Signal transduction. Exchange rate mechanisms.
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Animals↗
Biomedical subjects
J Downward
Publications and source records attributed to J Downward.
Aberrant regulation of ras proteins in malignant tumour cells from type 1 neurofibromatosis patients.
Defects in the NF1 gene have been implicated in the inherited disorder neurofibromatosis type 1, which is characterized by several developmental abnormalities including an increased frequency of benign and malignant tumours of neural crest origin (neurofibromas and neurofibrosarcomas respectively). The NF1 gene encodes a ubiquitous protein homologous to p120GAP, the GTPase-activating protein (GAP) for the products of the ras protooncogenes. When expressed in non-mammalian systems, the region of the NF1 gene homologous to p120GAP produces a protein with GAP-like activity. Here we present evidence that the ras proteins in malignant tumour cell lines from patients with type 1 neurofibromatosis are in a constitutively activated state, as judged by the guanine nucleotide bound to them, and are necessary for cellular proliferation. These cells contain p21ras and p120GAP that are both functionally wild type, but barely any functional NF1 protein. Our results show that the NF1 protein is normally essential for correct negative regulation of ras proteins in the cell, even in the presence of normal p120GAP, and they support the hypothesis that NF1 is a tumour-suppressor gene whose product acts upstream of ras.
Antibodies, Monoclonal↗
The growth factor IL-2 activates p21ras proteins in normal human T lymphocytes.
The T cell growth factor IL-2 induces T cell progression through the cell cycle and ultimately controls T cell mitosis. Here we show that the guanine nucleotide-binding proteins p21ras may be involved in IL-2 signal transduction pathways. IL-2 causes a rapid and prolonged activation of p21ras in both murine and human T cells. The concentration-dependence of IL-2-mediated stimulation of p21ras correlated with IL-2 stimulation of T cell proliferation, which indicates that p21ras activity can be controlled by signals generated via the interaction between IL-2 and its high affinity cellular receptor. These results suggest that p21ras may play a role in the regulation of T cell growth by IL-2.
Antigens, Differentiation, T-Lymphocyte↗
T lymphocyte activation signals.
Activation of T lymphocytes results in immediate biochemical changes including increases in intracellular calcium levels, activation of protein kinase C (PKC) and changes in tyrosine phosphorylation. In T cells recent studies have indicated that activation of the guanine nucleotide-binding proteins p21ras is mediated by PKC, which suggests that the p21ras proteins may regulate intracellular signalling events downstream of PKC. The p21ras proteins can be activated in T cells by signals generated by triggering of the T cell antigen receptor (TCR), the CD2 antigen and the interleukin 2 receptor. Experiments using a PKC pseudosubstrate inhibitor indicate that PKC does not mediate TCR-induced activation of p21ras. These results imply that an alternative signal transduction pathway not involving PKC can regulate the activity of p21ras proteins in T cells.
Humans↗
Regulatory mechanisms for ras proteins.
The proteins encoded by the ras proto-oncogenes play critical roles in normal cellular growth, differentiation and development in addition to their potential for malignant transformation. Several proteins that are involved in the control of the activity of p21ras have now been characterised. p120GAP stimulates the GTPase activity of p21ras and hence acts as a negative regulator of ras proteins. It may be controlled by tyrosine phosphorylation or association with tyrosine phosphorylated proteins. The neurofibromatosis type 1 (NF 1) gene also encodes a potential GTPase activating protein which is likely to be subject to a different control mechanism. Guanosine nucleotide exchange factors for p21ras have now been identified: these may be positive regulators of ras protein function. It appears that p21ras is subject to rapid regulation by several distinct mechanisms which are likely to vary in different cell types; the ras proteins are thereby able to act as very sensitive cellular monitors of the extracellular environment.
Animals↗
Interleukin (IL)-2 activation of p21ras in murine myeloid cells transfected with human IL-2 receptor beta chain.
The T cell growth factor interleukin-2 (IL-2) induces p21ras activation in T lymphocytes. To determine whether the IL-2 receptor (IL-2R) can regulate p21ras when expressed in a non-T cell environment we have examined the ability of IL-2 to activate p21ras in 32D murine myeloid progenitor cells transduced with human IL-2R beta chains. These cells are denoted beta 53 cells. 32D cells normally proliferate in response to IL-3 but the expression of the IL-2R beta chain confers IL-2 responsiveness to the cells. Our data show that IL-3 is able to activate p21ras in the parental 32D cells and both IL-2 and IL-3 can stimulate p21ras in the IL-2R-expressing beta 53 clone of 32D. In T lymphocytes, activation of protein kinase C (PKC) with phorbol esters is sufficient to stimulate p21ras. However, in 32D and beta 53 cells activation of PKC with phorbol esters does not result in p21ras activation even though these cells express functional PKC. It appears, therefore, that a PKC-mediated pathway for p21ras regulation exists in T lymphocytes but not in 32D cells. The IL-2R can couple to p21ras independently of the concomitant presence of the PKC pathway for p21ras regulation. These data imply that multiple intracellular mechanisms may exist to regulate p21ras and that cells of different lineages may differ with regard to p21ras regulation.
Animals↗
p21ras mediates control of IL-2 gene promoter function in T cell activation.
It has been shown previously in T cells that stimulation of protein kinase C or the T cell antigen receptor leads to a rapid and persistent activation of p21ras as measured by a dramatic increase in the amount of bound GTP. These stimuli are also known to induce the expression of the T lymphocyte growth factor, interleukin-2 (IL-2), an essential growth factor for the immune system. Receptor induced activation of p21ras has been demonstrated in several cell types but involvement of protein kinase C as an upstream activator of p21ras appears to be unique to T cells. In this study we show that p21ras acts as a component of the protein kinase C and T cell antigen receptor downstream signalling pathway controlling IL-2 gene expression. In the murine T cell line EL4, constitutively active p21ras greatly potentiates the phorbol ester and T cell receptor agonist induced production of IL-2 as measured both by biological assay for the cytokine and by the use of a reporter construct. Active p21ras also partially replaces the requirement for protein kinase C activation in synergizing with a calcium ionophore to induce production of IL-2. Furthermore, using a dominant negative mutant of ras, Ha-rasN17, we show that endogenous ras function is essential for induction of IL-2 expression in response to protein kinase C or T cell receptor stimulation. Activation of ras proteins is thus a necessary but not sufficient event in the induction of IL-2 synthesis. Ras proteins are therefore pivotal signalling molecules in T cell activation.
Animals↗
The regulation and function of p21ras in T cells.
In T cells, activation of the guanine-nucleotide-binding proteins encoded by the p21ras proto-oncogenes is a common response to triggering of the T-cell antigen receptor, the adhesion molecule CD2 and the receptor for the cytokine interleukin 2. This article by Julian Downward and colleagues describes the mechanisms of p21ras regulation and the potential function of p21ras in T cells, and discusses the evidence that multiple intracellular pathways may be involved in the coupling of cell surface receptors to p21ras.
GTPase-Activating Proteins↗
Ras regulation: putting back the GTP.
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Journal Article↗
Regulation of p21ras by GTPase activating proteins and guanine nucleotide exchange proteins.
Ras proteins play a critical role in controlling normal cellular growth and, when activated by mutation, in causing malignant transformation. Regulation of p21ras is achieved by GTPase activating proteins, which control the rate of hydrolysis of GTP to GDP, and also by GDP dissociation stimulators, which catalyze the exchange of guanine nucleotides. Several such proteins have now been identified and their control mechanisms characterized.
Animals↗
Role of protein kinase C in T-cell antigen receptor regulation of p21ras: evidence that two p21ras regulatory pathways coexist in T cells.
T-lymphocyte activation via the antigen receptor complex (TCR) results in accumulation of p21ras in the active GTP-bound state. Stimulation of protein kinase C (PKC) can also activate p21ras, and it has been proposed that the TCR effect on p21ras occurs as a consequence of TCR regulation of PKC. To test the role of PKC in TCR regulation of p21ras, a permeabilized cell system was used to examine TCR regulation of p21ras under conditions in which TCR activation of PKC was blocked, first by using a PKC pseudosubstrate peptide inhibitor and second by using ionic conditions that prevent phosphatidyl inositol hydrolysis and hence diacylglycerol production and PKC stimulation. The data show that TCR-induced p21ras activation is not mediated exclusively by PKC. Thus, in the absence of PKC stimulation, the TCR was still able to induce accumulation of p21ras-GTP complexes, and this stimulation correlated with an inactivation of p21ras GTPase-activating proteins. The protein tyrosine kinase inhibitor herbimycin could prevent the non-PKC-mediated, TCR-induced stimulation of p21ras. These data indicate that two mechanisms for p21ras regulation coexist in T cells: one PKC mediated and one not. The TCR can apparently couple to p21ras via a non-PKC-controlled route that may involve tyrosine kinases.
Amino Acid Sequence↗
Expression cloning and characterization of the TGF-beta type III receptor.
The rat TGF-beta type III receptor cDNA has been cloned by overexpression in COS cells. The encoded receptor is an 853 amino acid protein with a large N-terminal extracellular domain containing at least one site for glycosaminoglycan addition, a single hydrophobic transmembrane domain, and a 41 amino acid cytoplasmic tail with no obvious signaling motif. Introduction of the cDNA into COS cells and L6 myoblasts induces expression of a heterogenously glycosylated 280-330 kd protein characteristic of the type III receptor that binds TGF-beta 1 specifically. In L6 myoblasts lacking the endogenous type III receptor, expression of the recombinant receptor leads to an increase in the amount of ligand bound and cross-linked to surface type II TGF-beta receptors. This indicates that the type III receptor may regulate the ligand-binding ability or surface expression of the type II receptor.
Amino Acid Sequence↗
CD2 antigen mediated activation of the guanine nucleotide binding proteins p21ras in human T lymphocytes.
T cell stimulation via the TCR complex (TCR/CD3 complex) results in activation of the guanine nucleotide binding proteins encoded by the ras protooncogenes (p21ras). In the present study we show that the activation state of p21ras in T lymphocytes can also be controlled by triggering of the CD2 Ag. The activation state of p21ras is controlled by GTP levels on p21ras. In T cells stimulation of protein kinase C is able to induce an accumulation of "active" p21ras-GTP complexes due to an inhibitory effect of protein kinase C stimulation on the intrinsic GTPase activity of p21ras. The regulatory effect of protein kinase C on p21ras GTPase activity appears to be mediated via regulation of GAP, the GTPase activating protein of p21ras. In the present report, we demonstrate that the TCR/CD3 complex and the CD2 Ag control the accumulation of p21ras-GTP complexes via a regulatory effect on p21ras GTPase activity. The TCR/CD3 complex and CD2 Ag are also able to control the cellular activity of GAP. These data demonstrate that p21ras is part of the signal transduction responses controlled by the CD2 Ag, and reveal that the TCR/CD3 complex and CD2 Ag control the activation state of p21ras via a similar mechanism.
Antigens, CD↗
Stimulation of p21ras upon T-cell activation.
External signals that control the activity of proteins encoded by the ras proto-oncogenes have not previously been characterized. It is now shown that stimulation of the antigen receptor of T lymphocytes causes a rapid activation of p21ras. The mechanism seems to involve a decrease in the activity of GAP, the GTPase-activating protein, on stimulation of protein kinase C. In lymphocytes, p21ras may therefore be an important mediator of the action of protein kinase C.
GTPase-Activating Proteins↗
The ras superfamily of small GTP-binding proteins.
Considerable advances have recently been made in understanding the structure and function of the proteins encoded by the ras proto-oncogenes. In addition, a large number of ras-related small GTP-binding proteins with very diverse activities have now been identified. This review explores developments in this rapidly expanding field.
Animals↗
Identification of a nucleotide exchange-promoting activity for p21ras.
The biological activity of proteins encoded by the ras family of oncogenes is dependent on whether they are bound to GTP or GDP: the type of nucleotide bound is dependent on the rate of GTP hydrolysis (promoted by the GTPase-activating protein, GAP) and the rate of nucleotide exchange with cytosolic pools. A protein that stimulates the rate of exchange of guanine nucleotide on p21ras has been identified and characterized in cytoplasmic extracts of human placenta. The exchange-promoting protein runs on a gel filtration column with an apparent relative molecular weight of about 60,000. It is sensitive to heat and to trypsin. The exchange-promoting protein acts reversibly and does not cause degradation of p21ras. It is inactive towards the alpha subunit of a heterotrimeric GTP-binding protein (Go alpha) but acts on a large number of different mutant ras proteins, including transforming and effector mutants that are insensitive to the action of GAP. This protein, which we have termed REP (ras exchange-promoting), has the characteristics expected of a physiological activator of p21ras in cellular growth-signal-transduction pathways.
Chromatography, Gel↗
Proteolytic generation of constitutive tyrosine kinase activity of the human insulin receptor.
Structural modification induced by partial digestion with trypsin has been shown to stimulate the tyrosine kinase activity of the insulin receptor both in solution and in intact cells [Tamura, Fujita-Yamaguchi & Larner (1983) J. Biol. Chem. 258, 14749-14752; Goren, White & Kahn (1987) Biochemistry 26, 2374-2382; Leef & Larner (1987) J. Biol. Chem. 262, 14837-14842]. Furthermore, experiments involving deletion of sequences encoding the extracellular domain of the insulin receptor suggest that it may function as a protooncogene in fibroblasts [Wang et al., (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5725-5729]. To further understand the structural requirements that generate this activity, the major activated fragments generated in solution following trypsin digestion have been characterized here, one of which is shown to have a similar amino acid sequence to a transforming protein. Furthermore, treatment with trypsin of intact Chinese hamster ovary cells that overexpress the human insulin receptor stimulates both autophosphorylation of the receptor and 2-deoxyglucose uptake into the cells, but does not enhance receptor internalization. Unlike digestion in solution, no proteolysis or loss of activity of the activated insulin receptor beta-subunit could be detected using intact cells, even at high trypsin concentrations, despite the existence of extracellular sites that are readily cleaved by trypsin in the solubilized receptor. These studies provide further detail of a mechanism used during trypsinization of cells in culture which mimics activation of the insulin receptor and contributes to stimulation of growth.
Amino Acid Sequence↗