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T M Roberts

Publications and source records attributed to T M Roberts.

At least 109 records · Page 6Linked to original sources
PubMed1992

Tyrosine kinase triggering in thymocytes undergoing positive selection.

Developing T cells undergo distinct selection processes that determine the T cell receptor (TcR) repertoire. One of these processes is positive selection. Positive selection involves the differentiation to mature T cells of thymocytes bearing TcR capable of recognizing antigens in the context of self-major histocompatibility complex (MHC) molecules. To study the potential involvement of tyrosine phosphorylation in the mechanism of positive selection, we have analyzed the activities of the tyrosine kinases pp56lck and pp59fyn in thymocytes expressing a unique TcR specific for HY antigen+ H-2 Db. Thymocytes undergoing positive selection displayed higher kinase specific activities of pp56lck and pp59fyn than nonselecting thymocytes. Furthermore, these increases in kinase activities were found only in the CD4+CD8+ subpopulation of thymocytes, where the selection process is believed to occur. These data suggest that tyrosine phosphorylation is part of the intracellular signals involved in MHC class I-driven positive selection from CD4+CD8+ immature thymocytes to CD4-CD8+ mature cells.

Animals↗
PubMed1992

Polyomavirus middle T-antigen NPTY mutants.

A polyomavirus middle T-antigen (MTAg) mutant containing a substitution of Leu for Pro at amino acid 248 has previously been described as completely transformation defective (B. J. Druker, L. Ling, B. Cohen, T. M. Roberts, and B. S. Schaffhausen, J. Virol. 64:4454-4461, 1990). This mutant had no alterations in associated proteins or associated kinase activities compared with wild-type MTAg. Pro-248 lies in a tetrameric sequence, NPTY, which is reminiscent of the so-called NPXY sequence in the low-density-lipoprotein receptor. In the low-density-lipoprotein receptor, mutations in the NPXY motif but not in the surrounding amino acids abolish receptor function, apparently by decreasing receptor internalization (W. Chen, J. L. Goldstein, and M. S. Brown, J. Biol. Chem. 265:3116-3123, 1990). To determine whether this sequence represents a functional motif in MTAg as well, a series of single amino acid substitutions was constructed in this region of MTAg. All of the mutations of N, P, T, or Y, including the relatively conservative substitution of Ser for Thr at amino acid 249, resulted in a transformation-defective MTAg, whereas mutations outside of this sequence allowed mutants to retain near-wild-type transformation capabilities. Transformation-defective mutants with mutations in the NPTY region behaved similarly to the mutant with the original Pro-248-to-Leu-248 mutation when assayed for associated proteins and activities in vitro; that is, they retained a full complement of wild-type activities and associated proteins. Further, insertion of the tetrameric sequence NPTY downstream of the mutated motif restored transforming abilities to these mutants. Thus, the tetrameric sequence NPTY in MTAg appears to represent a well-defined functional motif of MTAg.

1-Phosphatidylinositol 4-Kinase↗
PubMed1992

The third subunit of protein phosphatase 2A (PP2A), a 55-kilodalton protein which is apparently substituted for by T antigens in complexes with the 36- and 63-kilodalton PP2A subunits, bears little resemblance to T antigens.

The small and middle T (tumor) antigens of polyomavirus have been shown previously to associate with the 36-kDa catalytic subunit and the 63-kDa regulatory subunit of protein phosphatase type 2A, apparently substituting for a normal third 55-kDa regulatory subunit (D.C. Pallas, L.K. Shahrik, B.L. Martin, S. Jaspers, T.B. Miller, D.L. Brautigan, and T.M. Roberts, Cell 60:167-176, 1990). To facilitate a comparison of the normal regulatory subunit and T antigens, we isolated a 2.14-kb cDNA clone encoding this 55-kDa subunit from a rat liver library. Using a probe from the coding region of this gene, we detected a major 2.4-kb mRNA transcript in liver and muscle RNAs. The 55-kDa protein phosphatase 2A subunit purified from rat skeletal muscle generates multiple species when analyzed on two-dimensional gels. Transcription and translation of the clone in vitro produced a full-length protein that comigrated precisely on two-dimensional gels with three of these species, indicating that the 55-kDa protein is apparently modified similarly in vivo and in reticulocyte lysates. Additional species in the purified preparation were not found in the translate, suggesting that there are probably two or more isoforms of this protein in rat muscle. Somewhat surprisingly, there was no clear homology with T-antigen amino acid sequences.

Amino Acid Sequence↗
PubMed1992

Transformation-defective mutants of polyomavirus middle T antigen associate with phosphatidylinositol 3-kinase (PI 3-kinase) but are unable to maintain wild-type levels of PI 3-kinase products in intact cells.

Middle T antigen (MT) of polyomavirus causes transformation by associating with a number of cellular proteins. The association with and activation of two such proteins, phosphatidylinositol 3-kinase (PI 3-kinase) and pp60c-src, appears to be necessary for transformation by MT. The tyrosine kinase activity of MT-associated pp60c-src is significantly increased when assayed in vitro, and levels of phosphotyrosine-containing proteins are elevated in vivo. Similarly, levels of the PI 3-kinase products phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] and phosphatiylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] are constitutively elevated in MT-transformed cells. However, the formation of a complete MT/cellular protein complex and the activation of tyrosine kinase are not sufficient to cause transformation, since the transformation-defective mutants 248m and dl1015 associate with all wild-type MT-associated proteins, including PI 3-kinase and pp60c-src, and neither mutant appears to be defective in MT-associated tyrosine kinase activity. Studies presented here compared (i) the amount of PI 3-kinase activity associated with the MT complex and (ii) levels of [3H]inositol incorporation into PI 3-kinase products in cells expressing mutant or wild-type MT. The results show that dl1015 is defective in both assays, whereas 248m is defective only for incorporation of [3H]inositol into PI(3,4,5)P2 and PI(3,4)P3. These findings identify a biochemical defect in the 248m mutant and corroborate previous results correlating transformation and elevated levels of PI 3-kinase products in vivo. In addition, they indicate that PI 3-kinase product levels are affected by factors other than simply the amount of PI 3-kinase activity associated with the MT complex.

1-Phosphatidylinositol 4-Kinase↗
PubMed1992

Structure and macromolecular assembly of two isoforms of the major sperm protein (MSP) from the amoeboid sperm of the nematode, Ascaris suum.

Ascaris sperm are amoeboid cells that crawl by extending pseudopods. Although amoeboid motility is generally mediated through an actin-based cytoskeleton, Ascaris sperm lack this system. Instead, their major sperm protein (MSP) forms an extensive filament system that appears to fulfil this function. Because their motility appears to be essentially the same as that of their actin-rich counterparts, Ascaris sperm offer a simple alternative system for investigation of the molecular mechanism of amoeboid movement. To examine the structure and composition of the cytoskeleton, we stabilized the extremely labile native MSP filaments by detergent lysis of sperm in the presence of either glutaraldehyde or polyethylene glycol (PEG). Biochemical analysis showed that the cytoskeleton contained two isoforms of MSP, designated alpha- and beta-, that we purified and sequenced. Both contain 126 amino acids and have an acetylated N-terminal alanine, but differ at four residues so that alpha-MSP is 142 Da larger and 0.6 pH unit more basic than beta-MSP. Neither isoform shares sequence homology with other cytoskeletal proteins. In ethanol, 2-methyl-2,4-pentanediol (MPD), and other water-miscible alcohols each isoform assembled into filaments 10 nm wide with a characteristic substructure repeating axially at 9 nm. These filaments were indistinguishable from native fibers isolated from detergent-lysed sperm. Pelleting assays indicated a critical concentration for assembly of 0.2 mM for both isoforms in 30% ethanol, but alpha-MSP formed filaments at lower solvent concentration than beta-MSP. When incubated in polyethylene glycol, both isoforms formed thin, needle-shaped crystals that appeared to be constructed from helical fibers, with a 9 nm axial repeat that matched that seen in isolated filaments. These crystals probably contained a parallel array of helical filaments, and may enable both the structure of MSP molecules and their mode of assembly into higher aggregates to be investigated to high resolution.

Actins↗
PubMed1992

Extracellular calcium mimics the actions of platelet-derived growth factor on mouse fibroblasts.

Microprecipitates of calcium phosphate (CaPO4) can substitute for platelet-derived growth factor (PDGF) to stimulate the growth of cultured 3T3 cells. In two-part complementation assays, CaPO4 behaves as a PDGF-like "competence factor"--that is, the mitogenic response to CaPO4 is enhanced synergistically by "progression factors" contained in platelet-poor plasma. In studies described here, we show that early cytoplasmic and intranuclear events in the mitogenic response to CaPO4 are equivalent to those induced by PDGF. However, no net increase in tyrosine kinase activity of either the PDGF-alpha or PDGF-beta receptor is seen following exposure to CaPO4. Our data suggest that calcium acts within the cell, regulating events which normally proceed from activation of PDGF receptors. Alternatively, microprecipitates of CaPO4 could act externally by activating a growth factor receptor which escapes detection with available reagents.

3T3 Cells↗
PubMed1991

Generation of a large library of point mutations in polyoma middle T antigen.

Polyoma middle T antigen (MTAg) transforms cells by associating with and activating a variety of intracellular proteins, including src family members and a phosphatidylinositol-3 kinase. In order to assist in the study of the relative importance of the various associated biochemical activities for transformation by polyomavirus MTAg, a library of MTAg mutants was constructed. Chemically mutagenized MTAg DNA was purified from wild-type DNA by separation on denaturing gradient gels and placed into a recombinant retrovirus vector. Utilizing the resultant library of MTAg-expressing retroviruses, fibroblast cell lines expressing retroviruses, fibroblast cell lines expressing individual MTAg mutants were generated and screened for a non-transformed morphology. Of the first seven non-transformed clones tested, all express the MTAg protein. We estimate that approximately 24% of the G418-resistant colonies contain a transformation-defective MTAg mutant. A more thorough evaluation of one such clone revealed four single base-pair changes as compared to wild-type. Further genetic dissection of this mutant reveals that substituting leucine for proline at amino acid 248 results in a completely transformation defective MTAg. The utility of this mutagenesis and screening procedure as well as the description of several new MTAg mutants is described. This library of mutations should be of general interest for studying the transforming ability of MTAg.

Animals↗
PubMed1991

Raf-1 is a potential substrate for mitogen-activated protein kinase in vivo.

MAP (mitogen-activated protein) kinase is shown to phosphorylate baculovirally expressed Raf-1 in vitro, generating one major tryptic phosphopeptide which co-migrated with a peptide from Raf-1 32P-labelled in situ. This peptide also undergoes an insulin-dependent increase in labelling. Thus the serine/threonine kinase Raf-1 may be a substrate for MAP kinase in vivo.

Animals↗
PubMed1991

Purification and initial characterization of the lymphoid-cell protein-tyrosine kinase p56lck from a baculovirus expression system.

The lymphocyte-specific protein-tyrosine kinase p56lck has been purified 90-fold to approximately 30% purity in 30% yield from a baculovirus expression system by a two-column purification procedure. At least two forms of p56lck were isolated, differing in the extent of phosphorylation and migrating as 56- and 59-kDa species on SDS/PAGE but as a single 56-kDa band after treatment with potato acid phosphatase. Autophosphorylation of purified p56lck occurred at a rate of 25 fmol/min to a maximum incorporation of approximately 2 mol of phosphate per mol of p56lck with tyrosine-394 (but not tyrosine-505) and other, unidentified tyrosine residue(s) being the major sites of phosphorylation in vitro. Phosphorylation of tyrosine-containing peptides was monitored using an automated HPLC system. Although peptide substrate Km values were in the 1-5 mM range, the Vmax for the 13-amino acid peptide RRLIEDAEYAARG (modified p60src autophosphorylation site) was 120 min-1 (350 min-1 when adjusted for p56lck purity), suggesting that the enzyme purified from recombinant baculovirus-infected Sf9 cells has a high catalytic turnover compared with other tyrosine kinases.

Amino Acid Sequence↗
PubMed1991

Presence of an SH2 domain in the actin-binding protein tensin.

The molecular cloning of the complementary DNA coding for a 90-kilodalton fragment of tensin, an actin-binding component of focal contacts and other submembraneous cytoskeletal structures, is reported. The derived amino acid sequence revealed the presence of a Src homology 2 (SH2) domain. This domain is shared by a number of signal transduction proteins including nonreceptor tyrosine kinases such as Abl, Fps, Src, and Src family members, the transforming protein Crk, phospholipase C-gamma 1, PI-3 (phosphatidylinositol) kinase, and guanosine triphosphatase-activating protein (GAP). Like the SH2 domain found in Src, Crk, and Abl, the SH2 domain of tensin bound specifically to a number of phosphotyrosine-containing proteins from v-src-transformed cells. Tensin was also found to be phosphorylated on tyrosine residues. These findings suggest that by possessing both actin-binding and phosphotyrosine-binding activities and being itself a target for tyrosine kinases, tensin may link signal transduction pathways with the cytoskeleton.

Actins↗
PubMed1991

Granulocyte-macrophage colony-stimulating factor and interleukin-3 induce rapid phosphorylation and activation of the proto-oncogene Raf-1 in a human factor-dependent myeloid cell line.

The product of the c-raf-1 proto-oncogene, Raf-1, is a 74,000-dalton cytoplasmic serine/threonine protein kinase that has been implicated as an intermediate in signal transduction mechanisms. In the human factor-dependent myeloid cell line MO7, both granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3) were found to induce rapid, dose-dependent phosphorylation of Raf-1, which resulted in altered Raf-1 mobility in sodium dodecyl sulfate-polyacrylamide gels. The increase in phosphorylation was due primarily to an increase in phosphoserine, with only a minor component (less than 2%) of phosphotyrosine. PMA (12-phorbol 13-myristic acid) also induced Raf-1 phosphorylation in MO7 cells, but the resulting alteration in electrophoretic mobility was different than that observed after GM-CSF or IL-3. GM-CSF and IL-3 rapidly and transiently increased Raf-1 kinase activity using Histone H1 as a substrate in an immune complex kinase assay in vitro. These results suggest that phosphorylation of Raf-1 could play a role in some aspect of GM-CSF and IL-3 signal transduction.

Cell Line↗
PubMed1991

Centripetal flow and directed reassembly of the major sperm protein (MSP) cytoskeleton in the amoeboid sperm of the nematode, Ascaris suum.

The cytoskeleton of the amoeboid spermatozoa of Ascaris suum consists of major sperm protein (MSP) filaments arranged into long, branched fiber complexes that span the length of the pseudopod and treadmill rearward continuously due to assembly and disassembly at opposite ends of the complexes (Sepsenwol et al., Journal of Cell Biology 108:55-66, (1989)). Examination by video-enhanced microscopy showed that this cytoskeletal flow is tightly coupled to sperm locomotion. The fiber complexes treadmilled rearward at the same rate (10-50 microns/min) as the cell crawled forward. Only fiber complexes with their plasmalemmal ends within a limited sector along the leading edge of the pseudopod underwent continuous assembly. Thus, the location of this sector, which occupies about 50% of the pseudopod perimeter, determined the direction of sperm locomotion. Treatment of sperm with agents that lower intracellular pH, such as weak acids and protonophores, caused the fiber complexes to disassemble completely in 4-5 sec. Removal of these compounds resulted in reassembly of the cytoskeleton in a pattern that mimicked treadmilling in intact sperm. The fiber complexes were reconstructed by assembly at their plasmalemmal ends so that within 30-60 sec the entire filament system reformed and the cell resumed locomotion. Both cytoskeletal reassembly and treadmilling required exogenous HCO3-. These results suggest that variation in intracellular pH may help regulate cytoskeletal treadmilling and thereby play a significant role in sperm locomotion.

Actin Cytoskeleton↗
PubMed1991

Characterization of an active, non-myristylated, cytoplasmic form of the lymphoid protein tyrosine kinase pp56lck.

pp56lck is a member of the src family of tyrosine kinases mainly expressed in T lymphocytes. Src tyrosine kinases have been implicated in the control of cell growth and differentiation in different cell types, but the mechanism of regulation of these enzymes is poorly understood. In order to characterize the distinct species of pp56lck, we have produced high yields of enzymatically active wild type pp56lck using the eukaryotic baculovirus expression system in Spodoptera frugiperda insect cells (Sf9). We find that the various species of baculoviral pp56lck are not only differentially phosphorylated (on serine and tyrosine residues) but also heterogeneously myristylated. Surprisingly a non-myristylated, very active form of bv-pp56lck is found in the cytoplasm of Sf9 cells. Fractionation of T cells reveals that cytoplasmic pp56lck exists in T lymphocytes as well.

Animals↗
PubMed1991

An Epstein-Barr virus protein associated with cell growth transformation interacts with a tyrosine kinase.

Epstein-Barr virus (EBV) encodes two integral membrane proteins in latently infected growth-transformed cells. One of these, LMP1, can transform rodent fibroblasts and induce markers of B-lymphocyte activation. The second, LMP2, colocalizes with LMP1 in a constitutive patch in the EBV-transformed B-lymphocyte plasma membrane. The experiments reported here demonstrate that LMP2 may biochemically interact with LMP1 and that LMP2 closely associates with and is an important substrate for a B-lymphocyte tyrosine kinase in EBV-transformed B lymphocytes or in B-lymphoma cells in which LMP2 is expressed by gene transfer. LMP2 is also serine and threonine phosphorylated. LMP2 localizes to a peripheral membrane (presumably plasma membrane) patch in transfected B-lymphoma cells and colocalizes with much of the cellular tyrosine-phosphorylated proteins. LMP2 undergoes tyrosine phosphorylation in anti-LMP2 or antiphosphotyrosine immunoprecipitates from transfected B-lymphoma cells or EBV-transformed B lymphocytes. The first 167 of the 497 amino acids of LMP2 retain full ability to associate with and act as a substrate for a tyrosine kinase. A 70-kDa phosphotyrosine cell protein associates with LMP2 in transfected cells or in EBV-transformed B lymphocytes and could be a mediator of the effects of LMP2.

Amino Acid Sequence↗
PubMed1991

Interleukin-2-triggered Raf-1 expression, phosphorylation, and associated kinase activity increase through G1 and S in CD3-stimulated primary human T cells.

To gain further insight into the role of Raf-1 in normal cell growth, c-raf-1 mRNA expression, Raf-1 protein production, and Raf-1-associated kinase activity in normal human T cells were analyzed. In contrast to the constitutive expression of Raf-1 in continuously proliferating cell lines, c-raf-1 mRNA and Raf-1 protein levels were barely detectable in freshly isolated G0 T lymphocytes. Previous work with fibroblasts has suggested that Raf-1 plays a signaling role in the G0-G1 phase transition. In T cells, triggering via the T-cell antigen receptor (TCR)-CD3 complex (TCR/CD3) resulted in an approximately fourfold increase in c-raf-1 mRNA. In addition, the promotion of G1 progression by interleukin 2 (IL-2) was associated with a 5- to 10-fold immediate/early induction of c-raf-1 mRNA, resulting in up to a 12-fold increase in Raf-1 protein expression. TCR/CD3 activation did not alter the phosphorylation state of Raf-1, whereas interleukin 2 receptor stimulation resulted in a rapid increase in the phosphorylation state of a subpopulation of Raf-1 molecules progressively increasing throughout G1. These findings were complemented by assays for Raf-1-associated kinase activity which revealed a gradual accumulation of serine and threonine autokinase activity in Raf-1 immunoprecipitates during G1, which remained elevated throughout DNA replication.

Antigens, CD↗
PubMed1990

Modulation of a Mr 175,000 c-neu receptor isoform in G8/DHFR cells by serum starvation.

The neu proto-oncogene product has been found to exist in two interconvertible forms in G8/DHFR mouse fibroblasts. The 185-kilodalton form (p185) present in growing cells is replaced by a 175-kilodalton form (p175) under conditions of serum starvation. This low molecular weight form accounts almost exclusively for the phosphotyrosine content of the receptor and is associated with increased tyrosine kinase activity. Addition of serum, platelet-derived growth factor or tumor promoter induces conversion of p175 to p185 within minutes, and this increase in molecular weight is associated with phosphorylation of serine and threonine; removal of serum growth factors is followed by replacement of p185 with p175 over several hours. Unlike G8/DHFR cells, the human breast cancer cell line SK-Br-3 expresses a high molecular weight neu/HER2 receptor with unchanged phosphotyrosine content in both serum-starved and serum-stimulated cultures. These findings indicate that activation of the neu proto-oncogene product in G8/DHFR cells may be regulated in part by protein kinase C-mediated receptor transmodulation rather than by ligand availability alone.

Animals↗
PubMed1990

Stimulation via CD3-Ti but not CD2 induces rapid tyrosine phosphorylation of a 68-kDa protein in the human Jurkat T cell line.

Tyrosine phosphorylation is an early biochemical event associated with surface receptor triggering in many cellular systems. In T lymphocytes, Ag receptor (CD3-Ti) stimulation results in tyrosine phosphorylation of the CD3 zeta subunit. The tyrosine kinase responsible for this modification after CD3-Ti triggering has not been identified. Here we reported that a 68-kDa T cell membrane-associated protein (pp68) in human Jurkat T cells is phosphorylated on tyrosine residues within 1 min after anti-CD3 mAb addition. This induced tyrosine phosphorylation is detected either by in vivo [32P]orthophosphate labeling of the Jurkat T cells or by in vitro [32P]ATP labeling after immunoprecipitation by antiphosphotyrosine antibody. In contrast, mAb stimulation via CD2 and CD4 structures does not induce phosphorylation of pp68. These data are among the first to provide evidence that CD3-Ti and CD2 activation pathways are distinct. Furthermore, they imply that pp68 is itself a tyrosine kinase and/or is a rapidly phosphorylated substrate of a tyrosine kinase.

Antigens, Differentiation, T-Lymphocyte↗