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T Hunter

Publications and source records attributed to T Hunter.

At least 163 records · Page 9Linked to original sources
PubMed1994

1001 protein kinases redux--towards 2000.

Recent genomic analysis indicates that the number of protein kinase genes in eukaryotes may be higher than previously thought, and vertebrate genomes may contain in the vicinity of 2000. In addition to the growing protein-serine/protein-tyrosine kinase superfamily, new types of protein kinase have been discovered that operate on different structural principles. The structures of four protein-serine kinases have been solved and have revealed a common structural core containing the highly conserved residues in the catalytic domain. These structures provide insights into substrate selectivity and protein kinase activation. New regulatory principles for protein kinase have been uncovered, including phosphorylation of residues within the catalytic domain by a second protein kinase, new second messengers and specific protein inhibitors. Evidence that protein kinase cascades play an important role in signal transduction has emerged. A number of fundamental cellular processes such as the cell cycle and transcription have been shown to require protein phosphorylation.

Animals↗
PubMed1994

Antibodies specific for the neuronal form of the Src protein elicited by an antigenized antibody.

To elicit antibodies directed specifically against the neuron-specific form of the c-src gene product, pp60c-src(+), we used an antigenized antibody comprising a decamer containing the amino acid sequence specific to pp60c-src(+) inserted into the third hypervariable loop of the heavy (H)-chain variable (V)-region. This was used to raise anti-idiotype antibodies reacting with the peptide epitope in rabbits. The antisera reacted with pp60c-src(+), as judged by immune blotting, immunoprecipitation, immune complex kinase assay, and indirect immunofluorescence staining, but did not react with the fibroblast form of the c-src gene product, pp60c-src. Antigenized antibody is a useful approach for producing antibodies able to distinguish between isoforms of the same gene product and specific for the neuronal form of the Src protein.

3T3 Cells↗
PubMed1994

Membrane localization of the kinase which phosphorylates p34cdc2 on threonine 14.

The key regulator of entry into mitosis is the serine/threonine kinase p34cdc2. This kinase is regulated both by association with cyclins and by phosphorylation at several sites. Phosphorylation at Tyr 15 and Thr 14 are believed to inhibit the kinase activity of cdc2. In Schizosaccharomyces pombe, the wee1 (and possibly mik1) protein kinase catalyzes phosphorylation of Tyr 15. It is not clear whether these or other, as yet unidentified, protein kinases phosphorylate Thr 14. In this report we show, using extracts of Xenopus eggs, that the Thr 14-directed kinase is tightly membrane associated. Specifically, we have shown that a purified membrane fraction, in the absence of cytoplasm, can promote phosphorylation of cdc2 on both Thr 14 and Tyr 15. In contrast, the cytoplasm can phosphorylate cdc2 only on Tyr 15, suggesting the existence of at least two distinctly localized subpopulations of cdc2 Tyr 15-directed kinases. The membrane-associated Tyr 15 and Thr 14 kinase activities behaved similarly during salt or detergent extraction and were similarly regulated during the cell cycle and by the checkpoint machinery that delays mitosis while DNA is being replicated. This suggests the possibility that a dual-specificity membrane-associated protein kinase may catalyze phosphorylation of both Tyr 15 and Thr 14.

Animals↗
PubMed1994

Budding and fission yeast casein kinase I isoforms have dual-specificity protein kinase activity.

We have examined the activity and substrate specificity of the Saccharomyces cerevisiae Hrr25p and the Schizosaccharomyces pombe Hhp1, Hhp2, and Cki1 protein kinase isoforms. These four gene products are isotypes of casein kinase I (CKI), and the sequence of these protein kinases predicts that they are protein serine/threonine kinases. However, each of these four protein kinases, when expressed in Escherichia coli in an active form, was recognized by anti-phosphotyrosine antibodies. Phosphoamino acid analysis of 32P-labeled proteins showed phosphorylation on serine, threonine, and tyrosine residues. The E. coli produced forms of Hhp1, Hhp2, and Cki1 were autophosphorylated on tyrosine, and both Hhp1 and Hhp2 were capable of phosphorylating the tyrosine-protein kinase synthetic peptide substrate polymer poly-E4Y1. Immune complex protein kinases assays from S. pombe cells showed that Hhp1-containing precipitates were associated with a protein-tyrosine kinase activity, and the Hhp1 present in these immunoprecipitates was phosphorylated on tyrosine residues. Although dephosphorylation of Hhp1 and Hhp2 by Ser/Thr phosphatase had little effect on the specific activity, tyrosine dephosphorylation of Hhp1 and Hhp2 caused a 1.8-to 3.1-fold increase in the Km for poly-E4Y1 and casein. These data demonstrate that four different CKI isoforms from two different yeasts are capable of protein-tyrosine kinase activity and encode dual-specificity protein kinases.

Amino Acid Sequence↗
PubMed1994

Regulation by protein-tyrosine phosphatase PTP2 is distinct from that by PTP1 during Dictyostelium growth and development.

We have cloned a gene encoding a second Dictyostelium discoideum protein-tyrosine phosphatase (PTP2) whose catalytic domain has approximately 30 to 39% amino acid identity with those of other PTPs and a 41% amino acid identity with D. discoideum PTP1. Like PTP1, PTP2 is a nonreceptor PTP with the catalytic domain located at the C terminus of the protein. PTP2 has a predicted molecular weight of 43,000 and possesses an acidic 58-amino-acid insertion 24 amino acids from the N terminus of the conserved catalytic domain. PTP2 transcripts are expressed at moderate levels in vegetative cells and are induced severalfold at the onset of development. Studies with a PTP2-lacZ reporter gene fusion indicate that PTP2, like PTP1, is preferentially expressed in prestalk and anterior-like cell types during the multicellular stages of development. PTP2 gene disruptants (ptp2 null cells) are not detectably altered in growth and show a temporal pattern of development similar to that of wild-type cells. ptp2 null slugs and fruiting bodies, however, are significantly larger than those of wild-type slugs, suggesting a role for PTP2 in regulating multicellular structures. D. discoideum strains overexpressing PTP2 from the PTP2 promoter exhibit growth rate and developmental abnormalities, the severity of which corresponds to the level of PTP2 overexpression. Strains with high overexpression of the PTP2 gene grow slowly on bacterial lawns and produce small cells in axenic medium. When development is initiated in these strains, cells are able to aggregate but then stop further morphogenesis for 6 to 8 h, after which time a variable fraction of these aggregates continue with normal timing, producing diminutive fruiting bodies. These disruption and overexpression phenotypes for PTP2 are distinct from the corresponding mutant PTP1 phenotypes. Immunoprobing PTP2 mutant strains during growth and development with antiphosphotyrosine antibodies reveals several changes in the tyrosine phosphorylation of proteins in PTP2 mutant strains compared with that in wild-type cells. These changes are different from those identified in the previously characterized corresponding PTP1 disruption and overexpression mutant strains. Thus, although PTP2 and PTP1 are nonreceptor PTPs with similar spatial patterns of expression, our findings suggest that they possess distinct regulatory functions in controlling D. discoideum growth and development.

Animals↗
PubMed1994

Protein kinase A and C site-specific phosphorylations of LAP (NF-IL6) modulate its binding affinity to DNA recognition elements.

LAP (NF-IL6 or C/EBP beta), is a liver transcriptional activator protein that confers liver-specific gene expression. Because LAP has a characteristic phosphoacceptor sequence for cAMP-dependent protein kinase A (PKA), we tested if in vitro phosphorylation of LAP by PKA modulates its interaction with specific DNA sequences. The major PKA phosphorylation site of LAP was identified as Ser105, which is a predicted PKA site. As expected, this PKA phosphorylation site disappears after mutation of Ser105 to Ala. Kinetic studies with LAP and LAP Asp105 (which mimics a phosphoserine residue) demonstrated that phosphorylation of Ser105 itself has no effect on DNA binding. Phosphorylation of other sites by PKA, identified in the region between Ser173 and Ser223 and at Ser240, by analysis of truncated and mutated LAP peptides, resulted in an inhibition of DNA binding. LAP was also phosphorylated by purified protein kinase C in vitro, and the major phosphoacceptor was shown to be Ser240 within the DNA-binding domain of LAP. Phosphorylation of LAP at this residue or introduction of a Ser240 to Asp mutation resulted in marked decrease in its binding to DNA. These results suggest that site-specific phosphorylations of LAP modulate transactivation of its target genes.

Albumins↗
PubMed1994

Image labeling. The need for a better look.

The important message in this editorial is for radiologists to critically examine how well images are labeled in their own department. If it is not satisfactory, then institute corrective measures. These can range from sophisticated computer programs for printing flashcards to merely sending the chief technologist all those films one comes across with unreadable labels. The quality of the image labeling should also be a consideration when purchasing CT, MRI, ultrasound, computed radiography and digital angiography equipment. The fact that you consider this important should be communicated to equipment manufacturers in the hope that they will pay more attention to it and offer more flexibility for each department to design its own labels. In any event, I feel consistently bad film labeling results in sloppy radiology with possible patient harm and unpleasant legal consequences for the radiologist.

Data Display↗
PubMed1994

The manganese superoxide dismutase gene of Caenorhabditis elegans.

We have cloned and sequenced a gene (sod-3) encoding manganese superoxide dismutase from the nematode Caenorhabditis elegans. The protein-coding region spans 943 bp including three intron sequences and encodes a protein of 227 amino acids (M(r) = 26,367) of which the first 33 amino acids are the presumed mitochondrial-targeting signal peptide. The deduced mature manganese-superoxide dismutase has 194 amino acids (M(r) = 22,193).

Amino Acid Sequence↗
PubMed1994

The copper/zinc superoxide dismutase gene of Caenorhabditis elegans.

The nucleotide and deduced amino acid sequences of the copper/zinc superoxide dismutase gene (sod-1) from the nematode Caenorhabditis elegans has been determined. The protein coding region is interrupted by three intron sequences covering 608 bp in total and encodes a protein of 158 amino acids (M(r) = 16,307).

Amino Acid Sequence↗
PubMed1993

Mutation of Tyr697, a GRB2-binding site, and Tyr721, a PI 3-kinase binding site, abrogates signal transduction by the murine CSF-1 receptor expressed in Rat-2 fibroblasts.

The receptor for the myeloid cell growth factor colony stimulating factor 1 (CSF-1) is a protein tyrosine kinase that is closely related to the PDGF receptor. Ligand binding results in kinase activation and autophosphorylation. Three autophosphorylation sites, Tyr697, Tyr706 and Tyr721, have been mapped to the kinase insert domain. Deletion of the entire kinase insert domain completely abrogates signal transduction by the CSF-1 receptor expressed in Rat-2 fibroblasts. To investigate the function of individual phosphorylation sites present in the CSF-1 receptor kinase insert domain, a number of phosphorylation site mutants were expressed in Rat-2 fibroblasts. Mutation of either Tyr697 or Tyr721 compromised signal transduction by the CSF-1 receptor. A mutant receptor, in which both Tyr697 and Tyr721 were replaced by phenylalanine, has lost all ability to induce changes in morphology or to increase cell growth rate in response to CSF-1. Tyr721 has been identified recently as the binding site for PI 3-kinase. Here we report that GRB2 associates with the CSF-1 receptor upon ligand binding. The phosphorylation on tyrosine of SHC and several other GRB2-associated proteins increased upon stimulation with CSF-1. Tyr697 was identified as a binding site for GRB2. We suggest that PI 3-kinase, GRB2 and some of the GRB2-associated proteins could play an important role in signal transduction by the CSF-1 receptor.

Adaptor Proteins, Signal Transducing↗
PubMed1993

Braking the cycle.

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CDC2-CDC28 Kinases↗
PubMed1993

Transactivation by NF-IL6/LAP is enhanced by phosphorylation of its activation domain.

One of the members of the bZIP family of transcriptional activators is NF-IL6/LAP (IL-6 DBP, C/EBP beta, CRP2). NF-IL6/LAP protein is highly expressed in liver nuclei, where it has been implicated as a master regulator of the acute-phase response, induced by interleukin-6 (IL-6) and other inflammatory mediators. Also, NF-IL6/LAP is involved in the activation of the IL-6 promoter in response to IL-1 and bacterial lipopolysaccharide. The control of NF-IL6/LAP expression and activity is complex and poorly understood. Under some conditions the NF-IL6/LAP gene is transcriptionally activated by IL-1 and lipopolysaccharide, whereas in other instances, its binding to cognate DNA sequences is enhanced by cytokines. Additionally, the ability of constitutively expressed NF-IL6/LAP to activate transcription is strongly augmented by IL-6, through an unknown signalling pathway. We now show that stimulation of the protein kinase C pathway increases the phosphorylation of Ser 105 within the activation domain of NF-IL6/LAP, and enhances its transcriptional efficacy.

Animals↗
PubMed1993

Cdc25M2 activation of cyclin-dependent kinases by dephosphorylation of threonine-14 and tyrosine-15.

Recent evidence has suggested that human cyclin-dependent kinase 2 (CDK2) is an essential regulator of cell cycle progression through S phase. CDK2 is known to complex with at least two distinct human cyclins, E and A. The kinase activity of these complexes peaks in G1 and S phase, respectively. The vertebrate CDC2/cyclin B1 complex is an essential regulator of the onset of mitosis and is inhibited by phosphorylation of CDC2 on Thr-14 and Tyr-15. In vitro, CDC2/cyclin B1 is activated by treatment with the members of the Cdc25 family of phosphatases. We found that, like CDC2, CDK2 is also phosphorylated on Thr-14 and Tyr-15 and that treatment of cyclin A or cyclin E immunoprecipitates with bacterially expressed Cdc25M2 (the mouse homolog of human CDC25B) increased the histone H1 kinase activity of these immune complexes 5- to 10-fold. Tryptic peptide mapping demonstrated that Cdc25M2 treatment of cyclin A or cyclin B1 immune complexes resulted in the specific dephosphorylation of Thr-14 and Tyr-15 on CDK2 or CDC2, respectively. Thus, we have confirmed that Cdc25 family members comprise a class of dual-specificity phosphatases. Furthermore, our data suggest that the phosphorylation and dephosphorylation of CDKs on Thr-14 and Tyr-15 may regulate not only the G2/M transition but also other transitions in the cell cycle and that individual cdc25 family members may regulate distinct cell cycle checkpoints.

Amino Acid Sequence↗