiency. Studies with the protein, clupeine Y1, as substrate indicated that the pseudorabies virus protein kinase can tolerate arginyl residues on the carboxyl-terminal side of its target residue when there are suitable amino-terminal arginyl determinants. In this respect the virus protein kinase resembled protein kinase C but differed from the cyclic AMP-dependent protein kinase which cannot tolerate such carboxyl-terminal basic residues. The relationship of substrate specificity with model peptides to the ability of the pseudorabies virus protein kinase to phosphorylate proteins in vitro and in vivo is discussed.

Amino Acid Sequence↗
PubMed1990

The activity of protein kinases from hamster fibroblasts towards a synthetic peptide based on a carboxy-terminal portion of ribosomal protein S6.

A synthetic decapeptide, S6(231-240), based on a region near the C-terminus of eukaryotic ribosomal protein S6, was used as a substrate for protein kinases (EC 2.7.1.37) from hamster fibroblasts stimulated with fresh medium. Consistent with the results of others using shorter peptides from this region, it was found that the cyclic AMP-dependent protein kinase preferentially phosphorylated the residue corresponding to Ser-235, whereas protein kinase C preferentially phosphorylated the residue corresponding to Ser-236 in this peptide. The peptide did not serve as a substrate for the growth-associated protein kinase from hamster fibroblasts that phosphorylated ribosomal protein S6 in 40S ribosomal subunits, but did serve as a substrate for a previously undetected protein kinase activity that was resolved from the latter by DEAE-cellulose chromatography. This S6(231-240) protein kinase activity did not phosphorylate ribosomal protein S6 in 40S ribosomal subunits, but is possibly a proteolytic fragment of the 40S ribosomal subunit S6 kinase as the latter activity acquired the ability to phosphorylate the decapeptide after partial tryptic proteolysis. The S6(231-240) protein kinase activity preferentially phosphorylated the residue corresponding to Ser-236 with an apparent Km of 15 microM. These results suggest that specific interactions with the ribosome may be required to activate the growth-associated ribosomal protein S6 kinase.

Amino Acid Sequence↗
PubMed1990

The structure of the pseudorabies virus genome at the end of the inverted repeat sequences proximal to the junction with the short unique region.

The complete nucleotide sequence is presented of the 2 x 67 kbp BamHI-EcoRV portion of the BamHI 10 fragment of the pseudorabies virus (PRV) genome (strain Ka) containing sequences upstream of the previously reported protein kinase gene, and completing the sequence of this 4008 bp fragment. It is predicted to contain a gene designated RSp40, homologous to gene US1 of herpes simplex virus type 1 (HSV-1), with the potential to encode a protein of 364 amino acids. Analysis of PRV mRNA synthesized in the presence and absence of cycloheximide indicated that, in contrast to its HSV-1 homologue, the PRV gene RSp40 does not specify an immediate-early mRNA. Between the RSp40 gene and the protein kinase gene are two reiterated sequences: one containing 11 tandem copies of a 35 nucleotide sequence and the other containing nine tandem copies of a 10 nucleotide sequence. The BamHI 10 and the BamHI 12 fragments of PRV contain the junctions between the short unique (US) and short repeat (RS) regions of the PRV genome. The nucleotide sequence of that portion of the BamHI 12 fragment containing US sequences was determined so that, by comparison with the nucleotide sequence of the BamHI 10 fragment, the junction between the US and RS regions could be defined. In BamHI 10 this was found to be at a point between the two reiterated sequences (which are in the RS region) and the protein kinase gene (which is in the US region). The organization of this region of the PRV genome is compared to that of other alphaherpesviruses.

Amino Acid Sequence↗
PubMed1990

The protein kinase encoded in the short unique region of pseudorabies virus: description of the gene and identification of its product in virions and in infected cells.

We have cloned and determined the nucleotide sequence of a gene, pk, that lies immediately upstream from the gene encoding glycoprotein X in the short unique region of the alphaherpesvirus, pseudorabies virus (PRV). The gene has the potential to encode a protein of 334 amino acids, and is related to gene US3 of herpes simplex virus type 1 (HSV-1), which has been shown to encode a protein kinase. The predicted amino acid sequence encoded by the PRV pk gene is homologous to the corresponding sequence encoded by the HSV-1 US3 gene in the C-terminal catalytic domain, but diverges markedly in the N-terminal domain. As with HSV-1, the mRNA for the pk gene appears to be 3' coterminal with that for the glycoprotein downstream. An antiserum was raised against a protein generated from the fusion of part of the PRV pk catalytic domain with Escherichia coli beta-galactosidase. This specifically reacted with a previously described physically homogeneous protein kinase, PRV-PK, isolated from hamster fibroblasts lytically infected with PRV. Although the majority of the PRV-PK is found in the cytoplasm, some was also detected in purified PRV virions by using the same antibody; a similar distribution was found for the HSV-1 protein kinase, using an antiserum raised against the corresponding HSV-1 fusion protein. When presented with heatinactivated virions, purified PRV-PK (in common with certain cellular protein kinases also present in the virion) was able to phosphorylate in vitro the major virion phosphoprotein phosphorylated in vivo.

Amino Acid Sequence↗
PubMed1989

Identification of induced protein kinase activities specific for the ribosomal proteins uniquely phosphorylated during infection of HeLa cells with vaccinia virus.

We have examined the ribosomal protein kinase activities in partially purified cytoplasmic extracts from HeLa cells infected with vaccinia virus. We found an activity or activities, absent from mock-infected cells, that was capable of phosphorylating the proteins S2 and S13 in vitro. The ribosomes phosphorylated in vitro exhibited the same multiple phosphorylation of S2 found in vivo, at least 3 phosphoryl residues being seen, and the same mono-phosphorylation of S13. Also as in vivo, ribosomal protein S2 contained phosphothreonine as well as phosphoserine, whereas S13 contained only phosphoserine. This strongly suggests that these new protein kinase activities are responsible for the ribosomal protein phosphorylations that occur during infection with vaccinia virus.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1988

Distinct type-1 protein phosphatases are associated with hepatic glycogen and microsomes.

The type-1 protein phosphatase associated with hepatic microsomes has been distinguished from the glycogen-bound enzyme in five ways. (1) The phosphorylase phosphatase/synthase phosphatase activity ratio of the microsomal enzyme (measured using muscle phosphorylase a and glycogen synthase (labelled in sites-3) as substrates) was 50-fold higher than that of the glycogen-bound enzyme. (2) The microsomal enzyme had a greater sensitivity to inhibitors-1 and 2. (3) Release of the catalytic subunit from the microsomal type-1 phosphatase by tryptic digestion was accompanied by a 2-fold increase in synthase phosphatase activity, whereas release of the catalytic subunit from the glycogen-bound enzyme decreased synthase phosphatase activity by 60%. (4) 95% of the synthase phosphatase activity was released from the microsomes with 0.3 M NaCl, whereas little activity could be released from the glycogen fraction with salt. (5) The type-1 phosphatase separated from glycogen by anion-exchange chromatography could be rebound to glycogen, whereas the microsomal enzyme (separated from the microsomes by the same procedure, or by extraction with NaCl) could not. These findings indicate that the synthase phosphatase activity of the microsomal enzyme is not explained by contamination with glycogen-bound enzyme. The microsomal and glycogen-associated enzymes may contain a common catalytic subunit complexed to microsomal and glycogen-binding subunits, respectively. Thiophosphorylase a was a potent inhibitor of the dephosphorylation of ribosomal protein S6, HMG-CoA reductase and glycogen synthase, by the glycogen-associated type-1 protein phosphatase. By contrast, thiophosphorylase a did not inhibit the dephosphorylation of S6 or HMG-CoA reductase by the microsomal enzyme, although the dephosphorylation of glycogen synthase was inhibited. The I50 for inhibition of synthase phosphatase activity by thiophosphorylase a catalysed by either the glycogen-associated or microsomal type-1 phosphatases, or for inhibition of S6 phosphatase activity catalysed by the glycogen-associated enzyme, was decreased 20-fold to 5-10 nM in the presence of glycogen. The results suggest that the physiologically relevant inhibitor of the glycogen-associated type-1 phosphatase is the phosphorylase a-glycogen complex, and that inhibition of the microsomal type-1 phosphatase by phosphorylase a is unlikely to play a role in the hormonal control of cholesterol or protein synthesis. Protein phosphatase-1 appears to be the principal S6 phosphatase in mammalian liver acting on the serine residues phosphorylated by cyclic AMP-dependent protein kinase.

Animals↗
PubMed1988

The tropomyosin mRNAs of mouse striated muscles: molecular cloning of beta-tropomyosin.

The nucleotide sequence corresponding to the complete coding region and much of the 5' and 3' untranslated regions of the skeletal muscle-specific mouse beta-tropomyosin mRNA was determined from overlapping cDNA clones isolated from a library of recombinants in pBR322. When one of these was used as a probe to detect tropomyosin mRNAs expressed after fusion of mouse myoblasts in culture, species were detected in addition to those corresponding to known tropomyosins of striated muscle. One of these was also detected in the leg muscle of 12 day-old mice. The identities of these species are uncertain, but they may correspond to alternatively spliced products of the same RNA transcripts that give rise to the predominant tropomyosin isoforms of striated muscle.

Amino Acid Sequence↗
PubMed1988

Mouse cytoskeletal gamma-actin: analysis and implications of the structure of cloned cDNA and processed pseudogenes.

The nucleotide sequence corresponding to almost the whole of a mouse gamma-cytoskeletal actin mRNA was determined from overlapping cloned DNA copies derived from brain mRNA. Several gamma-actin processed pseudogenes were isolated from a library of cloned DBA mouse genomic DNA, and the nucleotide sequences of these were determined and compared with that of the cDNA. This showed that two of these pseudogenes had arisen from a gene duplication or amplification event, and indicated that they had subsequently undergone partial correction against one another. The relative ages of the pseudogenes were estimated on the basis of their percentage divergence from the cDNA sequence and these were compared with an estimation based on the number of presumed silent mutations in the cDNA since each pseudogene had arisen. Consistent results were obtained, except in the case of one pseudogene which also showed an anomalous regional distribution of differences from the cDNA sequence. One way of accounting for the features of this anomalous pseudogene is by postulating that it is derived from a second functional gene for gamma-actin, different from that represented by the cDNA described here.

Actins↗
PubMed1988

Duplicated region of the mouse genome containing a cytoplasmic gamma-actin processed pseudogene associated with long interspersed repetitive elements.

The structures of two cloned recombinants of bacteriophage lambda and mouse genomic DNA (lambda mA14 and lambda mA36) were compared by electron microscopic analysis of various heteroduplex DNAs, restriction endonuclease mapping and nucleotide sequence determination. Each clone was shown to be derived from a distinct region of the mouse genome, but the two exhibited structural similarity over a region of at least 11,000 bases which included a cytoskeletal gamma-actin processed pseudogene of approximately 1800 bases. It is concluded that the two genomic regions were derived from a common ancestral region by duplication or amplification. The homologous regions of the two clones contained members of the long interspersed repetitive L1Md (long interspersed repeated sequence 1 of Mus domesticus) family lying in opposite orientation to one another, so that single-stranded DNA from the clones could form intra-molecular heteroduplexes. The complete nucleotide sequences of three L1Md members in lambda mA14 were determined. The longest of these (L1Md-14LH) had inserted into the gamma-actin processed pseudogene and, although it contained internal deletions, appeared to possess intact 5' and 3' ends. A second L1Md member (L1Md-14RH1) also appeared to have an intact 5' end but had lost most of its 3' portion, and a third member (L1Md-14RH2) was an internal fragment. The repeated sequence at the 5' ends of L1Md-14LH and L1Md-14RH1 showed these to be members of the L1Md-A family.

Actins↗