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Biomedical subjects

T Hunter

Publications and source records attributed to T Hunter.

At least 181 records · Page 10Linked to original sources
PubMed1993

G2 delay induced by nitrogen mustard in human cells affects cyclin A/cdk2 and cyclin B1/cdc2-kinase complexes differently.

We investigated the temporal regulation of cyclin A- and B1-dependent kinases in human lymphoma cells treated with nitrogen mustard (HN2) and pentoxifylline, to determine whether the activity of these complexes correlated with cell cycle arrest induced by DNA damage. Cells were synchronized in G1/S, treated with HN2, and then postincubated with pentoxifylline. HN2-induced a protracted delay in G2 phase. This delay correlated with suppression of cyclin B1- and cdc2-kinase activities, and stabilization of hyperphosphorylated-cdc2 in the presence of similar cyclin B1 levels to those found in mitosis. HN2 had no discernible effect on the S phase activity of cyclin A- or cdk2-immune complexes. Entry of control cells into mitosis correlated with destruction of cyclin A, disappearance of cyclin A-bound cdk2 and decreased cdk2 kinase activity. G2 delay induced by HN2 was associated with stabilization of cyclin A, increased abundance of cyclin A-bound cdk2, and increased cdk2 activity. Cyclin A was also associated with cdc2, which, contrary to complexes containing cdk2, were only activated upon entry into mitosis. Pentoxifylline abrogated cell cycle arrest induced by aphidicolin and HN2 in human lymphoma cells. Pentoxifylline also reverted the activity of cyclin A- and B1-kinases in HN2-treated cells to approximately that observed in controls. Our findings suggest that delayed entry into mitosis following DNA damage correlates with suppression of cyclin B1/cdc2 and cyclin A/cdc2 complexes, while maintaining cyclin A/cdc2 complexes in an active state. Furthermore, we found that pentoxifylline disrupts the signal transduction pathway that regulates these complexes when damaged DNA is present, resulting in abrogation of cell cycle arrest.

Antigen-Antibody Complex↗
PubMed1993

Increased production of the TrkB protein tyrosine kinase receptor after brain insults.

The protein-tyrosine kinases Trk, TrkB, and TrkC are signal-transducing receptors for a family of neurotrophic factors known as the neurotrophins. Here we show that seizures induced by hippocampal kindling lead to a rapid, transient increase of trkB mRNA and protein in the hippocampus. TrkB is a component of a high affinity receptor for brain-derived neurotrophic factor (BDNF). No change was detected in mRNAs for Trk or TrkC, components of the high affinity nerve growth factor or neurotrophin-3 receptors, respectively. trkB mRNA was also transiently increased in the dentate gyrus following cerebral ischemia and hypoglycemic coma; these treatments had no effect on trk and trkC mRNAs. The increase in trkB mRNA and protein showed the same time course and distribution as the increase in BDNF mRNA. These data suggest that BDNF and its receptor may play a local role within the hippocampus in kindling-associated neural plasticity and in neuronal protection following epileptic, ischemic, and hypoglycemic insults.

Animals↗
PubMed1993

Antiproliferative effects on keratinocytes of a range of clinically used drugs with calmodulin antagonist activity.

Thirty-two drugs, including some in use for a variety of clinical disorders, were examined for their ability to inhibit calmodulin activity in vitro. From these, 10 drugs were selected for their inhibition of calmodulin activity and examined for their ability to inhibit proliferation of rapidly dividing human keratinocytes. A significant correlation between antiproliferative activity and calmodulin antagonist potency was found. Of these drugs there were several, including miconazole, dequalinium chloride, bromocriptine and tamoxifen, whose use is well established and well documented. The potential use of these drugs (and others identified in this way) as antipsoriatic agents is discussed.

Calmodulin↗
PubMed1993

Characterization of a human protein threonine kinase isolated by screening an expression library with antibodies to phosphotyrosine.

We have studied the activity and substrate specificity of the catalytic domain of a protein kinase that was isolated in a screen of a human lambda gt11 fibroblast cDNA library with anti-phosphotyrosine antibodies. The sequence of this protein kinase would predict that it is a protein serine/threonine kinase, which at first seemed incongruent with the cloning method. However, recent reports indicate that some protein kinases can phosphorylate both tyrosine and serine/threonine residues. To determine whether this protein kinase, which we call PYT (for phosphotyrosine picked threonine kinase), was a dual-specificity protein kinase we investigated its substrate specificity when expressed in bacteria. The catalytic domain was active as a protein kinase when expressed from any of several promoters and when expressed as a TrpE fusion protein. All experiments that resulted in an active protein kinase, as judged by incorporation of 32P by metabolic labeling, also resulted in the generation of proteins that were recognized by anti-phosphotyrosine antibodies. Phosphoamino acid analyses of the metabolically labeled proteins that were recognized by the antibodies consistently yielded large amounts of phosphothreonine and only trace amounts of phosphotyrosine. We mapped the phosphorylation sites in the phosphorylated PYT protein and found only phosphothreonine; 90% of the radioactivity mapped to a threonine in the region autophosphorylated by many protein kinases. These data demonstrate that PYT is primarily a protein threonine kinase, but that it can phosphorylate tyrosine to a small extent, making it a potential dual-specificity protein kinase.

Amino Acid Sequence↗
PubMed1992

Increased levels of trkB mRNA and trkB protein-like immunoreactivity in the injured rat and cat spinal cord.

Expression of neurotrophins and neurotrophin receptors was examined with in situ hybridization and immunohistochemical techniques 10 days to 6 weeks after ventral or dorsal funiculus spinal cord lesions in adult rats and cats, lesions that have previously been shown to allow axon regrowth. Strongly elevated levels of trkB mRNA were seen in the scar tissue formed in the white matter after both types of lesions. Only small increases were detected for nerve growth factor, brain-derived neurotrophic factor, neurotrophin 3, neurotrophin 4, trk, and trkC mRNA in response to the injuries. trkB protein-like immunoreactivity was increased in the regions that showed elevated levels of trkB mRNA. EM localized this immunoreactivity to neurons, astrocytes, and leptomeningeal cells. Neurofilament immunolabeling and axonal tracing demonstrated that nerve fibers in the scar tissue were concentrated to areas that showed strong trkB protein-like immunoreactivity. The findings implicate a role for neurotrophin receptors in axonal sprouting and glial reactions in the injured spinal cord.

Animals↗
PubMed1992

Identification of the two major epidermal growth factor-induced tyrosine phosphorylation sites in the microvillar core protein ezrin.

In response to epidermal growth factor (EGF) the microvillar core protein ezrin is phosphorylated transiently to a high level on tyrosine residues in human epidermoid carcinoma A431 cells. Here we report the identification of the tyrosine phosphorylation sites in ezrin using bacterially expressed protein as a substrate for in vitro phosphorylation with the EGF receptor. The two major phosphotyrosine-containing peptides observed in vivo were also phosphorylated in vitro. By secondary digestions and site-directed mutagenesis tyrosines 145 and 353 were identified as the sites of phosphorylation. One of the sites, Tyr145, lies in the N-terminal region of homology that is common to the band 4.1-talin-ezrin protein family. This tyrosine residue and its vicinal amino acids are conserved throughout the family members, including radixin, moesin, and the two phosphotyrosine phosphatases, PTP H1 and PTP MEG, but not in band 4.1 or talin. Tyr353 is localized within the alpha-helical domain of ezrin and comparison of the protein sequences reveals that this site is unique to ezrin.

Amino Acid Sequence↗
PubMed1992

Casein kinase II is a negative regulator of c-Jun DNA binding and AP-1 activity.

c-Jun, a major component of the inducible transcription factor AP-1, is a phosphoprotein. In nonstimulated fibroblasts and epithelial cells, c-Jun is phosphorylated on a cluster of two to three sites abutting its DNA-binding domain. Phosphorylation of these sites inhibits DNA binding, and their dephosphorylation correlates with increased AP-1 activity. We show that two of these sites, Thr-231 and Ser-249, are phosphorylated by casein kinase II (CKII). Substitution of the third site, Ser-243, by Phe interferes with phosphorylation of the inhibitory sites in vivo and by purified CKII in vitro. Microinjection into living cells of synthetic peptides that are specific competitive substrates or inhibitors of CKII results in induction of AP-1 activity and c-Jun expression. Microinjection of CKII suppresses induction of AP-1 by either phorbol ester or an inhibitory peptide. These results suggest that one of the roles of CKII, a major nuclear protein kinase with no known functions, is to attenuate AP-1 activity through phosphorylation of c-Jun.

Amino Acid Sequence↗
PubMed1992

The budding yeast HRR25 gene product is a casein kinase I isoform.

The Saccharomyces cerevisiae HRR25 gene was identified as a regulator of DNA strand-break repair. HRR25 encodes a protein kinase that is closely related to bovine casein kinase I (CKI). CKI is a ubiquitous multipotential protein kinase. Rabbit polyclonal antibodies that recognize and immunoprecipitate Hrr25p have been generated and an immune complex protein kinase assay has been developed. The reaction depends upon HRR25 and shows that Hrr25p uses casein as a substrate. The identity between Hrr25p and bovine CKI suggests that Hrr25p is a yeast isoform of the CKI family and that CKIs may play a role in regulating DNA metabolism.

Amino Acid Sequence↗
PubMed1992

Induction of cyclin mRNA and cyclin-associated histone H1 kinase during liver regeneration.

Cyclins and cyclin-associated cdc kinases are key regulators of oocyte maturation (Maller, J. L. (1990) in The Biology and Medicine of Signal Transduction (Nishizuka, Y., Endo, M., and Tanaka, C., eds) pp. 323-328, Raven Press, New York), yeast cell cycles (Nurse, P. (1990) Nature 344, 503-508), DNA replication in cell-free systems (D'Urso, F., Marraccino, R. L., Marshak, R. R., and Roberts, J. M. (1990) Science 250, 786-791), and amphibian cell proliferative transitions (Hunt, T. (1991) Nature 350, 462-463). The extent to which these regulatory molecules participate in the growth control of differentiated epithelial cells like hepatocytes is unknown. Therefore, we investigated the expression of "G1" (E, C, and D) and "G2/M" (A, B1, and B2) cyclin mRNAs, the relative levels of cyclin A- and B1-associated histone H1-kinase activity, and the appearance of cyclin-associated kinases (p32/p33cdk2 and p33/p34cdc2) in regenerating rat liver and in control tissues from sham hepatectomized rats. To do this, we exploited a battery of human cyclin cDNAs and cyclin antisera that recognize rat molecules. The results suggest an apparent sequence of regeneration-specific changes: 1) elevated and induced expression of cyclins E (2.1 kilobases (kb)) and C (4 kb), and D mRNAs (4 kb), within 12 h, respectively; 2) induction of cyclins A (3.4 and 1.8 kb), B1 (2.5 and 1.8 kb), and B2 (1.9 kb) mRNAs at 24 h; 3) induction of cyclin A- and B1-associated nuclear histone H1 kinase at 24 h; and 4) enhanced levels of PSTAIRE-containing proteins of Mr approximately 32-33 and 33-34 kDa in nuclear extracts from 24-h regenerating liver that co-immunoprecipitate with cyclin A and B1 antisera, respectively. These observations provide an intellectual framework that unifies the biology of hepatocyte mitogenesis, proto-oncogene expression, and the machinery of the cell cycle.

Animals↗
PubMed1992

A cyclin A-protein kinase complex possesses sequence-specific DNA binding activity: p33cdk2 is a component of the E2F-cyclin A complex.

The E2F transcription factor has been found in association with the cyclin A protein, and this complex accumulates during the S phase of the cell cycle, suggesting that E2F may play a role in cell cycle control. In independent studies, cyclin A has been shown to be associated with two other proteins, the Rb-related p107 protein and the cdc2-related p33 cdk2 protein kinase. Through an analysis of the E2F-cyclin A complex, we now find that both the p107 protein and the cdc2-related p33cdk2 kinase are components of the previously described complex. Moreover, the complex possesses H1 kinase activity. These results thus define a cyclin A-cdk2 kinase complex that possesses sequence-specific DNA binding activity. This suggests that the cdk2 kinase may phosphorylate other DNA-bound substrates, and that one role of the E2F factor may be to localize this protein kinase to the DNA.

3T3 Cells↗
PubMed1992

Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3'-kinase SH2 domains: a model for SH2-mediated receptor-target interactions.

Efficient binding of active phosphatidylinositol (PI) 3'-kinase to the autophosphorylated macrophage colony stimulating factor receptor (CSF-1R) requires the noncatalytic kinase insert (KI) region of the receptor. To test whether this region could function independently to bind PI 3'-kinase, the isolated CSF-1R KI was expressed in Escherichia coli, and was inducibly phosphorylated on tyrosine. The tyrosine phosphorylated form of the CSF-1R KI bound PI 3'-kinase in vitro, whereas the unphosphorylated form had no binding activity. The p85 alpha subunit of PI 3'-kinase contains two Src homology (SH)2 domains, which are implicated in the interactions of signalling proteins with activated receptors. Bacterially expressed p85 alpha SH2 domains complexed in vitro with the tyrosine phosphorylated CSF-1R KI. Binding of the CSF-1R KI to PI 3'-kinase activity, and to the p85 alpha SH2 domains, required phosphorylation of Tyr721 within the KI domain, but was independent of phosphorylation at Tyr697 and Tyr706. Tyr721 was also critical for the association of activated CSF-1R with PI 3'-kinase in mammalian cells. Complex formation between the CSF-1R and PI 3'-kinase can therefore be reconstructed in vitro in a specific interaction involving the phosphorylated receptor KI and the SH2 domains of p85 alpha.

Amino Acids↗
PubMed1992

A single cyclin A gene and multiple cyclin B1-related sequences are dispersed in the mouse genome.

Cyclin activation of protein serine/threonine kinases plays a pivotal role in regulating the cell cycle. Multiple cyclins that fall into at least five classes, A, B, C, D, and E, have been identified. In some organisms, more than one member of a single cyclin class has been observed. To gain insight into the function of cyclin multiplicity, we determined the number of cyclin A- and B1-related sequences present in the mouse genome, the relationship between these cyclin-related sequences and previously described mutations in the mouse, and cyclin A and B1 mRNA expression in mouse embryos. By genetic mapping using human cyclin A and B1 probes, we identified 1 cyclin A gene located on chromosome 3 and 10 cyclin B1-related sequences located on chromosomes 4, 5, 7, 8, 13, 14, 15, and 17. Cyclin B1-related sequences map in the vicinity of the metaphase-arrest mutation oligosyndactyly (Os) and embryonic lethal mutations associated with the albino (c) locus and the t-complex. In Northern analysis, two cyclin A-related transcripts of 2.1 and 3.4 kb and three cyclin B1-related transcripts of 1.7, 2.1, and 2.7 kb were detected in embryonic stem cells and postimplantation embryos from Day 9.5 to 15.5 of development. Identification of multiple cyclin B1-related sequences in the mouse genome and multiple cyclin B1 mRNAs raises the possibility that seemingly redundant cyclin B genes might have developmental- and/or cell-type-specific functions.

Animals↗
PubMed1992

Dual-specificity protein kinases: will any hydroxyl do?

Protein kinases are classified by the target amino acid in their substrates. Those protein kinases that phosphorylate hydroxyamino acids comprise two groups, the protein-tyrosine and protein-serine/threonine kinases, which, until recently, had been thought to be mutually exclusive. However, several new protein kinases have been discovered that, by the criterion of primary structure, would be classified as protein-serine/threonine kinases but which, surprisingly, are able to phosphorylate tyrosine residues. Even more surprising, there are reports of protein kinases that are capable of phosphorylating both tyrosine and serine/threonine residues. We review and discuss recent developments concerning these 'dal-specificity' protein kinases.

Amino Acid Sequence↗
PubMed1992

Requirement for p34cdc2 kinase is restricted to mitosis in the mammalian cdc2 mutant FT210.

The mouse FT210 cell line is a temperature-sensitive cdc2 mutant. FT210 cells are found to arrest specifically in G2 phase and unlike many alleles of cdc2 and cdc28 mutants of yeasts, loss of p34cdc2 at the nonpermissive temperature has no apparent effect on cell cycle progression through the G1 and S phases of the division cycle. FT210 cells and the parent wild-type FM3A cell line each possess at least three distinct histone H1 kinases. H1 kinase activities in chromatography fractions were identified using a synthetic peptide substrate containing the consensus phosphorylation site of histone H1 and the kinase subunit compositions were determined immunochemically with antisera prepared against the "PSTAIR" peptide, the COOH-terminus of mammalian p34cdc2 and the human cyclins A and B1. The results show that p34cdc2 forms two separate complexes with cyclin A and with cyclin B1, both of which exhibit thermal lability at the non-permissive temperature in vitro and in vivo. A third H1 kinase with stable activity at the nonpermissive temperature is comprised of cyclin A and a cdc2-like 34-kD subunit, which is immunoreactive with anti-"PSTAIR" antiserum but is not recognized with antiserum specific for the COOH-terminus of p34cdc2. The cyclin A-associated kinases are active during S and G2 phases and earlier in the division cycle than the p34cdc2-cyclin B1 kinase. We show that mouse cells possess at least two cdc2-related gene products which form cell cycle regulated histone H1 kinases and we propose that the murine homolog of yeast p34cdc/CDC28 is essential only during the G2-to-M transition in FT210 cells.

Amino Acid Sequence↗
PubMed1992

A mouse cdc25 homolog is differentially and developmentally expressed.

The timing and activation of the p34cdc2 kinase in mammals is associated with dephosphorylation of phosphotyrosine and phosphothreonine residues on the p34cdc2 kinase. For fission yeast, the timing of mitosis is regulated by cyclic accumulation of cdc25, which promotes dephosphorylation of p34cdc2 and concomitant protein kinase activation. We report the identification and characterization of a structural and functional mouse homolog, Cdc25M2, of the cdc25 phosphatase. Cdc25M2 shows high sequence identity to the previously reported human homolog cdc25Hu2. Cdc25M2 can functionally complement for a Schizosaccharomyces pombe cdc25ts mutation, and when expressed in Escherichia coli and purified, Cdc25M2 is an active phosphatase. cdc25M2 mRNA shows variation in expression in different tissues in the mouse embryo and is expressed in a developmental and cell-cycle-dependent fashion. We suggest that the expression and accumulation of the cdc25 mitotic inducer may play a critical role in the regulation of mouse development.

Amino Acid Sequence↗
PubMed1992

Receptor protein tyrosine kinases and phosphatases.

It is clear that the number of receptor PTKs and PTPs encoded by a typical vertebrate genome is rather large. Although the signal pathways activated by the receptor PTKs may in many cases be common, specificity is provided by the ligand-binding domain and the availability of ligand. In addition, the precise spectrum of substrates that bind to and are phosphorylated by each receptor PTK can differ based on the number and nature of the autophosphorylation sites and on the repertoire of SH2-containing proteins and other substrates expressed in each cell type. It is also clear that receptor PTKs can activate multiple independent signaling pathways and that the output of these pathways can be integrated to provide a specific cellular response. The role of receptor PTPs in such integrated signaling networks is not yet obvious. In some cases, they may activate nonreceptor PTKs, whereas in other cases, they may counteract the effects of activated receptor and nonreceptor PTKs by dephosphorylating the PTKs themselves or their substrates. We know very little about the substrate specificity of PTPs, but in part this must be dictated by their subcellular location. It is possible that there are specific pairs of receptor PTKs and PTPs, which act in concert at the cell surface to activate and down-regulate specific signal pathways. Progress in understanding the function of receptor PTPs will depend on identifying ligands for receptor PTPs and then determining how ligand binding influences their activity.

Animals↗