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T M Roberts

Publications and source records attributed to T M Roberts.

At least 19 recordsLinked to original sources
PubMed1995

Downregulation of Lck-mediated signal transduction by tip of herpesvirus saimiri.

A protein, called tip, of herpesvirus saimiri associates with Lck in transformed T cells. To investigate the effects of complex formation on cellular signal transduction, we constructed human Jurkat-T-cell lines expressing tip. The expression of tip in Jurkat-T cells dramatically suppressed cellular tyrosine phosphorylation and surface expression of lymphocyte antigens. The expression of tip also blocked the induction of tyrosine phosphorylation by anti-CD3 stimulation. The expression of tip in fibroblast cells suppressed the transforming activity of oncogenic F505 Lck. Binding assays showed that the SH3 domain of Lck is sufficient to form a stable complex with tip in vitro. These results demonstrate that tip acts at an early stage of the T-cell signal transduction cascade by associating with Lck and downregulating Lck-mediated activation. Inhibition of Lck-mediated signal transduction by tip in T cells appears to be analogous to the inhibition of Lyn/Syk-mediated signal transduction in B cells by LMP2A of the B-cell-tropic Epstein-Barr virus.

Base Sequence↗
PubMed1992

Selective association between MHC class I-restricted T cell receptors, CDS, and activated tyrosine kinases on thymocytes undergoing positive selection.

Developing T cells undergo distinct selection processes that determine the TCR repertoire. Positive selection involves the differentiation of immature thymocytes capable of recognizing antigens complexed with self-MHC molecules to mature T cells. Besides the central role of TCR engagement by MHC in triggering selection; the interaction of CD8 and CD4 with MHC class I and class II, respectively; is thought to be important in regulating the selection process. To study potential mechanisms involved in positive selection of CD8+ cells, we have analyzed mice expressing a unique transgenic TCR. The transgenic receptor recognizes the HY male Ag in the context of the MHC class I molecule, H2-Db. We describe that CD8 and the TCR are selectively associated in thymocytes of mice expressing the restricting MHC, but not in thymocytes of mice expressing a nonrestricting MHC. pp56lck and pp59fyn, the tyrosine kinases associated with CD8 and TCR, respectively, were found to be present in this complex in an activated form. No comparable TCR-CD4 complex formation was found in thymuses undergoing positive selection to CD8+ cells. The formation of a multimolecular complex between CD8 and TCR, in which pp56lck and pp59fyn are activated, may initiate specific signaling programs involved in the maturation of CD8+ cells.

Animals↗
PubMed1992

Purification and biochemical characterization of non-myristoylated recombinant pp60c-src kinase.

To obtain sufficient material for the biochemical and biophysical study of pp60c-src, we have utilized a recombinant pp60c-src baculovirus lacking the myristoylation site at codon 2. On infection of Sf9 cells, this virus produced large amounts of soluble non-myristoylated pp60c-src. The use of non-myristoylated pp60c-src (1) increases production of pp60c-src compared with the wild-type protein, (2) facilitates purification, (3) yields a stable product and (4) allows biochemical studies in the absence of detergents. Up to 20 mg of pp60c-src of greater than 95% purity has been purified from 6 litres of Sf9 cells grown in a bioreactor. One major and multiple minor forms of pp60c-src were separated by Mono Q f.p.l.c. Isoelectric focusing of purified pp60c-src species revealed heterogeneity, some of which could be attributed to differences in the tyrosine phosphorylation state of the enzyme. Kinetic analysis of non-myristoylated pp60c-src kinase in the presence of Mg2+ gave Km values for angiotensin II and ATP of 2 mM and 30 microM respectively and a Vmax. of 620 nmol/min per mg. The kinetic constants and metal ion preferences of a number of copolymers and peptide substrates have been compared. Polylysine and poly(GLAT), which was not phosphorylated by the pp60c-src kinase, dramatically activated autophosphorylation of Tyr-416, suggesting a conformation modulation of pp60c-src by charged polymers. This finding implies that Tyr-527 dephosphorylation is not sufficient for full activation of pp60c-src in vitro.

Amino Acid Sequence↗
PubMed1992

Synthetic phosphopeptide immunogens yield activation-specific antibodies to the c-erbB-2 receptor.

We inoculated rabbits with synthetic phosphopeptides, duplicating a major autophosphorylation site of the c-erbB-2 protooncogene product. The rabbits produced antisera that, after reverse immunoaffinity purification, selectively recognize the erbB-2 protein in its enzymatically active configuration. These anti-phosphopeptide antisera identify a subset of erbB-2-positive human cell lines wherein the protein is constitutively active as a tyrosine kinase. Synthetic phosphopeptides incorporating informative protein phosphorylation sites may prove useful for generating antibodies that indicate the activation state of additional tyrosine kinases and perhaps other proteins phosphorylated on serine and threonine residues.

Animals↗
PubMed1992

Regulation of the reverse transcriptase of human immunodeficiency virus type 1 by dNTPs.

Reverse transcriptase (RNA-directed DNA polymerase, EC 2.7.7.49) of human immunodeficiency virus type 1 has been examined with respect to the steady-state kinetics of polymerization of dNTPs into product DNA. With dNTPs as variable substrate, the kinetics of polymerization deviated from standard Michaelis-Menten kinetics. Substrate inhibition was observed at high substrate concentrations and negative cooperativity was seen at lower substrate concentrations. Examination of incorporation of substrate dNMPs in the presence of nucleotides not complementing the template demonstrated that dNTPs may act as noncompetitive inhibitors, as well as substrate. The Ki of the enzyme for dNTPs was 104 microM. A working model is presented that accounts for the substrate inhibition. In this model, the reverse transcriptase is a multisubunit holoenzyme, where noncompetitive inhibition is mediated by one subunit binding nucleotide and down-regulating the enzymatically active 64-kDa subunit. With additional assumptions, this model can accommodate the negative cooperativity observed.

Allosteric Regulation↗
PubMed1992

Activation of mitogen-activated protein kinase kinase by v-Raf in NIH 3T3 cells and in vitro.

Mitogen-activated protein (MAP) kinases are 42- and 44-kD serine-threonine protein kinases that are activated by tyrosine and threonine phosphorylation in cells stimulated with mitogens and growth factors. MAP kinase and the protein kinase that activates it (MAP kinase kinase) were constitutively activated in NIH 3T3 cells infected with viruses containing either of two oncogenic forms (p35EC12, p3722W) of the c-Raf-1 protein kinase. The v-Raf proteins purified from cells infected with EC12 or 22W viruses activated MAP kinase kinase from skeletal muscle in vitro. Furthermore, a bacterially expressed v-Raf fusion protein (glutathione S-transferase-p3722W) also activated MAP kinase kinase in vitro. These findings suggest that one function of c-Raf-1 in mitogenic signaling is to phosphorylate and activate MAP kinase kinase.

3T3 Cells↗
PubMed1992

Both p21ras and pp60v-src are required, but neither alone is sufficient, to activate the Raf-1 kinase.

The raf genes encode a family of cytoplasmic proteins with intrinsic protein-serine/threonine kinase activity. The c-raf gene is the cellular homolog of v-raf, the transforming gene of murine sarcoma virus 3611. The constitutive kinase activity of the v-Raf protein has been implicated in transformation and mitogenesis. The activity of Raf-1, the protein product of the c-raf gene, is normally suppressed by a regulatory N-terminal domain. Activation of various tyrosine-kinase growth factor receptors results in activation of Raf-1 and its hyperphosphorylation. Further, Raf-1 has been shown to act either downstream or independently of the p21ras protein, as indicated by experiments involving microinjection of anti-Ras antibodies. To investigate the potential role of p21ras in the activation of Raf-1 by tyrosine kinases, we have used the baculovirus/Sf9 cell system to overproduce various wild-type and mutant forms of pp60src, p21ras, and Raf-1 proteins. We show that either pp60v-src or p21c-ras can independently activate the autokinase activity of Raf-1, but only to a limited extent. Surprisingly, both pp60v-src and p21c-ras are required to fully activate Raf-1. Analysis of the Raf-1 autokinase activity in vitro shows that Raf-1 autophosphorylation sites are distributed equally on serine and threonine residues. When Raf-1 is analyzed by immunoblotting, as previously reported for mammalian cell experiments, a marked increase in the apparent molecular weight of Raf-1 is seen only when it is coexpressed with both pp60v-src and p21ras.

Animals↗
PubMed1992

ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf-1, and RSK.

p21c-ras plays a critical role in mediating tyrosine kinase-stimulated cell growth and differentiation. However, the pathways through which p21c-ras propagates these signals remain unknown. We report that in PC12 cells, expression of a dominant inhibitory mutant of ras, c-Ha-ras(Asn-17), antagonizes growth factor- and phorbol ester-induced activation of the erk-encoded family of MAP kinases, the 85-92 kd RSKs, and the kinase(s) responsible for hyperphosphorylation of the proto-oncogene product Raf-1. In addition, we find that expression of the activated ras oncogene is sufficient to stimulate these events. These data indicate that ras mediates nerve growth factor receptor and protein kinase C modulation of MAP kinases, RSKs, and Raf-1.

Animals↗
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↗