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L A Feig

Publications and source records attributed to L A Feig.

At least 37 records · Page 2Linked to original sources
PubMed1997

Activation of the exchange factor Ras-GRF by calcium requires an intact Dbl homology domain.

Ras-GRF is a guanine nucleotide exchange factor that activates Ras proteins. Its activity on Ras in cells is enhanced upon calcium influx. Activation follows calcium-induced binding of calmodulin to an IQ motif near the N-terminus of Ras-GRF. Ras-GRF also contains a Dbl homology (DH) domain C-terminal to the IQ motif. In many proteins, DH domains act as exchange factors for Rho-GTPase family members. However, we failed to detect exchange activity of this domain on well characterized Rho family members. Instead, we found that mutations analogous to those that block exchange activity of Dbl prevented Ras-GRF activation by calcium/ calmodulin in vivo. All DH domains are followed immediately by a pleckstrin homology (PH) domain. We found that a mutation at a conserved site within the PH domain following the DH domain also prevented Ras-GRF activation by calcium in vivo. These results suggest that in addition to playing a role as activators of Rho proteins, DH domains can also contribute to the coupling of cellular signals to Ras activation.

Calcium↗
PubMed1996

Ral-GTPases mediate a distinct downstream signaling pathway from Ras that facilitates cellular transformation.

Ral proteins (RalA and RalB) comprise a distinct family of Ras-related GTPases (Feig and Emkey, 1993). Recently, Ral-GDS, the exchange factor that activates Ral proteins, has been shown to bind specifically to the activated forms of RasH, R-Ras and Rap1A, in the yeast two-hybrid system. Here we demonstrate that although all three GTPases have the capacity to bind Ral-GDS in mammalian cells, only RasH activates Ral-GDS. Furthermore, although constitutively activated Ra1A does not induce oncogenic transformation on its own, its expression enhances the transforming activities of both RasH and Raf. Finally, a dominant inhibitory form of RalA suppresses the transforming activities of both RasH and Raf. These results demonstrate that activation of Ral-GDS and thus its target, Ral, constitutes a distinct downstream signaling pathway from RasH that potentiates oncogenic transformation.

3T3 Cells↗
PubMed1996

Evidence for a Ras/Ral signaling cascade.

It is becoming clear that Ras proteins mediate their diverse biological functions by binding to, and participating in, the activation of multiple downstream targets. Recent work has identified nucleotide-exchange factors for Ral-GTPases as the newest members of the set of putative Ras 'effector molecules'. This new work has also detected two potential downstream targets of Ral proteins, a novel CDC42/Rac GTPase-activating protein and a phospholipase D.

Animals↗
PubMed1996

The N-terminal pleckstrin, coiled-coil, and IQ domains of the exchange factor Ras-GRF act cooperatively to facilitate activation by calcium.

We have recently shown that the neuronal exchange factor p140 Ras-GRF becomes activated in vivo in response to elevated calcium levels [C. L. Farnsworth, N. W. Freshney, L. B. Rosen, A. Ghosh, M. E. Greenberg, and L. A. Feig, Nature (London) 376:524-527, 1995]. Activation is mediated by calcium-induced calmodulin binding to an IQ domain near the N terminus of Ras-GRF. Here we show that the adjacent N-terminal pleckstrin homology (PH), coiled-coil, and IQ domains function cooperatively to allow Ras-GRF activation. Deletion of the N-terminal PH domain redistributes a large percentage of Ras-GRF from the particulate to the cytosolic fraction of cells and renders the protein insensitive to calcium stimulation. A similar cellular distribution and biological activity are observed when only the core catalytic domain is expressed. Although the PH domain is necessary for particulate association of Ras-GRF, it is not sufficient for targeting the core catalytic domain to this cellular location. This requires the PH domain and the adjacent coiled-coil and IQ sequences. Remarkably, this form of Ras-GRF is constitutively activated. The PH and coiled-coil domains must also perform an additional function, since targeting to the particulate fraction of cells is not sufficient to allow Ras-GRF activation by calcium. A Ras-GRF mutant containing the PH domain from Ras-GTPase-activating protein in place of its own N-terminal PH domain localizes to the particulate fraction of cells but does not respond to calcium. Similar phenotypes are seen with mutant Ras-GRFs containing point mutations in either the PH or coiled-coil domain. These findings argue that the N-terminal PH, coiled-coil, and IQ domains of Ras-GRF function together to connect Ras-GRF to multiple components in the particulate fractions of cells that are required for responsiveness of the protein to calcium signaling.

3T3 Cells↗
PubMed1995

Involvement of Ral GTPase in v-Src-induced phospholipase D activation.

An early response to the tyrosine kinase activity of v-Src is an increase in phospholipase D (PLD) activity, which leads to the generation of biologically active lipid second messengers, including phosphatidic acid, lysophosphatidic acid and diacylglycerol. We have recently demonstrated that v-Src-induced PLD activity is mediated by Ras, although Ras involvement was indirect, requiring a cytosolic factor for PLD activation. Ras interacts with and activates Ral-GDS, the exchange factor responsible for the activation of Ral GTPases. Here we report that this newly identified Ras/Ral signalling pathway mediates PLD activation by v-Src. PLD activity could be precipitated from v-Src-transformed cell lysates with immobilized RalA protein and with an anti-Ral antibody. A mutation to the region of RalA analogous to the 'effector domain' of Ras did not reduce the ability of RalA to complex with PLD, although deletion of a Ral-specific amino-terminal region did. Overexpression of RalA potentiated PLD activation by v-Src, and expression of dominant negative RalA mutants inhibited both v-Src- and v-Ras-induced PLD activity. Thus RalA is involved in the tyrosine kinase activation of PLD through its unique N terminus, and that PLD is a downstream target of a Ras/Ral GTPase cascade.

3T3 Cells↗
PubMed1995

Calcium activation of Ras mediated by neuronal exchange factor Ras-GRF.

Tyrosine kinase receptors stimulate the Ras signalling pathway by enhancing the activity of the SOS nucleotide-exchange factor. This occurs, at least in part, by the recruitment of an SOS-GRB2 complex to Ras in the plasma membrane. Here we describe a different signalling pathway to Ras that involves activation of the Ras-GRF exchange factor in response to Ca2+ influx. In particular, we show that the ability of Ras-GRF to activate Ras in vivo is markedly enhanced by raised Ca2+ concentrations. Activation is mediated by calmodulin binding to an IQ motif in Ras-GRF, because substitutions in conserved amino acids in this motif prevent both calmodulin binding to Ras-GRF and Ras-GRF activation in vivo. So far, full-length Ras-GRF has been detected only in brain neurons. Our findings implicate Ras-GRF in the regulation of neuronal functions that are influenced by Ca2+ signals.

Amino Acid Sequence↗
PubMed1995

Differential response of the Ras exchange factor, Ras-GRF to tyrosine kinase and G protein mediated signals.

Ras-GRF, a guanine-nucleotide exchange factor that activates Ras p21, was tested for its ability to couple to either tyrosine kinase or heterotrimeric G protein signal transduction pathways. Ras-GRF failed to bind the SH2 and SH3 containing adaptor protein Grb2, either in vitro or in vivo. Furthermore, Ras-GRF did not form a stable complex with activated EGF receptor. However, as has been shown previously (Cen et al., 1994), the presence of Ras-GRF in NIH3T3 cells enhanced the activation of Ras induced by serum stimulation. A similar effect was not observed with PDGF stimulation. Moreover, serum stimulation lead to the hyperphosphorylation of Ras-GRF. Both the serum induced super-activation of Ras, and the hyperphosphorylation of Ras-GRF were blocked by pretreatment of cells with the Gi,o inhibitor pertussis toxin, but not by pretreatment with the tyrosine kinase inhibitor genistein. These results suggest that Ras-GRF has the capacity to mediate Ras activation initiated by signals using heterotrimeric G proteins.

3T3 Cells↗
PubMed1995

The chemotactic response to PDGF-BB: evidence of a role for Ras.

The PDGF receptor-beta mediates both mitogenic and chemotactic responses to PDGF-BB. Although the role of Ras in tyrosine kinase-mediated mitogenesis has been characterized extensively, its role in PDGF-stimulated chemotaxis has not been defined. Using cells expressing a dominant-negative ras, we find that Ras inhibition suppresses migration toward PDGF-BB. Overexpression of either Ras-GTPase activating protein (Ras-GAP) or a Ras guanine releasing factor (GRF) also inhibited PDGF-stimulated chemotaxis. In addition, cells producing excess constitutively active Ras failed to migrate toward PDGF-BB, consistent with the observation that either excess ligand or excess signaling intermediate can suppress the chemotactic response. These results suggest that Ras can function in normal cells to support chemotaxis toward PDGF-BB and that either too little or too much Ras activity can abrogate the chemotactic response. In contrast to Ras overexpression, cells producing excess constitutively active Raf, a downstream effector of Ras, did migrate toward PDGF-BB. Cells expressing dominant-negative Ras were able to migrate toward soluble fibronectin demonstrating that these cells retained the ability to migrate. These results suggest that Ras is an intermediate in PDGF-stimulated chemotaxis but may not be required for fibronectin-stimulated cell motility.

3T3 Cells↗
PubMed1995

Involvement of Ras-related Rho proteins in the mechanisms of action of Clostridium difficile toxin A and toxin B.

Toxins A and B of Clostridium difficile are responsible for pseudomembranous colitis, a disease that afflicts a substantial number of hospitalized patients treated with antibiotics. A major effect of these proteins is the disruption of the actin cytoskeleton. Recently, I. Just, G. Fritz, K. Aktories, M. Giry, M. R. Popoff, P. Boquet, S. Hegenbarth, and C. von Eichel-Streiber (J. Biol. Chem. 269:10706-10712, 1994) implicated Rho proteins as cellular targets of C. difficile toxin B, since pretreatment of cells or purified Rho with toxin prevented subsequent ADP-ribosylation of Rho by exoenzyme C3. Moreover, they showed that overexpression of Rho proteins in cells suppressed cell rounding normally associated with exposure of cells to C. difficile toxin B. Here we expand these findings by showing directly that Rho proteins are covalently modified by both C. difficile toxins A and B. In addition, we demonstrate that the stability of toxin-modified Rho in NIH 3T3 cells is dramatically reduced. Finally, we show that C. difficile toxins A and B do not have similar effects on the closely related Rac and CDC42 GTP-binding proteins.

3T3 Cells↗
PubMed1995

Identification and characterization of Ral-binding protein 1, a potential downstream target of Ral GTPases.

Ral proteins constitute a distinct family of Ras-related GTPases. Although similar to Ras in amino acid sequence, Ral proteins are activated by a unique nucleotide exchange factor and inactivated by a distinct GTPase-activating protein. Unlike Ras, they fail to promote transformed foci when activated versions are expressed in cells. To identify downstream targets that might mediate a Ral-specific function, we used a Saccharomyces cerevisiae-based interaction assay to clone a novel cDNA that encodes a Ral-binding protein (RalBP1). RalBP1 binds specifically to the active GTP-bound form of RalA and not to a mutant Ral with a point mutation in its putative effector domain. In addition to a Ral-binding domain, RalBP1 also contains a Rho-GTPase-activating protein domain that interacts preferentially with Rho family member CDC42. Since CDC42 has been implicated in bud site selection in S. cerevisiae and filopodium formation in mammalian cells, Ral may function to modulate the actin cytoskeleton through its interactions with RalBP1.

Amino Acid Sequence↗
PubMed1994

Guanine-nucleotide exchange factors: a family of positive regulators of Ras and related GTPases.

The past year has seen remarkable advances in our understanding of how members of the Ras superfamily of GTPases are activated. Not only have new guanine-nucleotide exchange factors (GEFs) that activate these GTPases been identified, but in some cases, the mechanisms by which GEFs are connected to events at the cell surface have also been described. We have also come to appreciate the possibilities that GEFs may be multifunctional and that they may contribute to tissue-specific regulation of their cognate GTPase.

Animals↗
PubMed1994

Rapid regeneration of virus from cells infected with a retroviral vector.

Recombinant retroviral vectors usually encode the genes of interest in place of the viral structural genes, which must be provided in trans. These viruses are therefore defective for replication: infected cells cannot produce progeny virus. However, in some cases it may be desirable to generate virus from an infected cell clone displaying a phenotype of interest. We describe a rapid method for producing virus, which involves fusing the infected cells to fresh packaging cells. Stable producer lines are generated after fusion by co-selecting for the Ecogpt+ marker in the packaging cells and the G418 resistance (neor) marker in the infected cells. We have used this method to develop cell lines that produce retroviruses encoding a Leu 61-activated c-Ha-ras oncogene as well as a neor gene. The viruses confer oncogenic transformation on 95%-100% of infected target cells as assayed by altered morphology, focus formation and soft agar growth.

Animals↗
PubMed1992

Evidence for a Ras-dependent extracellular signal-regulated protein kinase (ERK) cascade.

The small GTP-binding protein Ras appears to be required for transformation and differentiation induced by tyrosine kinases. The Ras requirement may be limited to a few tyrosine kinase-regulated signaling pathways or may be universal for all tyrosine kinase actions. Because both Ras and the microtubule-associated protein 2 kinases ERK1 and ERK2 have been implicated in events that lead to neurite outgrowth, we explored the possibility that Ras and ERKs may lie on the same signaling pathway. Utilizing PC-12 rat adrenal pheochromocytoma cell lines that contain a dominant inhibitory Ras mutant (S17N-Ras(H)), we found that Ras was required for stimulation of the ERK cascade by nerve growth factor but apparently not by the heterotrimeric G protein activator AlF4-. Within this cascade, Ras appears to be upstream of an ERK activator, raising the intriguing possibility that Ras may directly regulate a serine/threonine protein kinase.

Aluminum↗
PubMed1992

Molecular cloning of cDNAs encoding a guanine-nucleotide-releasing factor for Ras p21.

The stimulation of a variety of cell surface receptors promotes the accumulation of the active, GTP-bound form of Ras proteins in cells. This is a critical step in signal transduction because inhibition of Ras activation by anti-Ras antibodies or dominant inhibitory Ras mutants blocks many of the effects of these receptors on cellular function. To reach the active GTP-bound state, Ras proteins must first release bound GDP. This rate-limiting step in GTP binding is thought to be catalysed by a guanine-nucleotide-releasing factor (GRF). Here we report the cloning of complementary DNAs from a rat brain library that encode a approximately 140K GRF for Ras p21 (p140Ras-GRF). Its carboxy-terminal region is similar to that of CDC25, a GRF for Saccharomyces cerevisiae RAS. This portion of Ras-GRF accelerated the release of GDP from RasH and RasN p21 in vitro, but not from the related RalA, or CDC42Hs GTP-binding proteins. A region in the amino-terminal end of Ras-GRF is similar to both the human breakpoint cluster protein, Bcr, and the dbl oncogene product, a guanine-nucleotide-releasing factor for CDC42Hs. An understanding of Ras-GRF function will enhance our knowledge of the many signal transduction pathways mediated by Ras proteins.

Amino Acid Sequence↗