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T M Roberts

Publications and source records attributed to T M Roberts.

At least 55 records · Page 3Linked to original sources
PubMed1997

Regulation of glycogen utilization, but not glucose utilization, by precontraction glycogen levels in vascular smooth muscle.

These experiments were designed to determine whether glycogenolysis was influenced by the glycogen concentration of vascular smooth muscle. Segments of hog carotid artery smooth muscle were allowed to synthesize variable amounts of 1-[13C]glucosyl units of glycogen. Artery segments were then isometrically contracted in the presence of 2-[13C]glucose. Prior to and after isometric contraction, measurements were made of tissue glycogen content and superfusate glucose and lactate concentrations. 2-[13C]Lactate and 3-[13C]lactate peak intensities in the superfusate were measured using 13C-NMR spectroscopy. The tissue glycogen content decreased exponentially during the 4.5 h of isometric contraction (R2 = 0.990), despite more than a 3-fold range of glycogen concentration prior to contraction. The extent of glycogen utilization during a 3 h isometric contraction varied linearly with the precontraction glycogen concentration (R2 = 0.727). Lactate production specifically from glycogen breakdown increased with an increase in precontraction glycogen concentration (R2 = 0.620). During a 3 h isometric contraction neither the glucose utilization (R2 = 0.007) nor lactate production specifically produced from glucose (R2 = 0.00002) varied with the precontraction glycogen concentration. It is concluded that the rate of glycogenolysis is determined by the content of glycogen during prolonged contractions. In addition, precontraction glycogen levels influence the pathway for glycogen utilization but not the pathway for glucose utilization. Therefore, glycolysis and glycogenolysis behave independently in vascular smooth muscle.

Animals↗
PubMed1997

DnaJ/hsp40 chaperone domain of SV40 large T antigen promotes efficient viral DNA replication.

The amino-terminal domain of SV40 large tumor antigen (TAg) is required for efficient viral DNA replication. However, the biochemical activity associated with this domain has remained obscure. We show here that the amino-terminal domain of TAg shares functional homology with the J-domain of DnaJ/hsp40 molecular chaperones. DnaJ proteins function as cofactors by regulating the activity of a member of the 70-kD heat shock protein family. Genetic analyses demonstrated that amino-terminal sequences of TAg comprise a novel J-domain that mediates a specific interaction with the constitutively expressed hsc70 and show that the J-domain is also required for efficient viral DNA replication in vivo. Furthermore, we demonstrated that the J-domain of two human DnaJ homologs, HSJ1 or DNAJ2, could substitute functionally for the amino-terminus of TAg in promoting viral DNA replication. Together, our findings suggest that TAg uses its J-domain to support SV40 DNA replication in a manner that is strikingly similar to the use of Escherichia coli DnaJ by bacteriophage lambda in DNA replication. However, TAg has evolved a more efficient strategy of DNA replication through an intrinsic J-domain to associate directly with a partner chaperone protein. Our observations provide evidence of a role for chaperone proteins in the process of eukaryotic DNA replication.

Amino Acid Sequence↗
PubMed1997

Erythropoietin activates Raf1 by an Shc-independent pathway in CTLL-EPO-R cells.

Stimulation of the erythropoietin receptor (EPO-R) or the interleukin-2 receptor (IL-2-R) by their respective ligands has been reported to activate tyrosine phosphorylation of the cytoplasmic protein, Shc. We have recently characterized a cell line, CTLL-EPO-R, that contains functional cell-surface receptors for both EPO and IL-2. Although stimulation with IL-2 or IL-15 resulted in the rapid, dose-dependent tyrosine phosphorylation of Shc, stimulation with EPO failed to activate Shc. EPO, IL-2, and IL-15 activated the tyrosine phosphorylation of the adaptor protein, Shp2, and the association of Shp2/Grb2/cytokine receptor complexes. In addition, EPO, IL-2, and IL-15 activated Raf1 and ERK2, demonstrating that the Raf1/MEK/MAP kinase pathway was activated. These results indicate that multiple biochemical pathways are capable of conferring a mitogenic signal in CTLL-EPO-R. EPO can activate the Raf1/MEK/MAP kinase pathway via Shc-dependent or Shc-independent pathways, and Shc activation is not required for EPO-dependent cell growth in CTLL-EPO-R.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Differential regulation of glucose and glycogen metabolism in vascular smooth muscle by exogenous substrates.

The aim of this study was to determine whether the pathways of glycolysis and glycogenolysis can be independently modulated by the provision of acetate or pyruvate as exogenous substrates. Hog carotid artery segments were allowed to replete glycogen stores to over 6 micromol/g of new 13C-labeled glycogen by incubation at 37 degrees C with 5 mM [1-13C]glucose for 6-16 h and then were isometrically contracted for 3 h with 80 mM KCl in the presence of 5 mM [2-13C]glucose and either 2 mM sodium acetate or 5 mM sodium pyruvate. Measurements were made of total lactate production, glucose utilization, glycogen utilization, isometric force, [2-13C]lactate and [3-13C]lactate production. Compared to experiments with glucose as the sole exogenous substrate, provision of pyruvate significantly decreased glucose utilization (by 28%) but insignificantly decreased glycogen utilization. In contrast, provision of acetate resulted in a statistically insignificant decrease in glucose utilization (by 23%) and an increase in glycogen utilization (by 20%). The fraction of [3-13C]pyruvate derived from glycogen that was converted to [3-13C]lactate was significantly decreased in the presence of acetate despite the enhanced glycogen utilization. Despite these alterations in cellular energy balance, isometric force generation and maintenance was similar for all experimental groups. This differential regulation of glycolysis and glycogenolysis may either reflect the compartmentation of these pathways or suggest a novel regulation of carbohydrate metabolism in vivo.

Acetates↗
PubMed1997

A PDGF-regulated immediate early gene response initiates neuronal differentiation in ventricular zone progenitor cells.

When exposed to platelet-derived growth factor (PDGF), uncommitted neuroepithelial cells from the developing cortex of embryonic day 14 (E14) rats develop into neurons. Outward signs of the neuronal phenotype are not observed for 4 days following exposure to PDGF. However, only a brief (2-3 hr) period of PDGF receptor activation is required to initiate neuronal development. During the window of receptor activation, RNA synthesis is essential, but protein synthesis is not. These observations indicate that specification of neuronal fate is mediated by an immediate early gene response.

Animals↗
PubMed1997

The DnaJ domain of polyomavirus large T antigen is required to regulate Rb family tumor suppressor function.

Tumor suppressors of the retinoblastoma susceptibility gene family regulate cell growth and differentiation. Polyomavirus large T antigens (large T) bind Rb family members and block their function. Mutations of large T sequences conserved with the DnaJ family affect large T binding to a cellular DnaK, heat shock protein 70. The same mutations abolish large T activation of E2F-containing promoters and Rb binding-dependent large T activation of cell cycle progression. Cotransfection of a cellular DnaJ domain blocks wild-type large T action, showing that the connection between the chaperone system and tumor suppressors is direct. Although they are inactive in assays dependent on Rb family binding, mutants in the J region retain the ability to associate with pRb, p107, and p130. This suggests that binding of Rb family members by large T is not sufficient for their inactivation and that a functional J domain is required as well. This work connects the DnaJ and DnaK molecular chaperones to regulation of tumor suppressors by polyomavirus large T.

3T3 Cells↗
PubMed1997

Mutation in the Jak kinase JH2 domain hyperactivates Drosophila and mammalian Jak-Stat pathways.

The Jak (Janus) family of nonreceptor tyrosine kinases plays a critical role in cytokine signal transduction pathways. In Drosophila melanogaster, the dominant hop(Tum-l) mutation in the Hop Jak kinase causes leukemia-like and other developmental defects. Previous studies have suggested that the Hop(Tum-l) protein might be a hyperactive kinase. Here, we report on the new dominant mutation hop(T42), which causes abnormalities that are similar to but more extreme than those caused by hop(Tum-l). We determined that Hop(T42) contains a glutamic acid-to-lysine substitution at amino acid residue 695 (E695K). This residue occurs in the JH2 (kinase-like) domain and is conserved among all Jak family members. We determined that Hop(Tum-1) and Hop(T42) both hyperphosphorylated and hyperactivated D-Stat when overexpressed in Drosophila cells. Moreover, we found that the hop(T42) phenotype was partially rescued by a reduction of wild-type D-stat activity. Finally, generation of the corresponding E695K mutation in murine Jak2 resulted in increased autophosphorylation and increased activation of Stat5 in COS cells. These results demonstrate that the mutant Hop proteins do indeed have increased tyrosine kinase activity, that the mutations hyperactivate the Hop-D-Stat pathway, and that Drosophila is a relevant system for the functional dissection of mammalian Jak-Stat pathways. Finally, we propose a model for the role of the Hop-D-Stat pathway in Drosophila hematopoiesis.

Amino Acid Sequence↗
PubMed1997

Inactivation of pRB-related proteins p130 and p107 mediated by the J domain of simian virus 40 large T antigen.

Inactivation of the retinoblastoma tumor suppressor protein (pRB) contributes to tumorigenesis in a wide variety of cancers. In contrast, the role of the two pRB-related proteins, p130 and p107, in oncogenic transformation is unclear. The LXCXE domain of simian virus 40 large T antigen (TAg) specifically binds to pRB, p107, and p130. We have previously shown that the N terminus and the LXCXE domain of TAg cooperate to alter the phosphorylation state of p130 and p107. Here, we demonstrate that TAg promotes the degradation of p130 and that the N terminus of TAg is required for this activity. The N terminus of TAg has homology to the J domain of the DnaJ family of molecular chaperone proteins. Mutants with mutations in the J-domain homology region of TAg are defective for altering p130 and p107 phosphorylation and for p130 degradation. A heterologous J-domain from a human DnaJ protein can functionally substitute for the N terminus of TAg in the effect on p107 and p130 phosphorylation and p130 stability. We further demonstrate that the J-domain homology region of TAg confers a growth advantage to wild-type mouse embryo fibroblasts (MEFs) but is dispensable in the case of MEFs lacking both p130 and p107. This indicates that p107 and p130 have overlapping growth-suppressing activities whose inactivation is mediated by the J domain of TAg.

Amino Acid Sequence↗
PubMed1996

The catalytic domain of pp56(lck), but not its regulatory domain, is sufficient for inducing IL-2 production.

The lymphoid src kinase pp56(lck) has been shown to be essential for the induction of different T lymphocyte responses, including CD4-mediated enhancement of Ag-induced T cell activation, early T cell differentiation, induction of IL-2 production, and cytotoxicity. It is assumed that pp56(lck) acts on these processes by phosphorylating substrates. However, it has been recently reported that the NH2 regulatory domain is sufficient to mediate CD4 accessory function. In this report we address the contribution of the regulatory and catalytic domains of pp56(lck) to another function of this enzyme independent of CD4: TCR-induced IL-2 production. Two pp56(lck) mutants lacking either the entire catalytic domain or the entire NH2 regulatory domain were generated, and their abilities to trigger transactivation of the TCR-regulated nuclear factor of activated T cells (NF-AT) region of the IL-2 promoter were compared. Only the catalytic, but not the NH2 regulatory, domain of pp56(lck) was able to induce NF-AT region transactivation on its own and to cooperate with other intracellular signals to trigger this response. Moreover, the catalytic domain of pp56(lck) was able to induce IL-2 cytokine production to an extent similar to that of wild-type pp56(lck). We conclude that different domains of the pp56(lck) molecule contribute to regulate distinct biologic functions. In fact, while the NH2 regulatory domain is sufficient to mediate CD4 accessory function, we show here that the catalytic domain of pp56(lck) is sufficient for induction of IL-2 production, mimicking TCR ligation.

Allosteric Site↗
PubMed1996

2.5 A resolution crystal structure of the motile major sperm protein (MSP) of Ascaris suum.

We have determined the structure of the Ascaris major sperm protein (MSP) to 2.5 A resolution using X-ray crystallography. The MSP polypeptide chain has an immunoglobulin-like fold based on a seven-stranded beta sandwich. In two strands, cis-proline residues impart distinctive kinks, and overall the structure most closely resembles that of the N-terminal domain of the bacterial chaperonin, PapD. In the C2 crystal form which we have solved here, two MSP chains are tightly associated in the asymmetric unit and are related by a non-crystallographic 2-fold rotation axis. This arrangement almost certainly represents the MSP dimer that is present in solution. Additionally, the arrangement of two MSP dimers at one of the crystallographic 2-fold axes in the 215 A unit cell suggests a possible mode for the assembly of MSP into the filaments which promote cell movement. This dimer-dimer association is based on a beta sheet extension mechanism between adjoining MSP monomers which resembles the interaction between PapD and its protein substrate.

Amino Acid Sequence↗
PubMed1996

The cytokine-activated tyrosine kinase JAK2 activates Raf-1 in a p21ras-dependent manner.

JAK2, a member of the Janus kinase superfamily was found to interact functionally with Raf-1, a central component of the ras/mitogen-activated protein kinase signal transduction pathway. Interferon-gamma and several other cytokines that are known to activate JAK2 kinase were also found to stimulate Raf-1 kinase activity toward MEK-1 in mammalian cells. In the baculovirus coexpression system, Raf-1 was activated by JAK2 in the presence of p21ras. Under these conditions, a ternary complex of p21ras, JAK2, and Raf-1 was observed. In contrast, in the absence of p21ras, coexpression of JAK2 and Raf-1 resulted in an overall decrease in the Raf-1 kinase activity. In addition, JAK2 phosphorylated Raf-1 at sites different from those phosphorylated by pp60v-src. In mammalian cells treated with either erythropoietin or interferon-gamma, a small fraction of Raf-1 coimmunoprecipitated with JAK2 in lysates of cells in which JAK2 was activated as judged by its state of tyrosine phosphorylation. Taken together, these data suggest that JAK2 and p21ras cooperate to activate Raf-1.

Animals↗
PubMed1996

Platelet-derived growth factor-induced formation of tensin and phosphoinositide 3-kinase complexes.

Tensin is an SH2 domain-containing cytoskeletal protein that binds to and caps actin filaments. Investigation of signal transduction mechanisms associated with tensin revealed the presence of phosphoinositide 3-kinase (PI 3-kinase) activity in tensin immunoprecipitates from platelet-derived growth factor-treated cells. Association of PI 3-kinase activity with tensin was transitory, and the amount of activity was approximately 1% of the total PI 3-kinase activity found in anti-phosphotyrosine (anti-pY) immunoprecipitates. In vitro, PI 3-kinase activity associated with the SH2 domain of tensin in a platelet-derived growth factor-dependent manner. The optimal phosphopeptide binding specificity of the SH2 domain of tensin was determined to be phospho-Y (E or D), N, (I, V, or F). Synthetic phosphopeptides containing the sequence YENI could specifically block the association of PI 3-kinase activity with tensin in a dose-dependent manner. These results suggest that PI 3-kinase interacts with the cytoskeleton via the SH2 domain of tensin and may play an important role in platelet-derived growth factor-induced cytoskeletal reorganization that is concomitant with cell migration and proliferation.

Amino Acid Sequence↗
PubMed1996

Intramolecular interactions of the regulatory domains of the Bcr-Abl kinase reveal a novel control mechanism.

BACKGROUND: The Abl nonreceptor tyrosine kinase is implicated in a range of cellular processes and its transforming variants are involved in human leukemias. The N-terminal regulatory region of the Abl protein contains Src homology domains SH2 and SH3 which have been shown to be important for the regulation of its activity in vivo. These domains are often found together in the same protein and biochemical data suggest that the functions of one domain can be influenced by the other. RESULTS: We have determined the crystal structure of the Abl regulatory region containing the SH3 and SH2 domains. In general, the individual domains are very similar to those of previously solved structures, although the Abl SH2 domain contains a loop which is extended so that one side of the resulting phosphotyrosine-binding pocket is open. In our structure the protein exists as a monomer with no intermolecular contacts to which a biological function may be attributed. However, there is a significant intramolecular contact between a loop of the SH3 domain and the extended loop of the SH2 domain. This contact surface includes the SH2 loop segment that is responsible for binding the phosphate moiety of phosphotyrosine-containing proteins and is therefore critical for orienting peptide interactions. CONCLUSIONS: The crystal structure of the composite Abl SH3-SH2 domain provides the first indication of how SH2 and SH3 domains communicate with each other within the same molecule and why the presence of one directly influences the activity of the other. This is the first clear evidence that these two domains are in contact with each other. The results suggest that this direct interaction between the two domains may affect the ligand binding properties of the SH2 domain, thus providing an explanation for biochemical and functional data concerning the Bcr-Abl kinase.

Amino Acid Sequence↗
PubMed1996

The motile major sperm protein (MSP) from Ascaris suum is a symmetric dimer in solution.

The major sperm protein (MSP) of Ascaris suum mediates amoeboid motility by forming an extensive intermeshed system of cytoskeletal filaments analogous to that formed by actin in many amoeboid cells. We have used a combination of biochemical and NMR methods to show that, in contrast to actin, MSP exist in solution as a symmetrical dimer. This result has important implications for the mechanism of both MSP filament assembly and the recognition of different MSP isoforms in vivo.

Actin Cytoskeleton↗
PubMed1996

A strategy for screening anti-tumor drugs utilizing oncogenes encoded in retroviral vectors.

A novel strategy for isolating potential anti-tumor drugs is presented. It is predicated on the idea that future anti-tumor drugs will be specific inhibitors of the signal-transduction pathways responsible for cell proliferation. Briefly, retroviral vectors are used to introduce focus-forming oncogenes into a test population of target cells, which are grown to confluence and treated with signal-transduction inhibitors. The inhibitors are screened for the ability to suppress the development of transformed foci without killing the confluent monolayer of non-transformed quiescent cells. For this work, a panel of inhibitors was first screened against the oncogene ras. The protein kinase C (PKC) inhibitor CGP 41251 and the protein tyrosine kinase (PTK) inhibitor CGP 45047 suppressed ras-induced focus formation and left a viable monolayer of quiescent cells. Focus inhibition was reversible; conversely, drug addition to developing foci retarded further expansion. CGP 41251 generally blocked proliferation of ras or control cells, suggesting that oncogenes cannot substitute for PKC. PTK inhibitors erbstatin and CGP 520 and phosphatase inhibitor okadaic acid failed to inhibit focus formation at concentrations toxic to the monolayer. Lavendustin A and CGP 47778A showed neither focus inhibition nor toxicity. In the complementary screen, a single inhibitor (CGP 41251) was tested against several oncogenes, including src, raf and polyomavirus middle T antigen. Focus formation by all oncogenes was suppressed. The strategy has several advantages over current drug-screening assays, and it can be adapted to large-scale screening with many drugs and many oncogenes.

3T3 Cells↗
PubMed1996

Reconstitution in vitro of the motile apparatus from the amoeboid sperm of Ascaris shows that filament assembly and bundling move membranes.

We have developed an in vitro motility system from Ascaris sperm, unique amoeboid cells that use filament arrays composed of major sperm protein (MSP) instead of an actin-based apparatus for locomotion. Addition of ATP to sperm extracts induces formation of fibers approximately 2 microns in diameter. These fibers display the key features of the MSP cytoskeleton in vivo. Each fiber consists of a meshwork of MSP filaments and has at one end a vesicle derived from the plasma membrane at the leading edge of the cell. Fiber growth is due to filament assembly at the vesicle; thus, fiber elongation results in vesicle translocation. This in vitro system demonstrates directly that localized polymerization and bundling of filaments can move membranes and provides a powerful assay for evaluating the molecular mechanism of amoeboid cell motility.

Amoeba↗
PubMed1995

Identification of Lck-binding elements in tip of herpesvirus saimiri.

A protein called Tip (tyrosine kinase interacting protein) of herpesvirus saimiri associates with Lck in virus-transformed human T cells and is an in vitro substrate for Lck kinase. Mutational analyses of a GST-Tip fusion protein revealed that binding to Lck requires putative SH3 binding sequences and a sequence homologous to the carboxyl terminus of Src-related kinases. These sequences are referred to as SH3-Binding (SH3B) and C-terminal Src-related Kinase Homology (CSKH) elements. Peptide fragments as short as 37 amino acids containing both SH3B and CSKH elements were sufficient to form a stable complex with Lck in vitro. Furthermore, these same sequences of Tip were necessary for in vivo association with Lck when Tip and Lck were expressed transiently in COS-1 cells or stably in Rat-1 cell lines. These results demonstrate that the CSKH element of Tip participates in the binding of sequences within Lck. Tip of herpesvirus saimiri has apparently acquired such CSKH and SH3B elements for the purpose of targeting cellular protein kinases. The interaction of Tip with Lck may influence Lck kinase activity or its binding to other cellular proteins and thereby alter Lck function in T cells infected by h. saimiri.

Amino Acid Sequence↗