Ras Guanyl Nucleotide Exchange Factor Research Articles

The Ras GTPase-activating protein UvGap1 orchestrates conidiogenesis and pathogenesis in the rice false smut fungus Ustilaginoidea virens.

Ras GTPase-activating proteins (Ras GAPs) act as negative regulators for Ras proteins and are involved in various signalling processes that influence cellular functions. Here, the function of four Ras GAPs, UvGap1 to UvGap4, was identified and analysed in Ustilaginoidea virens, the causal agent of rice false smut disease. Disruption of UvGAP1 or UvGAP2 resulted in reduced mycelial growth and an increased percentage of larger or dumbbell-shaped conidia. Notably, the mutant ΔUvgap1 completely lost its pathogenicity. Compared to the wild-type strain, the mutants ΔUvgap1, ΔUvgap2 and ΔUvgap3 exhibited reduced tolerance to H2 O2 oxidative stress. In particular, the ΔUvgap1 mutant was barely able to grow on the H2 O2 plate, and UvGAP1 was found to influence the expression level of genes involved in reactive oxygen species synthesis and scavenging. The intracellular cAMP level in the ΔUvgap1 mutant was elevated, as UvGap1 plays an important role in maintaining the intracellular cAMP level by affecting the expression of phosphodiesterases, which are linked to cAMP degradation in U. virens. In a yeast two-hybrid assay, UvRas1 and UvRasGef (Ras guanyl nucleotide exchange factor) physically interacted with UvGap1. UvRas2 was identified as an interacting partner of UvGap1 through a bimolecular fluorescence complementation assay and affinity capture-mass spectrometry analysis. Taken together, these findings suggest that the UvGAP1-mediated Ras pathway is essential for the development and pathogenicity of U. virens.

Whole-exome sequencing analysis of NSCLC reveals the pathogenic missense variants from cancer-associated genes

BackgroundNon-small-cell lung cancer (NSCLC) is the most common type of lung cancer. NSCLC accounts for 84% of all lung cancer cases. In recent years, advances in pathway understanding, methods for discovering novel genetic biomarkers, and new drugs designed to inhibit the signaling cascades have enabled clinicians to personalize therapy for NSCLC. ObjectivesThe primary aim of this study is to identify the genes associated with NSCLC that harbor pathogenic variants that could be causative for NSCLC. The second aim is to investigate their roles in different pathways that lead to NSCLC. MethodsWe examined exome-sequencing datasets from 54 NSCLC patients to characterize the variants associated with NSCLC. ResultsOur findings revealed that 17 variants in 14 genes were considered highly pathogenic, including CDKN2A, ERBB2, FOXP1, IDH1, JAK3, KMT2D, K-Ras, MSH3, MSH6, POLE, RNF43, TCF7L2, TP53, and TSC1. Gene set enrichment analysis revealed the involvement of transmembrane receptor protein tyrosine kinase activity, protein binding, ATP binding, phosphatidylinositol-4,5-bisphosphate 3-kinase, and Ras guanyl-nucleotide exchange factor activity. Pathway analysis of these genes yielded different cancer-related pathways, including colorectal, prostate, endometrial, pancreatic, PI3K-Akt signaling pathways, and signaling pathways regulating pluripotency of stem cells. Module 1 from protein-protein interactions (PPIs) identified genes that harbor pathogenic SNPs. Three of the most deleterious SNPs are ERBB2 (rs1196929947), K-Ras (rs121913529), and POLE (rs751425952). Interestingly, one patient has a pathogenic K-Ras variant (rs121913529) co-occurred with the missense variant (rs752054698) inTSC1 gene. ConclusionThis study maps highly pathogenic variants associated with NSCLC and investigates their contributions to the pathogenesis of NSCLC. This study sheds light on the potential applications of precision medicine in patients with NSCLC.

Abstract 2138: RasGRP3 mediates MAPK pathway activation in GNAQ mutant uveal melanoma

Abstract Uveal melanoma (UM) is the most common intraocular malignancy in adults and no effective treatment options are available for metastatic disease. Over 80% of UM show mutations in the Gαq family members GNAQ and GNA11. MAP-kinase pathway activation in part mediated by protein kinase C (PKC) has been shown as one critical contributing factor to GNAQ-mediated oncogenesis. However PKC inhibition alone does not completely suppress MAPK signaling. A more refined understanding of the signaling cascade linking MAPK signaling to mutant GNAQ or GNA11 is required to develop more effective strategies for targeted therapy. Among more than 10 different PKC isoforms, we identified both PKC δ and PKC ϵ to be required and sufficient to activate MAPK pathway in GNAQ mutant melanomas by using siRNA mediated knock-down and co-transfection of GNAQQ209L and specific PKC isoform cDNAs. Overexpression of GNAQQ209L in 293FT cells increased Ras-GTP level and knock down of three Ras isoforms in GNAQ mutant uveal melanoma cell lines decreased MAPK signaling. Microarray analysis of 5 different GNAQ/11 mutant and 5 NRAS/BRAF-mutant melanoma cells revealed RasGRP3, a Ras-guanyl nucleotide exchange factor (RasGEF), ranks at the top of 487 differentially expressed genes between (cut off : p<0.05, fold change >2 or <-2). Quantitative RT-PCR and western blot confirmed markedly (>100 fold) elevated RasGRP3 levels in GNAQ/11 mutant melanoma cells, while other RasGEFs were not significantly altered. Knock down of RasGRP3 decreased MAPK signaling and proliferation in GNAQ mutant melanoma cells, while it no effect on BRAF mutant cells. Mutating the PKC phosphorylation site of RasGRP3 (T133) partially attenuated RasGRP3-mediated MAPK signaling. Overexpression of PKC δ and PKC ϵ increased RasGRP3 T133 phosphorylation but not PKC α and PKC ζ. While PKC inhibition completely abrogated RasGRP3 T133 phosphorylation, it did not fully suppress RasGRP3-mediated MAPK signaling, indicating that its activating effect only partly requires PKC. We found that RasGRP3 activation mediated by its diacylglycerol(DAG) binding C1 domain provides an independent conduit of activation, which is augmented by PKC-mediated phosphorylation at T133. DAG is a second messenger that is released by phospholipase C, a direct effector of activated GNAQ/11. Finally, we found that the markedly elevated RasGRP3 expression in GNAQ mutant melanoma cell lines is a direct consequence of the oncogenic signaling and is mediated by PKC δ and ϵ. Hence, our data identify RasGRP3 as a critical signaling node linking oncogenic signaling downstream of GNAQ/11 to the Ras/Raf/MEK/ERK pathway through three mechanisms: phosphorylation by PKC δ and ϵ, binding of the second messenger DAG, and by upregulation of its protein level as a consequence of oncogenic GNAQ-mediated activation of PKC δ and PKC ϵ. The findings nominate RasGRP3 as a possible therapeutic target for cancers driven by oncogenic GNAQ/11. Citation Format: Xu Chen, Qiuxia Wu, Philippe Depeille, Jeroen P. Roose, Boris C. Bastian. RasGRP3 mediates MAPK pathway activation in GNAQ mutant uveal melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2138. doi:10.1158/1538-7445.AM2015-2138

AB0052 Tnfa influences rasgrp1 and rasgrp3 expression levels in pbmc, b and t cells.

BackgroundRheumatoid arthritis (RA) is the most common inflammatory arthritis. B and T lymphocytes play a central role in the pathophysiology of RA. RasGRP is a member of the CDC25 family...

Abstract 4292: RasGRP3 contributes to Ras signaling, growth, and migration of human lung cancer cell lines.

Abstract Elevated levels of Ras activity are a prominent feature in human lung cancer. In approximately 20% of lung cancer cases, Ras activation is achieved by mutation of the Ras proto-oncogene. Here, we explore the possible contribution of RasGRP3 to Ras signaling in lung cancer. RasGRP3 is a Ras guanyl nucleotide exchange factor which has been shown to be expressed in prostate tumor and melanoma cell lines, contributing to the level of Ras activation, cell proliferation, and tumor xenograft growth. Here, we report that RasGRP3 is expressed in multiple human lung cancer cell lines but not in normal human bronchial epithelial cells. Likewise, initial analysis reveals its expression in human lung tumors but not in normal lung tissue. Suppression of endogenous RasGRP3 expression in lung cancer cell lines showing reduced H-Ras-GTP and K-Ras-GTP formation as well as Akt phosphorylation downstream from epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA) stimulation. Additionally, suppression of RasGRP3 inhibited translocation of the NF-kB p50 and p65 subunits from the cytoplasm to the nuclear fraction and modulated several other transcriptional factors as well. Conversely, expression of exogenous RasGRP3 elevated H-Ras-GTP and K-Ras-GTP and activated AKT phosphorylation in the lung cancer H496 cell line, which itself only expresses RasGRP3 at low levels. These changes in signaling were mirrored at the cellular level. Downregulation of endogenous RasGRP3 inhibited cell proliferation, impeded cell migration and reduced colony formations in soft agar. Our results suggest that RasGRP3 may provide an addition targetable signaling element in lung cancer. Citation Format: Jing Yang, Timothy Esch, Dazhi Yang, Peter Blumberg. RasGRP3 contributes to Ras signaling, growth, and migration of human lung cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4292. doi:10.1158/1538-7445.AM2013-4292

Regulation of RasGRP1 Function in T Cell Development and Activation by Its Unique Tail Domain

The Ras-guanyl nucleotide exchange factor RasGRP1 plays a critical role in T cell receptor-mediated Erk activation. Previous studies have emphasized the importance of RasGRP1 in the positive selection of thymocytes, activation of T cells, and control of autoimmunity. RasGRP1 consists of a number of well-characterized domains, which it shares with its other family members; however, RasGRP1 also contains an ∼200 residue-long tail domain, the function of which is unknown. To elucidate the physiological role of this domain, we generated knock-in mice expressing RasGRP1 without the tail domain. Further analysis of these knock-in mice showed that thymocytes lacking the tail domain of RasGRP1 underwent aberrant thymic selection and, following TCR stimulation, were unable to activate Erk. Furthermore, the deletion of the tail domain led to enhanced CD4+ T cell expansion in aged mice, as well as the production of autoantibodies. Mechanistically, the tail-deleted form of RasGRP1 was not able to traffic to the cell membrane following stimulation, indicating a potential reason for its inability to activate Erk. While the DAG-binding C1 domain of RasGRP1 has long been recognized as an important factor mediating Erk activation, we have revealed the physiological relevance of the tail domain in RasGRP1 function and control of Erk signaling.

RasGRP1 and RasGRP3 expression in lymphocytes of rheumatoid arthritis patients

IntroductionRasGRP is a member of the CDC25 family of Ras guanyl nucleotide exchange factors. RasGRP1 is expressed in T and B cells whereas RasGRP3 is only expressed in B cells....

Regulation of Erk activation in T cells through the tail domain of RasGRP1 (160.5)

Abstract The Ras-guanyl nucleotide exchange factor RasGRP1 plays a critical role linking T cell receptor engagement to Erk activation. Previous studies have emphasized the importance of RasGRP1 for positive selection of thymocytes, activation of T cells, and control of autoimmunity. Unique from its other family members, RasGRP1 contains an approximately 600 nucleotide-long tail domain, the function of which is unknown. In order to determine the role of the tail domain in RasGRP1 function, we generated a knock-in mouse deleting this domain. Thymocytes containing the truncated form of RasGRP1 underwent aberrant selection processes and, following stimulation, were unable to activate Erk. Furthermore, the deletion of the tail domain negatively affected the ability of mature T cells to become activated and proliferate. Strikingly, these mice developed a severe autoimmune disease, marked by splenomegaly, high serum IgE levels, and the accumulation of T cells with an effector memory phenotype. Additionally, in an attempt to determine how the tail domain can regulate Erk activation, we observed that, in Jurkat T cells, the tail-deleted form of RasGRP1 demonstrated improper cellular localization following stimulation. While the DAG-binding C1 domain has long been viewed as the most important factor mediating RasGRP1 function, we have established an essential role for the tail domain, revealing a novel mechanism for the control of Erk activation through RasGRP1.

RasGRP1 Regulates Antigen-Induced Developmental Programming by Naive CD8 T Cells

Ag encounter by naive CD8 T cells initiates a developmental program consisting of cellular proliferation, changes in gene expression, and the formation of effector and memory T cells. The strength and duration of TCR signaling are known to be important parameters regulating the differentiation of naive CD8 T cells, although the molecular signals arbitrating these processes remain poorly defined. The Ras-guanyl nucleotide exchange factor RasGRP1 has been shown to transduce TCR-mediated signals critically required for the maturation of developing thymocytes. To elucidate the role of RasGRP1 in CD8 T cell differentiation, in vitro and in vivo experiments were performed with 2C TCR transgenic CD8 T cells lacking RasGRP1. In this study, we report that RasGRP1 regulates the threshold of T cell activation and Ag-induced expansion, at least in part, through the regulation of IL-2 production. Moreover, RasGRP1(-/-) 2C CD8 T cells exhibit an anergic phenotype in response to cognate Ag stimulation that is partially reversible upon the addition of exogenous IL-2. By contrast, the capacity of IL-2/IL-2R interactions to mediate Ras activation and CD8 T cell expansion and differentiation appears to be largely RasGRP1-independent. Collectively, our results demonstrate that RasGRP1 plays a selective role in T cell signaling, controlling the initiation and duration of CD8 T cell immune responses.

Chronic Immunodeficiency in Mice Lacking RasGRP1 Results in CD4 T Cell Immune Activation and Exhaustion

The Ras-guanyl nucleotide exchange factor RasGRP1 is an important link between TCR-mediated signaling and the activation of Ras and its downstream effectors. RasGRP1 is especially critical for the survival and differentiation of developing thymocytes whereas negative selection of thymocytes bearing an autoreactive TCR appears to be RasGRP1 independent. Despite apparently normal central tolerance, RasGRP1(-/-) mice spontaneously acquire an acutely activated and proliferating CD4 T cell population that exhibits characteristics of T cell exhaustion, including strong expression of programmed cell death-1. To elucidate the basis for RasGRP1(-/-) CD4 T cell immune activation, we initiated a series of adoptive transfer experiments. Remarkably, the copious amounts of cytokines and self-Ags present in hosts made lymphopenic through irradiation failed to induce the majority of RasGRP1(-/-) CD4 T cells to enter cell cycle. However, their infusion into either congenitally T cell- or T/B cell-deficient recipients resulted in robust proliferation and L-selectin down-regulation. These findings imply that the activation and proliferation of RasGRP1(-/-) CD4 T cells may be dependent on their residence in a chronically immunocompromised environment. Accordingly, bacterial and viral challenge experiments revealed that RasGRP1(-/-) mice possess a weakened immune system, exhibiting a T cell-autonomous defect in generating pathogen-specific T cells and delayed pathogen clearance. Collectively, our study suggests that chronic T cell immunodeficiency in RasGRP1(-/-) mice may be responsible for CD4 T cell activation, proliferation, and exhaustion.

RasGRP1 Transmits Prodifferentiation TCR Signaling That Is Crucial for CD4 T Cell Development

TCR signaling plays a governing role in both the survival and differentiation of bipotent double-positive thymocytes into the CD4(+) and CD8(+) single-positive T cell lineages. A central mediator of this developmental program is the small GTPase Ras, emitting cytoplasmic signals through downstream MAPK pathways and eventually affecting gene expression. TCR signal transduction orchestrates the activation of Ras by integrating at least two Ras-guanyl nucleotide exchange factors, RasGRP1 and Sos. In this study, we have characterized the relationship between RasGRP1 function and its potential roles in promoting ERK activity, cell survival, maturation, and lineage commitment. Investigations on RasGRP1(-/-) mice expressing a transgenic (Tg) MHC class II-restricted TCR revealed that the development of CD4 T cells expressing this Tg TCR is completely dependent on RasGRP1. Unexpectedly, a small number of functional CD8 single-positive thymocytes expressing the Tg MHC class II-restricted TCR exists in mutant mice. In addition, RasGRP1(-/-) double-positive thymocytes exhibit marked deficits in TCR-stimulated up-regulation of the positive selection marker CD69 and the antiapoptotic protein Bcl-2, whereas CD5 induction is unaffected. To evaluate the role of RasGRP1 in providing cellular survival signaling, we enforced Bcl-2 expression in RasGRP1(-/-) thymocytes. These studies demonstrate that RasGRP1 function cannot be fully complemented by Tg Bcl-2 expression. Therefore, we propose that RasGRP1 transmits differentiation signaling critically required for CD4 T cell development.

Activation of RasGRP3 by phosphorylation of Thr-133 is required for B cell receptor-mediated Ras activation.

The Ras signaling pathway plays a critical role in B lymphocyte development and activation, but its activation mechanism has not been well understood. At least one mode of Ras regulation in B cells involves a Ras-guanyl nucleotide exchange factor, RasGRP3. We demonstrate here that RasGRP3 undergoes phosphorylation at Thr-133 upon B cell receptor cross-linking, thereby resulting in its activation. Deletion of phospholipase C-gamma2 or pharmacological interference with conventional PKCs resulted in marked reduction in both Thr-133 phosphorylation and Ras activation. Moreover, mutation of Thr-133 in RasGRP3 alone severely impaired its ability to activate Ras in B cell receptor signaling. Hence, our data suggest that PKC, after being activated by diacylglycerol, phosphorylates RasGRP3, thereby contributing to its full activation.

RasGRP1 Transduces Low-Grade TCR Signals which Are Critical for T Cell Development, Homeostasis, and Differentiation

Two important Ras-guanyl nucleotide exchange factors, Sos and RasGRP1, control Ras activation in thymocytes. However, the relative contribution of these two exchange factors to Ras/ERK activation and their resulting impact on positive and negative selection is unclear. We have produced two lines of RasGRP1 −/− TCR transgenic mice to determine the effect of RasGRP1 in T cell development under conditions of defined TCR signaling. Our results demonstrate that RasGRP1 is crucial for thymocytes expressing weakly selecting TCRs whereas those that express stronger selecting TCRs are more effective at utilizing RasGRP1-independent mechanisms for ERK activation and positive selection. Analysis of RasGRP1 −/− peripheral T cells also revealed hitherto unidentified functions of RasGRP1 in regulating T cell homeostasis and sustaining antigen-induced developmental programming.

Presence of Ras guanyl nucleotide-releasing protein in striosomes of the mature and developing rat

Ras signal transduction pathways have been implicated as key regulators in neuroplasticity and synaptic transmission in the brain. These pathways can be modulated by Ras guanyl nucleotide exchange factors, (GEF) which activate Ras proteins by catalysing the exchange of GDP for GTP. Ras guanyl nucleotide-releasing protein (RasGRP), a recently discovered Ras GEF, that links diacylglycerol and probably calcium to Ras signaling pathways, is expressed in brain as well as in T-cells. Here, we have used a highly selective monoclonal antibody against RasGRP to localize this protein within the striatum and related forebrain structures of developing and adult rats. RasGRP immunolabeling was found to be widespread in the mature and developing rat forebrain. Most notably, it presented a prominent patchy distribution throughout the striatum at birth and at all postnatal ages examined. These patches were found to correspond with the striosomal compartment of the striatum, as identified by μ-opioid receptor labeling in the adult. RasGRP-immunoreactivity was also observed in the matrix-like compartment surrounding these patches/striosomes but appeared later in development and was always weaker than in the patches. In both striatal compartments, RasGRP was exclusively expressed by medium-sized spiny neurons and showed no preference for neurons that project either directly or indirectly to the substantia nigra. At the ultrastructural level, immunogold labeling of RasGRP was confined to the cell bodies and dendritic shafts of these output neurons. We conclude that the prominent expression of RasGRP in striosomes may be of significance for diacylglycerol signaling in the striatum, and could be of importance for the processing of limbic-related activity within the basal ganglia.

Cellular and subcellular localization of Ras guanyl nucleotide-releasing protein in the rat hippocampus

Ras guanyl nucleotide-releasing protein (RasGRP) is a recently discovered Ras guanyl nucleotide exchange factor that is expressed in selected regions of the rodent CNS, with high levels of expression in the hippocampus. Biochemical studies suggest that RasGRP can activate the Ras signal pathway in response to changes in diacylglycerol and possibly calcium. To investigate potential sites for RasGRP signaling, we have determined the cellular and subcellular localization of RasGRP protein in adult rat hippocampus, and have also examined the appearance of RasGRP mRNA and protein during hippocampal development. RasGRP immunoreactivity is predominately localized to those neurons participating in the direct cortico-hippocampo-cortical loop. In both hippocampal and entorhinal neurons, RasGRP protein appeared to be localized to both dendrites and somata, but not to axons. Electron microscopy of hippocampal pyramidal cells confirmed RasGRP immunoreactivity in neuronal cell bodies and dendrites, where it appeared to be associated with microtubules. The localization of RasGRP to dendrites suggests a role for this pathway in the regulation of dendritic function.Examination of developing hippocampal structures indicated that RasGRP mRNA and protein appear synchronously during the first 2 weeks of postnatal development as these neurons become fully mature. This result indicates that the RasGRP signal transduction pathway is not required during early hippocampal development, but is a feature of mature neurons during the later stages of development.

Protein Recruitment Systems for the Analysis of Protein-Protein Interactions

The yeast Saccharomyces cerevisiae serves as an excellent genetic tool for the analysis of protein-protein interactions. The most common system, used to date, is the two-hybrid system. Although proven very powerful, the two-hybrid system exhibits several inherent problems and limitations. Recently, two alternative systems have been described that take advantage of the fact that localization of signal transduction effectors to the inner leaflet of the plasma membrane is absolutely necessary for yeast viability. These effectors can either be the Ras guanyl nucleotide exchange factor or Ras itself. The yeast strain used in both systems is a temperature-sensitive mutant in the yeast Ras guanyl nucleotide exchange factor, CDC25. Membrane localization of these effectors is achieved via protein-protein interaction. Each system can be used to test interaction between known protein pairs, as well as for isolation of novel protein interactions. Described here are the scientific and technical steps to be considered for both protein recruitment systems.

Distribution of ras guanyl releasing protein (RasGRP) mRNA in the adult rat central nervous system.

In the nervous system, Ras signal transduction pathways are involved in cellular differentiation, neuronal survival and synaptic plasticity. These pathways can be modulated by Ras guanyl nucleotide exchange factors (Ras GEFs), which activate Ras protein by catalyzing the exchange of GDP for GTP. RasGRP, a recently discovered Ras GEF is expressed in brain as well as in T cells. In addition to the catalytic domain which catalyzes dissociation of Ras-GDP, RasGRP has a pair of calcium-binding EF hands and a diacylglycerol binding domain. The structure of RasGRP suggests that it serves to link calcium and lipid messengers to Ras signaling pathways. We have used an RNase protection assay to detect RasGRP mRNA in various regions of the rat brain and we have determined the cellular distribution of RasGRP mRNA by in situ hybridization. RasGRP mRNA is widely distributed and is present in both interneurons and projection neurons but not confined to any neuronal type or neurotransmitter phenotype. The presence of RasGRP mRNA in archicortical neurons suggests that this pathway may be important in phylogenetically older regions of the CNS. The restriction of RasGRP mRNA to subsets of neurons suggests that activation of Ras by RasGRP has a specific function in certain neuronal types. We did not detect RasGRP in glial cells.

A novel in vivo assay for the analysis of protein-protein interaction.

The Ras Recruitment System (RRS) is a method for identification and isolation of protein-protein interaction. The method is based on translocation of cytoplasmic mammalian Ras protein to the inner leaflet of the plasma membrane through protein-protein interaction. The system is studied in a temperature-sensitive yeast strain where the yeast Ras guanyl nucleotide exchange factor is inactive at 36 degrees C. Protein-protein interaction results in cell growth at the restrictive temperature. We developed a gene reporter assay for the analysis of protein-protein interaction in mammalian cells. Ras activation in mammalian cells induces the mitogen-activated kinase cascade (MAPK), which can be monitored using Ras-dependent reporter genes. This greatly extends the usefulness of the system and provides a novel assay for protein-protein interaction in mammalian cells.

A novel in vivo assay for the analysis of protein-protein interaction

The Ras Recruitment System (RRS) is a method for identification and isolation of protein-protein interaction. The method is based on translocation of cytoplasmic mammalian Ras protein to the inner leaflet of the plasma membrane through protein-protein interaction. The system is studied in a temperature-sensitive yeast strain where the yeast Ras guanyl nucleotide exchange factor is inactive at 36 degrees C. Protein-protein interaction results in cell growth at the restrictive temperature. We developed a gene reporter assay for the analysis of protein-protein interaction in mammalian cells. Ras activation in mammalian cells induces the mitogen-activated kinase cascade (MAPK), which can be monitored using Ras-dependent reporter genes. This greatly extends the usefulness of the system and provides a novel assay for protein-protein interaction in mammalian cells.

Insulin-induced Desensitization of Extracellular Signal-regulated Kinase Activation Results from an Inhibition of Raf Activity Independent of Ras Activation and Dissociation of the Grb2-SOS Complex

Previous studies have suggested that the interaction between the small adaptor protein Grb2 with the Ras guanyl nucleotide exchange factor SOS is functionally important in the regulation of the Ras activation/inactivation cycle. To examine the relationship between the Grb2-SOS complex and Ras activation, we observed that insulin stimulation results in a rapid but transient activation of Ras and the extracellular-signal regulated kinase (ERK) followed by dissociation of the Grb2-SOS complex. Although treatment with the phorbol myristate acetate resulted in ERK activation and complete dissociation of the Grb2-SOS complex, there was no effect on subsequent insulin-stimulated Ras activation. Similarly, insulin stimulation followed by insulin removal resulted in a time-dependent restoration of the Grb2-SOS complex but which was significantly slower than the recovery of insulin-stimulated Ras activation. In addition, although insulin was able to activate Ras under these conditions, there was a complete desensitization of Raf and ERK activation. This apparent homologous desensitization of insulin action was specific for Raf and ERK as the insulin re-stimulation of insulin receptor autophosphorylation and protein kinase B activation were unaffected. Together, these data demonstrate the presence of a pathway independent of the Grb2-SOS complex that can lead to Ras activation but that the desensitization of Raf accounts for the homologous desensitization of ERK.