Stacey L. Stang

RasGRP is essential for mouse thymocyte differentiation and TCR signaling.

The Ras signaling pathway plays a critical role in thymopoiesis and T cell activation, but the mechanism of Ras regulation is controversial. At least one mode of Ras regulation in T cells involves the messenger diacylglycerol (DAG). RasGRP, a Ras activator with a DAG-binding C1 domain, is expressed in T cells and thymocytes. Here we show that thymi of RasGRP-null mutant mice have approximately normal numbers of immature thymocytes but a marked deficiency of mature, single-positive (CD4+CD8− and CD4−CD8+) thymocytes. In Ras signaling and proliferation assays, mutant thymocytes showed a complete lack of response to DAG analogs or T cell receptor (TCR) stimulation by antibodies. Thus, TCR and DAG are linked through RasGRP to Ras signaling.

T cell anergy is reversed by active Ras and regulated by diacylglycerol kinase

T cell anergy has been correlated with defective signaling by the GTPase Ras, but causal and mechanistic data linking defective Ras activity with T cell anergy are lacking. Here we used adenoviral transduction to genetically manipulate nonproliferating T cells and show that active Ras restored interleukin 2 production and mitogen-activated protein kinase signaling in T cells that were made anergic in vitro or in vivo. Diacylglycerol kinases (DGKs), which negatively regulate Ras activity, were upregulated in anergic T cells, and a DGK inhibitor restored interleukin 2 production in anergic T cells. Both anergy and DGK-α overexpression were associated with defective translocation of the Ras guanine nucleotide–exchange factor RasGRP1 to the plasma membrane. Our data support a causal function for excess DGK activity and defective Ras signaling in T cell anergy.

RasGRP1 and RasGRP3 Regulate B Cell Proliferation by Facilitating B Cell Receptor-Ras Signaling

The RasGRPs are a family of Ras activators that possess diacylglycerol-binding C1 domains. In T cells, RasGRP1 links TCR signaling to Ras. B cells coexpress RasGRP1 and RasGRP3. Using Rasgrp1 and Rasgrp3 single and double null mutant mice, we analyzed the role of these proteins in signaling to Ras and Erk in B cells. RasGRP1 and RasGRP3 both contribute to BCR-induced Ras activation, although RasGRP3 alone is responsible for maintaining basal Ras-GTP levels in unstimulated cells. Surprisingly, RasGRP-mediated Ras activation is not essential for B cell development because this process occurs normally in double-mutant mice. However, RasGRP-deficient mice do exhibit humoral defects. Loss of RasGRP3 led to isotype-specific deficiencies in Ab induction in immunized young mice. As reported previously, older Rasgrp1−/− mice develop splenomegaly and antinuclear Abs as a result of a T cell defect. We find that such mice have elevated serum Ig levels of several isotypes. In contrast, Rasgrp3−/− mice exhibit hypogammaglobulinemia and show no signs of splenomegaly or autoimmunity. Double-mutant mice exhibit intermediate serum Ab titers, albeit higher than wild-type mice. Remarkably, double-mutant mice exhibit no signs of autoimmunity or splenomegaly. B cell proliferation induced by BCR ligation with or without IL-4 was found to be RasGRP1- and RasGRP3-dependent. However, the RasGRPs are not required for B cell proliferation per se, because LPS-induced proliferation is unaffected in double-mutant mice.

Hypothermic stress leads to activation of Ras-Erk signaling

The small GTPase Ras is converted to the active, GTP-bound state during exposure of vertebrate cells to hypothermic stress. This activation occurs more rapidly than can be accounted for by spontaneous nucleotide exchange. Ras-guanyl nucleotide exchange factors and Ras GTPase-activating proteins have significant activity at 0 degrees C in vitro, leading to the hypothesis that normal Ras regulators influence the relative amounts of Ras-GTP and Ras-GDP at low temperatures in vivo. When hypothermic cells are warmed to 37 degrees C, the Raf-Mek-Erk protein kinase cascade is activated. After prolonged hypothermic stress, followed by warming to physiologic temperature, cultured fibroblasts assume a rounded morphology, detach from the substratum, and die. All of these biologic responses are attenuated by pharmacologic inhibition of Mek. Previously, it had been found that low temperature blocks acute growth factor signaling to Erk. In the present study, we found that this block occurs at the level of Raf activation. Temperature regulation of Ras signaling could help animal cells respond appropriately to hypothermic stress, and Ras-Erk signaling can be manipulated to improve the survival of cells in cold storage.

Interaction of activated Ras with Raf-1 alone may be sufficient for transformation of rat2 cells.

v-H-ras effector mutants have been assessed for transforming activity and for the ability of the encoded proteins to interact with Raf-1-, B-Raf-, byr2-, ralGDS-, and CDC25-encoded proteins in the yeast two-hybrid system. Transformation was assessed in rat2 cells as well as in a mutant cell line, rv68BUR, that affords a more sensitive transformation assay. Selected mutant Ras proteins were also examined for their ability to interact with an amino-terminal fragment of Raf-1 in vitro. Finally, possible cooperation between different v-H-ras effector mutants and between effector mutants and overexpressed Raf-1 was assessed. Ras transforming activity was shown to correlate best with the ability of the encoded protein to interact with Raf-1. No evidence for cooperation between v-H-ras effector mutants was found. Signaling through the Raf1-MEK-mitogen-activated protein kinase cascade may be the only effector pathway contributing to RAS transformation in these cells.

RAS signalling is abnormal in a c-raf1 MEK1 double mutant.

A mutant rat cell clone that suppresses the transformation defects of RAS effector loop substitutions is heterozygous for mutations in c-raf1 and MEK1. The mutant cells can be transformed by many otherwise defective RAS effector mutants, including RAS genes with the effector regions of distantly related GTPases, even though the encoded RAS proteins do not interact with either the mutant or wild-type RAF in Saccharomyces cerevisiae. While the significance of the c-raf1 mutation is unclear, the MEK1 mutation increases MEK1 activity and leads to activation of mitogen-activated protein kinase. The mutant MEK1 is coupled to the epidermal growth factor pathway but exhibits decreased physical interaction with RAF. When overexpressed, the MEK1 mutation is transforming and causes hyperphosphorylation of RAF. Signalling from RAS to MEK1 may be mediated by something other than RAF alone, but signalling through MEK1 is probably sufficient for RAS transformation.

Ras activation in response to phorbol ester proceeds independently of the EGFR via an unconventional nucleotide-exchange factor system in COS-7 cells.

Ras is a major mediator of PE (phorbol ester) effects in mammalian cells. Various mechanisms for PE activation of Ras have been reported [Downward, Graves, Warne, Rayter and Cantrell (1990) Nature (London) 346, 719-723; Shu, Wu, Mosteller and Broek (2002) Mol. Cell. Biol. 22, 7758-7768; Roose, Mollenauer, Gupta, Stone and Weiss (2005) Mol. Cell. Biol. 25, 4426-4441; Grosse, Roelle, Herrlich, Höhn and Gudermann (2000) J. Biol. Chem. 275, 12251-12260], including pathways that target GAPs (GTPase-activating proteins) for inactivation and those that result in activation of GEFs (guanine nucleotide-exchange factors) Sos (son of sevenless homologue) or RasGRP (RAS guanyl releasing protein). However, a biochemical link between PE and GAP inactivation is missing and GEF stimulation is hard to reconcile with the observation that dominant-negative S17N-Ras does not compromise Ras-dependent ERK (extracellular-signal-regulated kinase) activation by PE. We have addressed this controversy and carried out an in-depth biochemical study of PE-induced Ras activation in COS-7 cells. Using a cell-permeabilization approach to monitor nucleotide exchange on Ras, we demonstrate that PE-induced Ras-GTP accumulation results from GEF stimulation. Nucleotide exchange stimulation by PE is prevented by PKC (protein kinase C) inhibition but not by EGFR [EGF (epidermal growth factor) receptor] blockade, despite the fact that EGFR inhibition aborts basal and PE-induced Shc (Src homology and collagen homology) phosphorylation and Shc-Grb2 (growth-factor-receptor-bound protein 2) association. In fact, EGFR inhibition ablates basal nucleotide exchange on Ras in growth-arrested COS-7 cells. These data disclose the existence of two separate GEF systems that operate independently from each other to accomplish PE-dependent formation of Ras-GTP and to maintain resting Ras-GTP levels respectively. We document that COS-7 cells do not express RasGRP and present evidence that the PE-responsive GEF system may involve PKC-dependent phosphorylation of Sos. More fundamentally, these observations shed new light on enigmatic issues such as the inefficacy of S17N-Ras in blocking PE action or the role of the EGFR in heterologous agonist activation of the Ras/ERK pathway.

Mutations in conserved regions 1, 2, and 3 of Raf-1 that activate transforming activity

To investigate the role of Raf‐1 in v‐Ha‐ras transformation, we have isolated and characterized a number of Raf‐1 mutants that display increased transforming activity in Rat2 fibroblasts. A dipeptide deletion (Δ144–145) in the cysteine‐rich domain (CRD) of conserved region (CR) 1 increased the interaction between Raf‐1 and v‐Ha‐ras effector loop mutants in the yeast two‐hybrid system, supporting the proposal that the CRD serves as a secondary ras‐binding domain. Many activating mutations were located in CR2. Two representative CR2 mutants (Δ250–258 and S257L) displayed increased interaction with v‐Ha‐ras effector loop mutants and with mitogen‐activated protein kinase/extracellular signal–regulated kinase (ERK) kinase (MEK) 1 in the two‐hybrid system. One novel mutation in CR3 was recovered; G361S affected the third glycine of the GXGXXG protein kinase motif involved in ATP binding. Expression of G361S Raf‐1 in Rat2 fibroblasts activated MEK and ERK. The CR1, CR2, and CR3 activating mutations, when combined in cis, cooperated in transforming Rat2 fibroblasts. Conversely, Raf‐1 transforming activity was decreased when the S257L or G361S mutation was combined in cis with the R89E substitution, which disrupts ras‐Raf interaction. This mutant analysis provides additional information about the distinct functions of individual Raf‐1 regions and documents a novel genetic mechanism for activating an oncogenic kinase.

ras effector loop mutations that dissociate p120GAP and neurofibromin interactions.

ras proteins are positively regulated by nucleotide exchange factors and negatively regulated by GTPase‐activating proteins (GAPs). Two GAPs have been found in mammalian cells, p120GAP and neurofibromin, the product of the type 1 neurofibromatosis (NF1) gene. A library of substitutions in the effector loop region of ras in an Escherichia coli plasmid expression system was screened for c‐Ha‐ras species with altered GAP interactions. Several substitutions preferentially disrupted the interaction of ras with p120GAP as compared with the interaction with the recombinant GAP‐related domain of neurofibromin (NF1‐GRD). The most extreme example, Tyr32His, encoded a ras species that was unaffected by p120GAP but was stimulated normally by NF1‐GRD. Tyr32His was weakly transforming in Rat2 cells. Tyr32His ras was primarily GDP‐bound in quiescent Rat2 cells, although it rapidly associated with GTP after treatment of cells with epidermal growth factor. These results show that the NF1 product has less stringent requirements than p120GAP for ras effector domain structure and that negative regulation of ras can be achieved in rat fibroblasts by the product of NF1.