Guanine Nucleotide Releasing Factor Research Articles

Treatment of Chronic Lymphocytic Leukemia Patients with Lenalidomide Induces Down-Regulation of miR342-3p Associated with Over-Expression of Tumor Suppressor RASSF4,

Abstract 3885Lenalidomide has promising clinical activity in patients with chronic lymphocytic leukemia (CLL). Unlike other anti-leukemia drugs, lenalidomide is not cytotoxic for CLL cells in vitro. Similar to CD154, lenalidomide can enhance CLL-cell expression of immune co-stimulatory molecules, formation of immunologic synapse, activation of NK-cells, and generation of anti-tumor immunity. Furthermore, lenalidomide repeatedly can enhance expression of CD154, which we had observed was functionally deficient in patients with CLL. However the exact mechanism of action of lenalidomide is still under investigation.Herein, we studied the gene expression profile and microRNA (miR) of CLL cells collected from 20 patients before and at day 8 and day 15 of treatment with 2.5–5 mg of lenalidomide in the CRC014 trial.We observed significant changes in expression level of 54 genes at day 8 versus pre-treatment samples. We identified significant changes in expression level of 189 genes at day 15 versus pre-treatment samples. This included 44 of the 54 (81%) genes noted at day 8. Forty genes were expressed at significantly higher levels at day 8 and day 15 of lenalidomide treatment. We noted that 7 (17%) of these genes were related to Ras pathway and its downstream signaling pathways (i.e. NF-KappaB pathway): Ras association (RalGDS/AF-6) domain family member 4 (RASSF4), a member of RAS oncogene family (RAB13), Ras protein-specific guanine nucleotide releasing factor 1 (RASGRF1), GTPase IMAP family member 6 (GIMAP6), GTP-binding protein ras homolog gene family member S (RND1), kinase suppressor of Ras 2 (KSR2) and toll-like receptor adaptor molecule 2 (TICAM2). Ras signaling affects many cellular functions, which includes cell proliferation, apoptosis, migration, fate specification, and differentiation. In the resting cells, Ras is tightly bound to GDP (Guanosine Diphosphate), which is exchanged for GTP (Guanosine Triphosphate) upon binding to activated cell membrane receptors. In the GTP-bound form, Ras interacts with a broad range of effector proteins to induce a diverse array of biological consequences. Although typically associated with enhanced growth and transformation, activated Ras also may induce growth antagonistic effects such as senescence or apoptosis. Some of the growth-inhibitory properties of Ras are mediated via the RASSF family of Ras effector/tumor suppressors. RASSF4 is the fifth member of this family and it binds directly to activated K-Ras in a GTP-dependent manner via the effector domain, thus exhibiting the basic properties of a Ras effector. Overexpression of RASSF4 induces Ras-dependent apoptosis in 293-T cells and inhibits the growth of human tumor cell lines. Although broadly expressed in normal tissue, RASSF4 is frequently down-regulated by promoter methylation in human tumor cells and primary tumors.However changes in miR expression also may affect the level of gene expression. Therefore we analyzed miRs expression by microarray in pre treatment, day 8, and day 15 CLL samples. We observed significant changes in expression levels of 33 miRs between day 8 and pre treatment samples. We identified significant changes in expression levels of 11 miRs between day 15 and pre treatment samples. Of the 33 miRs differentially expressed at day 8, only 5 were up-regulated whereas the remaining 28 were down-regulated. Interestingly, among these 28 down-regulated miRs, 5 miRs (miR-103, miR-16, miR-30a, miR-30b and miR-342-3p) target RASSF4. Noteworthy, miR-342-3p was one of the 3 miRs (miR-26a, miR-138 and miR-342-3p) down-regulated both at day 8 and at day 15, suggesting that the down-regulation of such miR has a key role in the overexpression of RASSF4 leading to Ras-dependent apoptosis.Further studies are ongoing to elucidate lenalidomide action on CLL cells via RASSF4 overexpression. This study demonstrates that treatment with lenalidomide can induce down-regulation of miRs associated with changes in gene expression by CLL cells, leading to over-expression of RASSF4 and other Ras or GTPase related proteins that can induce growth antagonistic effects and account in part for the activity of this drug in CLL. Disclosures:James:Celgene: Research Funding. Neuberg:Celgene: Research Funding. Corral:Celgene: Employment. Kipps:Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbott Industries: Research Funding; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding.

RASGRF1 (Ras protein-specific guanine nucleotide-releasing factor 1)

Review on RASGRF1 (Ras protein-specific guanine nucleotide-releasing factor 1), with data on DNA, on the protein encoded, and where the gene is implicated.

Effect of curcumin on irradiated and estrogen-transformed human breast cell lines

Curcumin (diferuloyl methane) is a well known antioxidant that exerts antiproliferative and apoptotic effects. Curcumin effect was evaluated in a breast cancer model that was developed using the immortalized breast epithelial cell line MCF-10F after exposure to low doses of high LET (linear energy transfer) α particles (150 keV/µm) of radiation, and subsequently cultured in the presence of 17β-estradiol (estrogen). This model consisted of human breast epithelial cells in different stages of transformation: i) MCF-10F; ii) Estrogen cell line; iii) a malignant Alpha3 cell line; iv) a malignant and tumorigenic, Alpha5 cell line; and v) a cell line derived from Alpha5 injected into the nude mice that gave rise to Tumor2 cell line. Curcumin decreased anchorage-independent growh in transformed breast cancer cell lines in comparison to their counterparts and increased the percentage of cells from G₀/G₁ with a concomitant increase in G₂/M phases, as well as a decrease in PCNA and Rho-A protein expression. Among the oncogenes, c-Ha-Ras and Ras homologous A (Rho-A) are important cell signaling factors for malignant transformation and to reach their active GTP bound state, Ras proteins must first release bound GDP mediated by a guanine nucleotide releasing factor (GRF). Then curcumin decrease RasGRF1 protein expression in malignant cell lines. Further, differential expression levels of cleaved (ADP) ribose polymerase 1 (PARP-1) and phosphorylated histone H2AX (γ-H2AX) were observed after curcumin treatment. It seems that PARP-1 similar to H2AX, confers cellular protection against radiation and estrogen-induced DNA damage mediated by curcumin. Therefore, targeting either PARP-1 or H2AX may provide an effective way of maximizing the therapeutic value of antioxidants for cancer prevention.

Uncovering the Proteome Response of the Master Circadian Clock to Light Using an AutoProteome System

In mammals, the suprachiasmatic nucleus (SCN) is the central circadian pacemaker that governs rhythmic fluctuations in behavior and physiology in a 24-hr cycle and synchronizes them to the external environment by daily resetting in response to light. The bilateral SCN is comprised of a mere ~20,000 neurons serving as cellular oscillators, a fact that has, until now, hindered the systematic study of the SCN on a global proteome level. Here we developed a fully automated and integrated proteomics platform, termed AutoProteome system, for an in-depth analysis of the light-responsive proteome of the murine SCN. All requisite steps for a large-scale proteomic study, including preconcentration, buffer exchanging, reduction, alkylation, digestion and online two-dimensional liquid chromatography-tandem MS analysis, are performed automatically on a standard liquid chromatography-MS system. As low as 2 ng of model protein bovine serum albumin and up to 20 μg and 200 μg of SCN proteins can be readily processed and analyzed by this system. From the SCN tissue of a single mouse, we were able to confidently identify 2131 proteins, of which 387 were light-regulated based on a spectral counts quantification approach. Bioinformatics analysis of the light-inducible proteins reveals their diverse distribution in different canonical pathways and their heavy connection in 19 protein interaction networks. The AutoProteome system identified vasopressin-neurophysin 2-copeptin and casein kinase 1 delta, both of which had been previously implicated in clock timing processes, as light-inducible proteins in the SCN. Ras-specific guanine nucleotide-releasing factor 1, ubiquitin protein ligase E3A, and X-linked ubiquitin specific protease 9, none of which had previously been implicated in SCN clock timing processes, were also identified in this study as light-inducible proteins. The AutoProteome system opens a new avenue to systematically explore the proteome-wide events that occur in the SCN, either in response to light or other stimuli, or as a consequence of its intrinsic pacemaker capacity.

A role for Ras signaling in modulating mammalian aging by the GH/IGF1 axis

The discovery that mutations in single genes can modulate aging was not only fascinating but it provided researchers with animal models with which to study aging in cohorts of different aging rates [1-2]. According to the GenAge database [3], at the time of writing, genetic manipulations in 68 genes have been shown to affect lifespan in mice. Among mouse genes in which mutations extend lifespan, a number are part of the growth hormone/insulin-like growth factor 1 (GH/IGF1) axis. As such, it is widely acknowledged that decreased GH/IGF1signaling in mice can extend lifespan, while increased GH levels may accelerate aging [4-5]. Although the GH/IGF1axis is one of the major pathways regulating aging in mammals, its precise downstream mechanisms remain a subject of debate and scrutiny. In this year's March issue of AGING, Borras et al. show that mice deficient in Rasgrf1, a guanine nucleotide–releasing factor for Ras, exhibit a significant increase (~20%) in average and maximum lifespan. This increase in lifespan was independent of cancer mortality and was accompanied by a retardation in age–related functional and physiological decline [6]. Ras homologues have been associated with lifespan modulation in yeast [7], so it seems that Ras signaling is another example of an evolutionary conserved pathway modulating aging. Rasgrf1 is unique among Ras family members, however, as it is only expressed in a few specific tissues, in particular in pancreatic β–cells and in some regions of the brain. Thus far, Rasgrf1 has been primarily associated with learning and memory [6,8-9]. Because of its expression in the brain, Rasgrf1 has been shown to regulate the synthesis and release of GH, and previous studies showed that Rasgrf1 deficient mice have lower levels of GH [9-10]. In line with these previous studies, and given that GH impacts on IGF1 levels, Rasgrf1 deficient mice showed lower IGF1 levels [6]. Decreased GH/IGF1 signaling results in stunted growth and reduced adult body size, which has been associated with life-extension in numerous models [11]. Borras et al. and other authors also observed that Rasgrf1deficient mice are smaller than controls [6,9-10]. Therefore, it is likely that decreased GH/IGF1 signaling is the mechanism by which Rasgrf1 impacts on aging. Changes in GH/IGH1 have also been hypothesized to be related to the mechanisms of caloric restriction [4-5], which is in line with the observation by Borras et al., that Rasgrf1 deficient mice have metabolic profiles similar to mice under caloric restriction. Because Rasgrf1 mutants have altered GH/IGF1 signaling, I had predicted that they would be long-lived [12]. Moreover, it was reported that bi-maternal mice are long-lived and, because Rasgrf1is an imprinted gene expressed from the paternal allele in neonates, it is possible that bi-maternal mice are long-lived due to Rasgrf1 effects [12-13]. Although conducted in different labs and strains, bi–maternal mice had a similar increase in average lifespan (28%), though a more modest increase in maximum lifespan (~5%). Rasgrf1 deficient mice are therefore another model to study the modulation of aging by the GH/IGF1 axis. Interestingly, mice expressing Rasgrf1from both alleles have increased body size and higher IGF1 levels [9]. Although the lifespan of these animals is unknown, they could be seen as a complementary model to the Rasgrf1 knock-outs. Consequently, since they are available with both increased GH (biallelic Rasgrf1 mice) and decreased GH levels (Rasgrf1 knock outs), the different Rasgrf1 mutant mice may be an excellent new model to study aging and its modulation by the GH/IGF1 axis.

Ras protein–specific guanine nucleotide–releasing factor 1 (RASGRF1)

Ras protein–specific guanine nucleotide–releasing factor 1 (RASGRF1)

Involvement of Grb2 Adaptor Protein in Nucleophosmin-Anaplastic Lymphoma Kinase (NPM-ALK)-mediated Signaling and Anaplastic Large Cell Lymphoma Growth

Most anaplastic large cell lymphomas (ALCL) express oncogenic fusion proteins derived from chromosomal translocations or inversions of the anaplastic lymphoma kinase (ALK) gene. Frequently ALCL carry the t(2;5) translocation, which fuses the ALK gene to the nucleophosmin (NPM1) gene. The transforming activity mediated by NPM-ALK fusion induces different pathways that control proliferation and survival of lymphoma cells. Grb2 is an adaptor protein thought to play an important role in ALK-mediated transformation, but its interaction with NPM-ALK, as well as its function in regulating ALCL signaling pathways and cell growth, has never been elucidated. Here we show that active NPM-ALK, but not a kinase-dead mutant, bound and induced Grb2 phosphorylation in tyrosine 160. An intact SH3 domain at the C terminus of Grb2 was required for Tyr(160) phosphorylation. Furthermore, Grb2 did not bind to a single region but rather to different regions of NPM-ALK, mainly Tyr(152-156), Tyr(567), and a proline-rich region, Pro(415-417). Finally, shRNA knockdown experiments showed that Grb2 regulates primarily the NPM-ALK-mediated phosphorylation of SHP2 and plays a key role in ALCL cell growth.

Overexpression of Rasgrf1, a Guanine Nucleotide Exchange Factor in Chronic Lymphocytic Leukemia.

Abstract 1253 Poster Board I-275 We performed microarray expression analysis to identify genes which are differentially expressed in chronic lymphocytic leukemia (CLL) cells. A number of genes from the cohort of maximally upregulated genes were members of the Ras superfamily of proteins function which function as switches between active GTP-complexed and inactive GDP-complexed states. One of the genes upregulated on the microarray analysis in all the CLL cells as compared to normal B cells was RASGRF1. This ras protein specific guanine nucleotide release factor stimulates the dissociation of GDP and activates Ras signaling pathway. To confirm our microarray analysis results, we performed real time PCR analysis on 35 CLL specimens and observed a 10-50 fold upregulation of rasgrf1 RNA as compared to human peripheral blood B cells. This high expression is consistently observed in all CLL specimens regardless of stage indicating that this upregulation is an early important event in the pathogenesis of CLL. Rasgrf1 protein expression was also higher in CLL cells as compared to normal B cells by western blot analysis. Interestingly the rasgrf1 gene is genomically imprinted during development in certain tissues and the expression is downregulated. Demethylation of normal peripheral blood B cells by treatment with azacytidine resulted in increased expression of rasgrf1 with no change in expression of rasgrf1 in CLL cells implying imprinting in normal B cells. Thus malignant transformation of B cells to CLL cells results in a loss of genomic imprinting and an aberrant upregulation of rasgrf1 in CLL cells. One important downstream effect of rasgrf1 upregulation is ERK activation via Ras-MAPK pathway. B cell receptor signaling in CLL results in activation of a number of pro-survival pathways including ERK which is more active in poor prognosis, unmutated immunoglobulin heavy chain CLL. Our results indicate that ERK phosphorylation is also increased in CLL specimens with rasgrf1 overexpression. Disclosures No relevant conflicts of interest to declare.

Differences in flexibility underlie functional differences in the Ras activators son of sevenless and Ras guanine nucleotide releasing factor 1.

The Ras-specific nucleotide exchange factor Son of sevenless (Sos) is inactive without Ras bound to a distal allosteric site. In contrast, the catalytic domain of Ras guanine nucleotide releasing factor 1 (RasGRF1) is active intrinsically. By substituting residues from RasGRF1 into Sos, we have generated mutants of Sos with basal activity, partially relieved of their dependence on allosteric activation. We have performed molecular dynamics simulations showing how Ras binding to the allosteric site leads to a bias toward the active conformation of Sos. The trajectories show that Sos fluctuates between active and inactive conformations in the absence of Ras and that the activating mutations favor conformations of Sos that are more permissive to Ras binding at the catalytic site. In contrast, unliganded RasGRF1 fluctuates primarily among active conformations. Our results support the premise that the catalytic domain of Sos has evolved an allosteric activation mechanism that extends beyond the simple process of membrane recruitment.

The Ras guanine nucleotide exchange factor RasGRF1 promotes matrix metalloproteinase-3 production in rheumatoid arthritis synovial tissue

IntroductionFibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients share many similarities with transformed cancer cells, including spontaneous production of matrix metalloproteinases (MMPs). Altered or chronic activation of proto-oncogenic Ras family GTPases is thought to contribute to inflammation and joint destruction in RA, and abrogation of Ras family signaling is therapeutic in animal models of RA. Recently, expression and post-translational modification of Ras guanine nucleotide releasing factor 1 (RasGRF1) was found to contribute to spontaneous MMP production in melanoma cancer cells. Here, we examine the potential relationship between RasGRF1 expression and MMP production in RA, reactive arthritis, and inflammatory osteoarthritis synovial tissue and FLS.MethodsExpression of RasGRF1, MMP-1, MMP-3, and IL-6 was detected in synovial tissue by immunohistochemistry and stained sections were evaluated by digital image analysis. Expression of RasGRF1 in FLS and synovial tissue was also assessed by immunoblotting. Double staining was performed to detect proteins in specific cell populations, and cells producing MMP-1 and MMP-3. RasGRF1 expression was manipulated in RA FLS by cDNA transfection and gene silencing, and effects on MMP-1, TIMP-1, MMP-3, IL-6, and IL-8 production measured by ELISA.ResultsExpression of RasGRF1 was significantly enhanced in RA synovial tissue, and detected in FLS and synovial macrophages in situ. In cultured FLS and synovial biopsies, RasGRF1 was detected by immunoblotting as a truncated fragment lacking its negative regulatory domain. Production of MMP-1 and MMP-3 in RA but not non-RA synovial tissue positively correlated with expression of RasGRF1 and co-localized in cells expressing RasGRF1. RasGRF1 overexpression in FLS induced production of MMP-3, and RasGRF1 silencing inhibited spontaneous MMP-3 production.ConclusionsEnhanced expression and post-translational modification of RasGRF1 contributes to MMP-3 production in RA synovial tissue and the semi-transformed phenotype of RA FLS.

C3G overexpression in glomerular epithelial cells during anti-GBM-induced glomerulonephritis

The guanine nucleotide exchange factor C3G, along with the CrkII adaptor protein, mediates GTP activation of the small GTPase proteins Rap1 and R-Ras, facilitating their activation of downstream signaling pathways, which had been found to be important in the pathogenesis of glomerulonephritis. We found that expression of C3G protein was upregulated in glomerular epithelial cells in an experimental model of accelerated anti-GBM antibody-induced glomerulonephritis expression. To determine the consequence of its increased expression, we transfected C3G (using adenoviral constructs) into cultured glomerular epithelial cells and measured the activated forms (i.e., GTP-bound) forms of Rap1 and R-Ras. Activation of Rap1 was not affected by C3G; however, the basal level of GTP-bound R-Ras was decreased. Further, C3G over-expression enhanced the activation of R-Ras in response to endothelin. Overexpression of C3G also led to a significant reduction in glomerular epithelial cell spreading and decreased the cells' E-cadherin expression and augmented their migration. We found that C3G was overexpressed in accelerated anti-GBM antibody-induced glomerulonephritis and suggest that this modulates glomerular epithelial cell morphology and behavior.

Translational responses of NR2B overexpression in the cerebral cortex of transgenic mice: A liquid chromatography-based proteomic approach

The N-methyl- d-aspartate (NMDA) receptor 2B subunit (NR2B) is important for long-term potentiation (LTP) and synaptic plasticity. The NR2B transgenic mice exhibited larger LTP in the hippocampal CA1 region and enhanced behavioral performance in several learning and memory tasks. In this study, we applied two-dimensional liquid chromatography-based proteomic approach to examine the expression levels of cerebral cortical proteins from 6-month NR2B transgenic (Tg) and their wild-type (WT) mice that were maintained on the same genetic background. Proteins were separated according to the pI in the first dimension using a chromatofocusing column and the hydrophobicity in the second dimension using a nonporous reversed-phase silica column. The DeltaVue software was applied to examine the differential expression of protein samples. Twenty six differentially expressed proteins were identified by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF/TOF) mass spectrometry, including glutamine synthetase (GS), guanine nucleotide-releasing factor 1, carbonic anhydrase, clathrin light chain B (Lcb), enolase 1, ATP synthase, cytochrome c, THO complex 4, and M-phase phosphoprotein 1. The findings were further corroborated in an independent group of NR2B Tg and WT mice by Western blot analysis of two selected proteins. The results revealed a unique profile of cortical proteins in the NR2B transgenic mice. A close association of functional activation of NR2B with the excitatory neurotransmission and neuroplasticity has been discussed.

Discovery of Epigenetically Silenced Genes by Methylated DNA Immunoprecipitation in Colon Cancer Cells

CpG island promoter hypermethylation of tumor suppressor genes is a common hallmark of human cancer, and new large-scale epigenomic technologies might be useful in our attempts to define the complete DNA hypermethylome of tumor cells. Here, we report a functional search for hypermethylated CpG islands using the colorectal cancer cell line HCT-116, in which two major DNA methyltransferases, DNMT1 and DNMT3b, have been genetically disrupted (DKO cells). Using methylated DNA immunoprecipitation methodology in conjunction with promoter microarray analyses, we found that DKO cells experience a significant loss of hypermethylated CpG islands. Further characterization of these candidate sequences shows CpG island promoter hypermethylation and silencing of genes with potentially important roles in tumorigenesis, such as the Ras guanine nucleotide-releasing factor (RASGRF2), the apoptosis-associated basic helix-loop transcription factor (BHLHB9), and the homeobox gene (HOXD1). Hypermethylation of these genes occurs in premalignant lesions and accumulates during tumorigenesis. Thus, our results show the usefulness of DNMT genetic disruption strategies combined with methylated DNA immunoprecipitation in searching for unknown hypermethylated candidate genes in human cancer that might aid our understanding of the biology of the disease and be of potential translational use.

Germ Line Gain of Function with SOS1 Mutation in Hereditary Gingival Fibromatosis

Mutation of human SOS1 is responsible for hereditary gingival fibromatosis type 1, a benign overgrowth condition of the gingiva. Here, we investigated molecular mechanisms responsible for the increased rate of cell proliferation in gingival fibroblasts caused by mutant SOS1 in vitro. Using ectopic expression of wild-type and mutant SOS1 constructs, we found that truncated SOS1 could localize to the plasma membrane, without growth factor stimuli, leading to sustained activation of Ras/MAPK signaling. Additionally, we observed an increase in the magnitude and duration of ERK signaling in hereditary gingival fibromatosis gingival fibroblasts that was associated with phosphorylation of retinoblastoma tumor suppressor protein and the up-regulation of cell cycle regulators, including cyclins C, D, and E and the E2F/DP transcription factors. These factors promote cell cycle progression from G(1) to S phase, and their up-regulation may underlie the increased gingival fibroblast proliferation observed. Selective depletion of wild-type and mutant SOS1 through small interfering RNA demonstrates the link between mutation of SOS1, ERK signaling, cell proliferation rate, and the expression levels of Egr-1 and proliferating cell nuclear antigen. These findings elucidate the mechanisms for gingival overgrowth mediated by SOS1 gene mutation in humans.

A Ras-induced conformational switch in the Ras activator Son of sevenless.

The Ras-specific guanine nucleotide-exchange factors Son of sevenless (Sos) and Ras guanine nucleotide-releasing factor 1 (RasGRF1) transduce extracellular stimuli into Ras activation by catalyzing the exchange of Ras-bound GDP for GTP. A truncated form of RasGRF1 containing only the core catalytic Cdc25 domain is sufficient for stimulating Ras nucleotide exchange, whereas the isolated Cdc25 domain of Sos is inactive. At a site distal to the catalytic site, nucleotide-bound Ras binds to Sos, making contacts with the Cdc25 domain and with a Ras exchanger motif (Rem) domain. This allosteric Ras binding stimulates nucleotide exchange by Sos, but the mechanism by which this stimulation occurs has not been defined. We present a crystal structure of the Rem and Cdc25 domains of Sos determined at 2.0-A resolution in the absence of Ras. Differences between this structure and that of Sos bound to two Ras molecules show that allosteric activation of Sos by Ras occurs through a rotation of the Rem domain that is coupled to a rotation of a helical hairpin at the Sos catalytic site. This motion relieves steric occlusion of the catalytic site, allowing substrate Ras binding and nucleotide exchange. A structure of the isolated RasGRF1 Cdc25 domain determined at 2.2-A resolution, combined with computational analyses, suggests that the Cdc25 domain of RasGRF1 is able to maintain an active conformation in isolation because the helical hairpin has strengthened interactions with the Cdc25 domain core. These results indicate that RasGRF1 lacks the allosteric activation switch that is crucial for Sos activity.

Age-dependent Participation of Ras-GRF Proteins in Coupling Calcium-permeable AMPA Glutamate Receptors to Ras/Erk Signaling in Cortical Neurons

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors (AMPARs) are ligand-gated sodium channels. Through their ability to mediate the majority of rapid excitatory transmission in the central nervous system, these neurotransmitter receptors have been shown to influence synaptic plasticity. Some of these receptors are also calcium-permeable (CP), and they also have been implicated in regulating synaptic plasticity, particularly in interneurons where their concentration is highest. However, the biochemical pathways emanating from CP-AMPARs that mediate these effects have not been well characterized. In this paper, we show that CP-AMPARs are the predominant AMPAR class responsible for activating the Ras/Erk kinase signaling cascade and the cAMP-response element-binding protein (CREB) transcription factor in the cortex of mature mice. Activation of Ras and Erk, but not CREB, occurs through the calcium/calmodulin regulated Ras-GRF1 and Ras-GRF2 exchange factors, which form AMPA-induced complexes with CP-AMPARs but not calcium-impermeable (CI) AMPARs in vivo. Furthermore, we show that CP-AMPARs are also the major AMPAR type to activate Ras/Erk signaling in pubescent mice; however, at this developmental stage Ras-GRF (guanine nucleotide-releasing factor) proteins are not involved. Finally, in neonatal animals CI-AMPARs, but not CP-AMPARs, are the predominant AMPAR type that activates Ras-Erk signaling and CREB in cortical neurons. This occurs indirectly through activation of L-type voltage-dependent calcium channels, an event that is also Ras-GRF-independent. Thus, Ras/Erk signaling and CREB activity induced by AMPARs occur through age-dependent mechanisms that likely make unique developmentally dependent contributions to synaptic function.

Inactivation of Crk SH3 domain-binding guanine nucleotide-releasing factor (C3G) in cervical squamous cell carcinoma

C3G, a Crk SH3 domain-binding guanine nucleotide-releasing factor functions as a guanine nucleotide exchange factor for Rap1. It is activated via the Crk adaptor protein and plays an important role in transducing signals from receptors on the cell surface to the nucleus via the Ras/Raf/MAPK signal transduction pathway. However, since the experimental data result in pleiotropic effects in the cascade manner, its precise function remains unclear. Here we examined the C3G expression in cervical squamous cell carcinomas and found a marked decrease in the expression of C3G in a high incidence of said samples. In addition, we also demonstrated frequent hypermethylation of C3G, which resulted in an inactivation mechanism of the gene. Clinical and pathologic data failed to show any relationship between the human papillomavirus infection and the down-regulation of C3G. These results indicate that inactivation of C3G by de novo methylation plays an important role in the development of cervical squamous cell carcinoma.

Requirement of phosphatidylinositol-4,5-bisphosphate for HERC1-mediated guanine nucleotide release from ARF proteins

HERC1 is a giant multidomain protein involved in membrane trafficking through its interaction with vesicle coat proteins such as clathrin and ARF. Previously, it has been shown that the RCC1-like domain 1 (RLD1) of HERC1 stimulates guanine nucleotide dissociation on ARF1 and Rab proteins. In this study, we have analyzed whether HERC1 may also regulate ARF6 activity. We show that HERC1, through its RLD1, stimulates GDP release from ARF6 but, unexpectedly, it inhibits GDP/GTP exchange on ARF6 under conditions where ARNO stimulates it. Furthermore, we demonstrate that the activity of HERC1 as a guanine nucleotide release factor requires the presence of PI(4,5)P2 bound to HERC1’s RLD1. In agreement with this, we find that purified HERC1 contains PI(4,5)P2 bound to the RLD1.

Amplification, up-regulation and over-expression of C3G (CRK SH3 domain-binding guanine nucleotide-releasing factor) in non-small cell lung cancers.

The Ras-CRK-Rap1 cellular signal-transduction system is regulated by guanine nucleotide exchange factors (GEFs). Transcription of C3G on chromosome 9q34 and a key member of the GEF gene family is activated by the CRK-adaptor protein; the C3G product is a CRK SH3 domain-binding guanine nucleotide-releasing factor. We document here the amplification of C3G in five of 18 primary non-small cell lung cancers examined and its increased expression in 18 of 28 tumors in comparison to corresponding non-cancerous lung tissues. Immunohistochemical staining revealed prominent C3G protein in the cytoplasm of cancer cells, associated with faint staining at the nucleolar membrane, but C3G was not detectable in normal bronchial mucoepithelial cells or in broncholoalveolar cells of the bronchial/bronchiolar ducts or alveoli. These data indicate that amplification and increased expression of the C3G gene may play some role in human lung carcinogenesis through derangement of the CRK-Rap1 signaling pathway.

P35/cyclin-dependent kinase 5 phosphorylation of ras guanine nucleotide releasing factor 2 (RasGRF2) mediates Rac-dependent Extracellular Signal-regulated kinase 1/2 activity, altering RasGRF2 and microtubule-associated protein 1b distribution in neurons.

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed kinase the activity of which is dependent on association with its neuron-specific activators, p35 and p39. Cdk5 activity is critical for the proper formation of cortical structures and lamination during development. In the adult nervous system, Cdk5 function is implicated in cellular adhesion, dopamine signaling, neurotransmitter release, and synaptic activity. In addition, Cdk5 is also involved in "cross-talk" with other signal transduction pathways. To further examine its involvement in cross-talk with other pathways, we identified proteins that interacted with p35 using the yeast two-hybrid system. We report here that p35 associates with Ras guanine nucleotide releasing factor 2 (RasGRF2) in coimmunoprecipitation and colocalization studies using transfected cell lines as well as primary cortical neurons. Additionally, Cdk5 phosphorylates RasGRF2 both in vitro and in vivo, leading to a decrease in Rac-guanidine exchange factor activity and a subsequent reduction in extracellular signal-regulated kinase 1/2 activity. We show that p35/Cdk5 phosphorylates RasGRF2 on serine737, which leads to an accumulation of RasGRF2 in the neuronal cell bodies coinciding with an accumulation of microtubule-associated protein 1b. The membrane association of p35 and subsequent localization of Cdk5 activity toward RasGRF2 and Rac provide insights into important cellular signaling processes that occur at the membrane, resulting in downstream effects on signal transduction cascades.