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Biomedical subjects

T Hunter

Publications and source records attributed to T Hunter.

At least 145 records · Page 8Linked to original sources
PubMed1995

Apoptosis is induced in BHK cells by the tsBN462/13 mutation in the CCG1/TAFII250 subunit of the TFIID basal transcription factor.

A temperature-sensitive (ts) mutant of the BHK21 cell line derived from golden hamsters, tsBN462 has a mutation in the gene encoding the largest subunit of the TFIID complex, TAFII250/p230/CCG1, and arrests in the G1 phase at the nonpermissive temperature, 39.5 degrees C. We found that tsBN462 cells underwent apoptosis following growth arrest at 39.5 degrees C, suggesting a role for CCG1 as a repressor of apoptosis. By electron microscopic observation, tsBN462 cells at 39.5 degrees C showed characteristic features of apoptosis. Apoptosis was not suppressed by expression of Bc1-2 or the adenovirus E1B 19 kDa protein. Cell death was suppressed completely by expression of wild-type CCG1 and partially by wild-type p53, a growth suppressor protein. Cell cycle arrest induced by p53 may help survival of tsBN462 cells at 39.5 degrees C. Apoptosis was accelerated in SV40 large T antigen-transformed tsBN462 cells at 39.5 degrees C where SV40 large T antigen formed a complex with p53, implying that the apoptosis of tsBN462 cells at 39.5 degrees C occurred in a p53-independent manner. Our results suggest that CCG1/TAFII250 is required for the expression of factors regulating apoptosis.

Adenovirus E1B Proteins↗
PubMed1995

The NIMA kinase: a mitotic regulator in Aspergillus nidulans and vertebrate cells.

CDC2 has been shown to regulate entry into mitosis in eukaryotic cells. However, in Aspergillus nidulans, activation of CDC2 itself is not sufficient to trigger mitosis if another mitotic protein kinase, NIMA, is not activated. Superficially, NIMA and CDC2 have analogous functions and are regulated in a similar manner. NIMA activity is tightly regulated during the cell cycle. Overexpression of NIMA induces germinal vesicle breakdown in Xenopus oocytes and promotes premature entry into mitosis in all eukaryotic cells examined, whereas dominant-negative mutant NIMA causes a specific G2 arrest in Aspergillus nidulans and human cells, as is the case for CDC2. However, NIMA and CDC2 have quite distinct primary sequence substrate specificities. Furthermore, the regulatory mechanisms that govern the cell cycle-dependent abundance, activity and localization are largely intramolecular for NIMA but intermolecular for CDC2. More importantly, a NIMA-like pathway is also required for the G2/M transition in vertebrate cells. Thus, NIMA may represent a new essential eukaryotic cell cycle regulator, although its homologues in other species are yet to be identified.

Amino Acid Sequence↗
PubMed1995

Multiple serine phosphorylation sites on the 30 kDa TMV cell-to-cell movement protein synthesized in tobacco protoplasts.

p30, the protein required for cell-to-cell movement of tobacco mosaic virus (TMV), has a slightly reduced mobility on SDS-polyacrylamide gels when isolated by immunoprecipitation from TMV-infected protoplasts compared with that of p30 translated from viral RNA in vitro. Further investigation established a probable cause for the difference in mobility between the two: protoplasts incorporate [32P]orthophosphate into p30 at multiple sites, predominantly as phosphoserine. Tryptic peptide mapping reveals at least five internal phosphopeptides in p30, besides the C-terminal tryptic phosphopeptide already reported, involving at least two distinct domains of the protein (at residues 61-114 and residues 212-231), which may be substrates for different protein kinases. These structural results are consistent with a three-domain model for the TMV movement protein with two regulatory domains similar to that recently proposed on genetic grounds for dianthovirus movement proteins.

Peptide Mapping↗
PubMed1995

Redistribution of the CDK inhibitor p27 between different cyclin.CDK complexes in the mouse fibroblast cell cycle and in cells arrested with lovastatin or ultraviolet irradiation.

The cyclin-dependent kinase (CDK) inhibitor p27 binds and inhibits the kinase activity of several CDKs. Here we report an analysis of the behavior and partners of p27 in Swiss 3T3 mouse fibroblasts during normal mitotic cell cycle progression, as well as in cells arrested at different stages in the cycle by growth factor deprivation, lovastatin treatment, or ultraviolet (UV) irradiation. We found that the level of p27 is elevated in cells arrested in G0 by growth factor deprivation or contact inhibition. In G0, p27 was predominantly monomeric, although some portion was associated with residual cyclin A.Cdk2. During G1, all of p27 was associated with cyclin D1.Cdk4 and was then redistributed to cyclin A.Cdk2 as cells entered S phase. The loss of the monomeric p27 pool as cyclins accumulate in G1 is consistent with the in vivo and in vitro data showing that p27 binds better to cyclin.CDK complexes than to monomeric CDKs. In growing cells, the majority of p27 was associated with cyclin D1 and the level of p27 was significantly lower than the level of cyclin D1. In cells arrested in G1 with lovastatin, cyclin D1 was degraded and p27 was redistributed to cyclin A.Cdk2. In contrast to p21 (which is a p27-related CDK inhibitor and is induced by UV irradiation), the level of p27 was reduced after UV irradiation, but because cyclin D1 was degraded more rapidly than p27, there was a transient increase in binding of p27 to cyclin A.Cdk2. These data suggest that cyclin D1.Cdk4 acts as a reservoir for p27, and p27 is redistributed from cyclin D1.Cdk4 to cyclin A.Cdk2 complexes during S phase, or when cells are arrested by growth factor deprivation, lovastatin treatment, or UV irradiation. It is likely that a similar principle of redistribution of p27 is used by the cell in other instances of cell cycle arrest.

3T3 Cells↗
PubMed1995

How do protein kinases discriminate between serine/threonine and tyrosine? Structural insights from the insulin receptor protein-tyrosine kinase.

The eukaryotic protein kinases that directly phosphorylate proteins are divided into two major classes: those that phosphorylate tyrosine and those that phosphorylate serine and threonine. Until recently, the similarities between these two classes of enzymes, which now total more than 400, were based primarily on sequence alignments. A recent report of the structure of the kinase domain (IRK) of the insulin receptor protein-tyrosine kinase now allows the features of these two families to be compared at the structural level. We review here this first tyrosine-specific protein kinase structure, and compare and contrast it to the structure of the serine/threonine-specific cAMP-dependent protein kinase. Although the general fold of the polypeptide backbone is conserved as predicted, unique features at the IRK active site provide a basis for understanding the differences in specificity for the phosphate acceptor amino acid. The structure of this inactive, dephosphorylated protein-tyrosine kinase also defines for the first time how activation might be achieved.

Adenosine Triphosphate↗
PubMed1995

The nonreceptor protein-tyrosine kinase CSK complexes directly with the GTPase-activating protein-associated p62 protein in cells expressing v-Src or activated c-Src.

CSK is a predominantly cytosolic protein-tyrosine kinase (PTK) that negatively regulates Src family PTKs by phosphorylation of a conserved tyrosine near their C termini. Little is known about how CSK itself is regulated. On the basis of immunofluorescence studies, a model has been proposed that when c-Src is activated, it is redistributed to podosomes, in which substrates become phosphorylated, creating binding sites for CSK. CSK is recruited to these sites of c-Src activation via its SH2 and SH3 domains and is then in a position to downregulate c-Src activity (B. W. Howell and J. A. Cooper, Mol. Cell. Biol. 14:5402-5411, 1994). To identify phosphotyrosine (P.Tyr)-containing proteins that may mediate translocation of CSK due to c-Src activation, we have examined the whole spectrum of P.Tyr-containing proteins that associate with CSK in v-Src NIH 3T3 cells by anti-P.Tyr immunoblotting. Nine P.Tyr-containing proteins coimmunoprecipitated with CSK from v-Src NIH 3T3 cells. One of these, an approximately 62-kDa protein, also associated with CSK in NIH 3T3 cells treated with vanadate prior to lysis and in NIH 3T3 cells expressing an activated c-Src mutant. This 62-kDa protein was shown to be identical to the GTPase-activating protein (GAP)-associated p62 (GAP-A.p62) protein. The interaction between CSK and GAP-A.p62 could be reconstituted in vitro with glutathione S-transferase fusion proteins containing full-length CSK or the CSK SH2 domain. Furthermore, our data show that CSK interacts directly with GAP.A-p62 and that the complex between the two proteins is localized in subcellular membrane or cytoskeletal fractions. Our results suggest that GAP-A.p62 may function as a docking protein and may mediate translocation of proteins, including GAP and CSK, to membrane or cytoskeletal regions upon c-Src activation.

3T3 Cells↗
PubMed1995

Ankylosed spines are prone to fracture.

Fracture of an ankylosed spine is often overlooked. Because the force that damages an ankylosed spine is frequently slight, patients do not realize they are injured. Doctors can miss the fracture for the same reason and because patients already have a history of back pain. Plain radiographs sometimes fail to demonstrate the fracture site.

Adult↗
PubMed1995

Minimizing synkinesis during rehabilitation of the paralyzed face: preliminary assessment of a new small-movement therapy.

Neuromuscular rehabilitation can reduce the severity of chronic facial paralysis, but complete recovery is frequently impeded by synkinesis. We evaluated whether or not such synkinesis could be minimized by preventing its possible reinforcement during rehabilitation. We compared "standard" therapy, which uses the appearance of synkinesis to guide rehabilitation, with a new "small-movement" therapy, which uses smaller movements that should minimize possible subthreshold reinforcement of synkinesis. Ten subjects who had had facial paralysis for 0.5 to 27 years were randomly assigned to either therapy group. Blinded assessments were performed before and after ten 1-hour treatments given over a 1-month interval. Facial movements in both groups were significantly more symmetric after treatment. Although synkinesis tended to be reduced in the small-movement group, this reduction was not significant. The new therapy was at least as good as the standard one, and it may be better. Further studies are required to demonstrate this.

Adolescent↗
PubMed1995

Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification.

The eukaryotic protein kinases make up a large superfamily of homologous proteins. They are related by virtue of their kinase domains (also known as catalytic domains), which consist of approximately 250-300 amino acid residues. The kinase domains that define this group of enzymes contain 12 conserved subdomains that fold into a common catalytic core structure, as revealed by the 3-dimensional structures of several protein-serine kinases. There are two main subdivisions within the superfamily: the protein-serine/threonine kinases and the protein-tyrosine kinases. A classification scheme can be founded on a kinase domain phylogeny, which reveals families of enzymes that have related substrate specificities and modes of regulation.

Amino Acid Sequence↗
PubMed1994

The differential localization of human cyclins A and B is due to a cytoplasmic retention signal in cyclin B.

We have shown previously that human cyclins A and B1 are localized differentially in the cell during interphase; cyclin A is nuclear and cyclin B1 is a cytoplasmic protein. To understand the basis of this difference we created deletion mutants and various chimeras between the two types of cyclin and expressed them in tissue culture cells by transient transfection. We find that the N-terminus of cyclin B1 contains a 42 amino acid region that is sufficient to retain the normally nuclear cyclin A in the cytoplasm. Conversely, deleting the cytoplasmic retention signal region from cyclin B1 causes the protein to become nuclear. Although the cytoplasmic retention signal region is outside the cyclin box, its sequence is well conserved in human cyclin B2, and is both necessary and sufficient to keep cyclin B2 in the cytoplasm. Thus we propose that the subcellular distribution of the B-type cyclins is determined primarily by a small region of the N-terminus which targets the cyclin--CDK complexes to particular structures in the cytoplasm.

Amino Acid Sequence↗
PubMed1994

p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21.

Using a yeast interaction screen to search for proteins that interact with cyclin D1-Cdk4, we identified a 27 kDa mouse protein related to the p21 cyclin-Cdk inhibitor. p27 interacts strongly with D-type cyclins and Cdk4 in vitro and more weakly with cyclin E and Cdk2. In mouse fibroblasts, p27 is associated predominantly with cyclin D1-Cdk4. Recombinant p27 is a potent inhibitor of cyclin D1-Cdk4 and cyclin A-Cdk2 protein kinase activity and a weaker inhibitor of cyclin B1-Cdc2. Overexpression of p27 in Saos-2 cells causes G1 arrest. p27 protein levels do not change as serum-stimulated quiescent mouse fibroblasts progress through the cell cycle. p27 is identical to p27Kip1, a cyclin-Cdk inhibitor present in TGF beta-treated cells. p27 has the hallmarks of a negative regulator of G1 progression and may mediate TGF beta-induced G1 arrest.

3T3 Cells↗
PubMed1994

Phosphorylation of receptor protein-tyrosine phosphatase alpha on Tyr789, a binding site for the SH3-SH2-SH3 adaptor protein GRB-2 in vivo.

Receptor protein-tyrosine phosphatase alpha (RPTP alpha) is a transmembrane protein with a short extracellular domain (123 amino acids) and two cytoplasmically localized protein-tyrosine phosphatase (PTP) domains. Here we report that RPTP alpha is constitutively phosphorylated on tyrosine in NIH 3T3 mouse fibroblasts. The in vivo tyrosine phosphorylation site was localized to the C-terminus of RPTP alpha by phosphopeptide mapping experiments using in vivo and in vitro 32P-labeled RPTP alpha. The identity of this site as Tyr789, located five residues from the C-terminus, was confirmed by site-directed mutagenesis. Transient overexpression of c-Src together with RPTP alpha in human embryonic kidney 293 cells increased phosphorylation of Tyr789, suggesting that c-Src may phosphorylate RPTP alpha in vivo. RPTP alpha had autodephosphorylation activity in vitro. When expressed in 293 cells the level of Tyr789 phosphorylation was higher in a non-functional mutant of RPTP alpha than in wild type RPTP alpha, indicating that RPTP alpha may have autodephosphorylation activity in vivo as well. The sequence on the C-terminal side of Tyr789 (YANF) fits the consensus binding site for the SH3-SH2-SH3 adaptor protein GRB2 (YXNX). We show that RPTP alpha, but not a mutant of RPTP alpha with a Tyr-->Phe mutation at position 789, bound to GRB2 in vitro. In addition, RPTP alpha co-immunoprecipitated with GRB2 from NIH 3T3 cells, demonstrating that GRB2 bound to RPTP alpha in vivo. The guanine nucleotide releasing factor for the Ras GTPase, Son of sevenless (Sos), which associates with GRB2 via its SH3 domains, was not detected in RPTP alpha immunoprecipitates. Our results suggest a role for RPTP alpha in attenuation of GRB2-mediated signaling.

3T3 Cells↗
PubMed1994

atpk1, a novel ribosomal protein kinase gene from Arabidopsis. I. Isolation, characterization, and expression.

Two protein kinase genes (atpk1 and atpk2) were isolated from Arabidopsis thaliana genomic DNA with a probe generated by polymerase chain reaction (PCR) using oligonucleotide primers encoding conserved eukaryotic protein kinase sequences. atpk1 and atpk2 are organized in a head-to-tail tandem array on chromosome 3 and have about 80% nucleotide sequence identity. atpk1 encodes a hydrophilic polypeptide of 465 amino acids, M(r) = 52,554. The centrally located catalytic domain contains all the conserved residues characteristic of eukaryotic protein kinases, with greatest similarity to the catalytic domains of 70-kDa ribosomal S6 protein kinase, protein kinase C, and protein kinase A. The C-terminal 75 residues also show homology to protein kinase C and S6 protein kinase. In contrast, the N-terminal 130 residues have no homology to any known protein, and thus may represent a new class of protein kinase regulatory domain. Other motifs found in the Atpk1 protein include two putative autophosphorylation sites, a pseudosubstrate site, two acidic domains, a lysine-rich domain, and two putative PEST sequences, which may contribute to the regulation of protein kinase activity. RNA-blot hybridization showed that atpk1 encoded a 1.8-kb mRNA. Analysis of atpk1 promoter/beta-glucuronidase reporter gene fusions in transgenic plants showed that atpk1 was expressed in all tissues and at all developmental stages, with the strongest expression observed in metabolically active tissues, suggesting that atpk1 is involved in the control of plant growth and development. The first intron of atpk1 functions as an enhancer in atpk1 expression.

Amino Acid Sequence↗
PubMed1994

atpk1, a novel ribosomal protein kinase gene from Arabidopsis. II. Functional and biochemical analysis of the encoded protein.

The Arabidopsis Atpk1 protein expressed in insect cells and plant cells exhibited multiple sizes consisting mainly of two doublets: p70 (68 and 70 kDa) and p85 (82 and 85 kDa). Extraction of p85 from cells required the presence of SDS, suggesting that p85 is associated with less soluble subcellular components. p70 was extracted by nonionic detergent without SDS, indicating that this form is cytoplasmic. p70 expressed in either Arabidopsis or insect cells underwent serine-specific autophosphorylation, indicating that Atpk1 is a protein-serine kinase. A point mutation (lysine 163 to arginine) in the ATP-binding site of the catalytic domain substantially diminished activity when expressed in insect cells. A 14-kDa protein (p14) was co-immunoprecipitated with p70 from insect cells expressing wild-type Atpk1 and was phosphorylated in immune complex kinase assays with Atpk1, suggesting it is a homolog of a natural substrate of Atpk1. Two plant ribosomal proteins (14 and 16 kDa) can be phosphorylated by the Atpk1 protein kinase, and we propose that Atpk1 is a novel ribosomal protein kinase. A 60-kDa form of Atpk1 derived from the insect cell-expressed p70 was more highly phosphorylated than p70 in in vitro kinase assays, suggesting a negative regulatory domain can be removed by proteolysis.

Animals↗
PubMed1994

Identification of TrkB autophosphorylation sites and evidence that phospholipase C-gamma 1 is a substrate of the TrkB receptor.

The TrkB receptor protein-tyrosine kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3. In response to brain-derived neurotrophic factor and neurotrophin-3 treatment, TrkB expressed exogenously in Rat-2 cells is rapidly phosphorylated on tyrosine residues. At least 2 regions of TrkB contain phosphorylated tyrosines. The major sites of autophosphorylation are in the region containing Tyr-670, Tyr-674, and Tyr-675, which lies in the kinase domain and corresponds by sequence homology to the Tyr-416 autophosphorylation site in p60c-Src. Tyr-785, which lies just to the COOH-terminal side of the kinase domain in a relatively short tail characteristic of the Trk family of protein-tyrosine kinase receptors, is also phosphorylated in response to neurotrophin-3 treatment. The sequence around Tyr-785 fits a consensus sequence for binding phospholipase C-gamma 1. The simplest interpretation of these results is that, in response to neurotrophin binding, at least two and perhaps all three of the tyrosines in the Tyr-670/674/675 region are autophosphorylated independently, and Tyr-785 is autophosphorylated in vivo. Following activation of TrkB, phospholipase C-gamma 1 is phosphorylated on Tyr-783, Tyr-771, and Tyr-1254. Phospholipase C-gamma 1 also forms a complex with TrkB in response to neurotrophin-3 treatment, consistent with the possibility that one of the TrkB autophosphorylation sites provides a binding site for the phospholipase C-gamma 1 SH2 domains, as is the case for other receptor protein-tyrosine kinases. We conclude that phospholipase C-gamma 1 is directly phosphorylated by TrkB. Since phosphorylation of Tyr-783 and Tyr-1254 results in activation of phospholipase C-gamma 1, we predict that neurotrophin-3 leads to activation of phospholipase C-gamma 1 following binding to TrkB in Rat-2 cells.

Amino Acid Sequence↗