Ras-mitogen Activated Protein Kinase Pathway Research Articles

Novel effects of Ras-MAPK pathogenic variants on the developing human brain and their link to gene expression and inhibition abilities

The RASopathies are genetic syndromes associated with pathogenic variants causing dysregulation of the Ras/mitogen-activated protein kinase (Ras-MAPK) pathway, essential for brain development, and increased risk for neurodevelopmental disorders. Yet, the effects of most pathogenic variants on the human brain are unknown. We examined: (1) How Ras-MAPK activating variants of PTPN11/SOS1 protein-coding genes affect brain anatomy. (2) The relationship between PTPN11 gene expression levels and brain anatomy, and (3) The relevance of subcortical anatomy to attention and memory skills affected in the RASopathies. We collected structural brain MRI and cognitive-behavioral data from 40 pre-pubertal children with Noonan syndrome (NS), caused by PTPN11 (n = 30) or SOS1 (n = 10) variants (age 8.53 ± 2.15, 25 females), and compared them to 40 age- and sex-matched typically developing controls (9.24 ± 1.62, 27 females). We identified widespread effects of NS on cortical and subcortical volumes and on determinants of cortical gray matter volume, surface area (SA), and cortical thickness (CT). In NS, we observed smaller volumes of bilateral striatum, precentral gyri, and primary visual area (d’s < −0.8), and extensive effects on SA (d’s > |0.8|) and CT (d’s > |0.5|) relative to controls. Further, SA effects were associated with increasing PTPN11 gene expression, most prominently in the temporal lobe. Lastly, PTPN11 variants disrupted normative relationships between the striatum and inhibition functioning. We provide evidence for the effects of Ras-MAPK pathogenic variants on striatal and cortical anatomy as well as links between PTPN11 gene expression and cortical SA increases, and striatal volume and inhibition skills. These findings provide essential translational information on the Ras-MAPK pathway’s effect on human brain development and function.

Combining SOS1 and MEK Inhibitors in a Murine Model of Plexiform Neurofibroma Results in Tumor Shrinkage.

Individuals with neurofibromatosis type 1 develop rat sarcoma virus (RAS)-mitogen-activated protein kinase-mitogen-activated and extracellular signal-regulated kinase (RAS-MAPK-MEK)-driven nerve tumors called neurofibromas. Although MEK inhibitors transiently reduce volumes of most plexiform neurofibromas in mouse models and in neurofibromatosis type 1 (NF1) patients, therapies that increase the efficacy of MEK inhibitors are needed. BI-3406 is a small molecule that prevents Son of Sevenless (SOS)1 interaction with Kirsten rat sarcoma viral oncoprotein (KRAS)-GDP, interfering with the RAS-MAPK cascade upstream of MEK. Single agent SOS1 inhibition had no significant effect in the DhhCre;Nf1 fl/fl mouse model of plexiform neurofibroma, but pharmacokinetics (PK)-driven combination of selumetinib with BI-3406 significantly improved tumor parameters. Tumor volumes and neurofibroma cell proliferation, reduced by MEK inhibition, were further reduced by the combination. Neurofibromas are rich in ionized calcium binding adaptor molecule 1 (Iba1)+ macrophages; combination treatment resulted in small and round macrophages, with altered cytokine expression indicative of altered activation. The significant effects of MEK inhibitor plus SOS1 inhibition in this preclinical study suggest potential clinical benefit of dual targeting of the RAS-MAPK pathway in neurofibromas. SIGNIFICANCE STATEMENT: Interfering with the RAS-mitogen-activated protein kinase (RAS-MAPK) cascade upstream of mitogen activated protein kinase kinase (MEK), together with MEK inhibition, augment effects of MEK inhibition on neurofibroma volume and tumor macrophages in a preclinical model system. This study emphasizes the critical role of the RAS-MAPK pathway in controlling tumor cell proliferation and the tumor microenvironment in benign neurofibromas.

AACE2021-A-1102: Growth Hormone Deficiency in Patient with De Novo Mutation in SHOC2 Causing Noonan Syndrome-Like Disorder with Loose Anagen Hair, and Castleman Syndrome

Clinical manifestations of Noonan syndrome (NS) include short stature, distinctive facial features, webbed/wide neck, cryptorchidism, and congenital heart disease, notably pulmonic stenosis. NS is also recognised to predispose to malignancy – particularly hematological disease. Almost half of all NS cases are due to PTPN11 mutation. Other common mutations include SOS1 and RAF. Several rare mutations and NS subtypes are recognised. All of the known genes encode proteins within the RAS-Mitogen Activated Protein Kinase pathway.

Mutational analysis of Ras hotspots in patients with urothelial carcinoma of the bladder.

BACKGROUNDMutational activation of Ras genes is established as a prognostic factor for the genesis of a constitutively active RAS-mitogen activated protein kinase pathway that leads to cancer. Heterogeneity among the distribution of the most frequent mutations in Ras isoforms is reported in different patient populations with urothelial carcinoma of the bladder (UCB).AIMTo determine the presence/absence of mutations in Ras isoforms in patients with UCB in order to predict disease outcome.METHODSThis study was performed to determine the mutational spectrum at the hotspot regions of H-Ras, K-Ras and N-Ras genes by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing followed by their clinical impact (if any) by examining the relationship of mutational spectrum with clinical histopathological variables in 87 UCB patients.RESULTSNone of the 87 UCB patients showed point mutations in codon 12 of H-Ras gene; codon 61 of N-Ras gene and codons 12, 13 of K-Ras gene by PCR-RFLP. Direct DNA sequencing of tumor and normal control bladder mucosal specimens followed by Blastn alignment with the reference wild-type sequences failed to identify even one nucleotide difference in the coding exons 1 and 2 of H-Ras, N-Ras and K-Ras genes in the tumor and control bladder mucosal specimens.CONCLUSIONOur findings on the lack of mutations in H-Ras, K-Ras and N-Ras genes could be explained on the basis of different etiological mechanisms involved in tumor development/progression, inherent genetic susceptibility, tissue specificity or alternative Ras dysfunction such as gene amplification and/or overexpression in a given cohort of patients.

Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer

Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer

Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer.

Neurofibromin is a tumor suppressor encoded by the NF1 gene, which is mutated in Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to aberrant signaling through the RAS–mitogen-activated protein kinase pathway due to disruption of the neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of most of the neurofibromin protein; to date, biochemical and structural data exist only for its GAP domain and a region containing a Sec-PH motif. To better understand the role of this large protein, here we carried out a series of biochemical and biophysical experiments, including size-exclusion chromatography–multiangle light scattering (SEC-MALS), small-angle X-ray and neutron scattering, and analytical ultracentrifugation, indicating that full-length neurofibromin forms a high-affinity dimer. We observed that neurofibromin dimerization also occurs in human cells and likely has biological and clinical implications. Analysis of purified full-length and truncated neurofibromin variants by negative-stain EM revealed the overall architecture of the dimer and predicted the potential interactions that contribute to the dimer interface. We could reconstitute structures resembling high-affinity full-length dimers by mixing N- and C-terminal protein domains in vitro. The reconstituted neurofibromin was capable of GTPase activation in vitro, and co-expression of the two domains in human cells effectively recapitulated the activity of full-length neurofibromin. Taken together, these results suggest how neurofibromin dimers might form and be stabilized within the cell.

Phenotypic spectrum of Costello syndrome individuals harboring the rare HRAS mutation p.Gly13Asp.

Costello syndrome is part of the RASopathies, a group of neurocardiofaciocutaneous syndromes caused by deregulation of the RAS mitogen-activated protein kinase pathway. Heterozygous mutations in HRAS are responsible for Costello syndrome, with more than 80% of the patients harboring the specific p.Gly12Ser variant. These individuals show a homogeneous phenotype. The clinical characteristics of the Costello syndrome individuals harboring rarer HRAS mutations are less understood, due to the small number of reported cases. Here, we describe the phenotypic spectrum of five additional individuals with HRAS c.38G>A; p.Gly13Asp, including one with somatic mosaicism, and review five previously described cases. The facial and hair abnormalities of the HRAS p.Gly13Asp individuals differ from the typical pattern observed in those showing the common HRAS (p.Gly12Ser) mutation, with less coarse facial features and slow growing, sparse hair with abnormal texture, the latter resembling the pattern observed in Noonan syndrome-like disorder with loose anagen hair and individuals harboring another amino acid substitution in HRAS (p.Gly13Cys). Although some individuals with HRAS p.Gly13Asp developed papillomata and vascular proliferation lesions, no malignant tumors occurred, similar to what was reported for individuals harboring the HRAS p.Gly13Cys. The fact that no malignant tumors were described in these individuals does not allow definitive conclusions about the risk for cancer development. It remains to be determined if substitutions of amino acid 13 in HRAS (p.Gly13Asp and p.Gly13Cys) increase the risk of tumor development.

Cyclophilin B Supports Myc and Mutant p53-Dependent Survival of Glioblastoma Multiforme Cells

Glioblastoma multiforme is an aggressive, treatment-refractory type of brain tumor for which effective therapeutic targets remain important to identify. Here, we report that cyclophilin B (CypB), a prolyl isomerase residing in the endoplasmic reticulum (ER), provides an essential survival signal in glioblastoma multiforme cells. Analysis of gene expression databases revealed that CypB is upregulated in many cases of malignant glioma. We found that suppression of CypB reduced cell proliferation and survival in human glioblastoma multiforme cells in vitro and in vivo. We also found that treatment with small molecule inhibitors of cyclophilins, including the approved drug cyclosporine, greatly reduced the viability of glioblastoma multiforme cells. Mechanistically, depletion or pharmacologic inhibition of CypB caused hyperactivation of the oncogenic RAS-mitogen-activated protein kinase pathway, induction of cellular senescence signals, and death resulting from loss of MYC, mutant p53, Chk1, and Janus-activated kinase/STAT3 signaling. Elevated reactive oxygen species, ER expansion, and abnormal unfolded protein responses in CypB-depleted glioblastoma multiforme cells indicated that CypB alleviates oxidative and ER stresses and coordinates stress adaptation responses. Enhanced cell survival and sustained expression of multiple oncogenic proteins downstream of CypB may thus contribute to the poor outcome of glioblastoma multiforme tumors. Our findings link chaperone-mediated protein folding in the ER to mechanisms underlying oncogenic transformation, and they make CypB an attractive and immediately targetable molecule for glioblastoma multiforme therapy.

Legius Syndrome, an Update.Molecular Pathology of Mutations in SPRED1

Multiple café-au-lait macules (CALMs) are the hallmark of Von Recklinghausen disease, or neurofibromatosis type 1 (NF1). In 2007 we reported that some individuals with multiple CALMs have a heterozygous mutation in the SPRED1 gene and have NF1-like syndrome, or Legius syndrome. Individuals with Legius syndrome have multiple CALMs with or without freckling, but they do not show the typical NF1-associated tumors such as neurofibromas or optic pathway gliomas. NF1-associated bone abnormalities and Lisch nodules are also not reported in patients with Legius syndrome. Consequently, individuals with Legius syndrome require less intense medical surveillance than those with NF1. The SPRED1 gene was identified in 2001 and codes for a protein that downregulates the RAS-mitogen activated protein kinase (RAS-MAPK) pathway; as does neurofibromin, the protein encoded by the NF1 gene. It is estimated that about 1-4% of individuals with multiple CALMs have a heterozygous SPRED1 mutation. Mutational and clinical data on 209 patients with Legius syndrome are tabulated in an online database (http://www.lovd.nl/SPRED1). Mice with homozygous knockout of the Spred1 gene show learning deficits and decreased synaptic plasticity in hippocampal neurons similar to those seen in Nf1 heterozygous mice, underlining the importance of the RAS-MAPK pathway for learning and memory. Recently, specific binding between neurofibromin and SPRED1 was demonstrated. SPRED1 seems to play an important role in recruiting neurofibromin to the plasma membrane.

Ink4a/Arf loss promotes tumor recurrence following Ras inhibition

Aberrant activation of rat sarcoma (Ras) signaling contributes to the development of a variety of human cancers, including gliomas. To determine the dependence of high-grade gliomas on continued Ras signaling, we developed a doxycycline-regulated Kirsten Ras (KRas) glioma mouse model. We previously demonstrated that KRas is required for the maintenance of glioblastoma multiforme tumors arising in the context of activated Akt signaling in vivo; inhibition of KRas expression resulted in apoptotic tumor regression and significantly increased survival. We utilized a well-established glioma mouse model to determine the reliance of gliomas on continued KRas signaling in the context of Ink4a/Arf deficiency, a common occurrence in human gliomas. Despite the dependency of primary gliomas on continued KRas signaling, a significant percentage of tumors progressed to a KRas-independent state in the absence of Ink4a/Arf expression, demonstrating that these tumor suppressors play a critical role in the suppression of glioma recurrence. While even advanced stages of gliomas may remain dependent upon KRas signaling for maintenance and growth, our findings demonstrate that loss of Ink4a/Arf facilitates the acquisition of oncogene independence and tumor recurrence. Furthermore, reactivation of the Ras mitogen-activated protein kinase pathway in the absence of virally delivered KRas expression is a common mechanism of recurrence in this context.

Activated platelet-derived growth factor β receptor and Ras–mitogen-activated protein kinase pathway in natural bovine urinary bladder carcinomas

Bovine papillomavirus types 1 or 2 (BPV-1/2) are involved in the aetiopathogenesis of bovine urinary bladder cancer. BPV-1/2 E5 activates the platelet-derived growth factor β receptor (PDGFβR). The aim of this study was to analyse the Ras/mitogen-activated protein kinase (MAPK) pathway in relation to activation of PDGFβR in natural bovine urinary bladder carcinomas. Co-immunoprecipitation and Western blot analysis demonstrated that recruitment of growth factor receptor bound protein 2 (GRB-2) and Sos-1 to the activated PDGFβR was increased in carcinomas compared to normal tissues. Higher grade bovine urinary bladder carcinomas were associated with activation of Ras, but not with activation of downstream mitogen-activated protein kinase/extracellular signal-regulated kinase (Mek 1/2) or extracellular signal-regulated kinase (Erk 1/2).

Promoter hypermethylation and expression of sprouty 2 in endometrial carcinoma

Sprouty 2 is a key antagonist regulator of receptor tyrosine kinases, and downstream signaling pathways, like fibroblastic growth factor (FGF) and Ras-mitogen-activated protein kinase (RAS-MAPK). By controlling these pathways, sprouty 2 is involved in regulation of cell proliferation, differentiation, and angiogenesis. Alterations in fibroblastic growth factor receptor (FGFR) and members of the RAS-MAPK pathway are frequent in endometrial carcinoma. The expression of sprouty 2 has been found to be decreased in several types of human cancer, by mechanisms of promoter methylation. In the present study, we have assessed the expression of sprouty 2 in endometrial carcinoma, in correlation with sprouty 2 promoter methylation. Sprouty 2 immunohistochemical expression was assessed using 3 different tissue microarrays: one constructed from paraffin blocks of 80 samples of normal endometrium and 2 tissue microarrays containing samples of 157 endometrial carcinoma (1 tissue microarray constructed with 95 endometrial carcinomas previously studied for microsatellite instability and alterations in phosphatase and tensin homolog (PTEN), k-ras, and b-catenin, and 1 tissue microarray containing 62 endometrial carcinoma, which were also subjected to sprouty 2 promoter methylation analysis). The immunohistochemical expression of sprouty 2 was correlated with cellular proliferation (Ki67) and clinicopathologic data. Sprouty 2 promoter methylation was assessed by methylation-specific polymerase chain reaction, with DNA obtained from fresh-frozen samples of endometrial carcinoma and corresponding normal tissues, and correlated with promoter methylation of RAS association domain family-1A (RASSF1A). A highly significant decrease in sprouty 2 immunoexpression was seen in the proliferative phase of normal endometrium (P < .001). Differences were detected between types I and II endometrial carcinoma, but they were not statistically significant. Reduced immunoexpression of sprouty 2 was seen in 19.85% of endometrial carcinoma and was strongly and inversely associated with increased cell proliferation (Ki67; r = -0.367; P = .001). Sprouty 2 promoter methylation was detected in 31 (53.4%) of 58 endometrial carcinomas. Results from our study show that alterations in sprouty 2 may be involved in endometrial carcinogenesis by controlling cell proliferation.

Noonan, Costello and cardio–facio–cutaneous syndromes: dysregulation of the Ras–MAPK pathway

A class of developmental disorders caused by dysregulation of the Ras-induced mitogen-activated protein kinase (MAPK) cascade (the Ras-MAPK pathway) has emerged. Three of these disorders - Noonan, Costello and cardio-facio-cutaneous syndromes - have overlapping phenotypic features characterised by distinctive facial dysmorphia, cardiac defects, musculoskeletal and cutaneous abnormalities, and neurocognitive delay. The germline mutations associated with these disorders are in genes that encode proteins of the Ras-MAPK pathway. In vitro studies have determined that the overwhelming majority of these mutations result in increased signal transduction down the pathway, but usually to a lesser degree than somatic mutations in the same genes that are associated with cancer. The Ras-MAPK pathway is essential in the regulation of the cell cycle, differentiation, growth and senescence, so it is not surprising that germline mutations that affect its function have profound effects on development. Here we review the clinical consequences of the known molecular lesions associated with Noonan syndrome, Costello syndrome and cardio-facio-cutaneous syndrome, and explore possible therapeutic modalities for treatment.

Chromatin organization and nuclear microenvironments in cancer cells

Nuclear morphometric descriptors such as nuclear size, shape, DNA content and chromatin organization are used by pathologists as diagnostic markers for cancer. However, our knowledge of events resulting in changes in nuclear shape and chromatin organization in cancer cells is limited. Nuclear matrix proteins, which include lamins, transcription factors (Sp1) and histone modifying enzymes (histone deacetylases), and histone modifications (histone H3 phosphorylation) have roles in organizing chromatin in the interphase nucleus, regulating gene expression programs and determining nuclear shape. Histone H3 phosphorylation, a downstream target of the Ras-mitogen activated protein kinase pathway, is involved in neoplastic transformation. This article will review genetic and epigenetic events that alter chromatin organization in cancer cells and the role of the nuclear matrix in determining nuclear morphology.

Stimulation of the Ras-MAPK pathway leads to independent phosphorylation of histone H3 on serine 10 and 28

The Ras-mitogen activated protein kinase (Ras-MAPK) pathway plays an integral role in the formation of human malignancies. Stimulation of this pathway results in phosphorylation of histone H3 at serines 10 and 28 and expression of immediate-early genes. Phosphorylated (serine 10) H3, which is also acetylated on lysine 14, is associated with immediate-early genes. In this report, we investigated the relationship between these two H3 phosphorylation events in parental and ras-transformed fibroblasts. Immunoblot analyses of two-dimensional gel patterns demonstrated that all three H3 variants were phosphorylated after stimulation of the Ras-MAPK pathway and during mitosis. Following stimulation of the Ras-MAPK pathway, H3 phosphorylated on serines 10 and 28 was excluded from regions of highly condensed chromatin and was present in increased levels in ras-transformed cells. Although H3 phosphorylated at serine 10 or 28 was dynamically acetylated, H3 phosphorylated at serine 28 had a higher steady state of acetylation than that of H3 phosphorylated at serine 10. When visualized with indirect immunofluorescence, most foci of phosphorylated serine 28 H3 did not co-localize with foci of H3 phosphorylated on serine 10 or phosphoacetylated on serine 10 and lysine 14, suggesting that these two phosphorylation events act separately to promote gene expression.

Nitrogen-containing bisphosphonate, YM529/ONO-5920 (a novel minodronic acid), inhibits RANKL expression in a cultured bone marrow stromal cell line ST2

Increase in bone resorption by osteoclasts can cause metabolic bone diseases, such as osteoporosis. Recent attention has been paid to the receptor activator of the NF-κB ligand (RANKL), an accelerator of osteoclast differentiation. RANKL is expressed on the bone marrow-derived stromal cell membrane and induces the differentiation of osteoclasts by binding to RANK expressed on the osteoclast precursor cell membrane. Since the inhibition of RANKL expression can lead to the inhibition of osteoclastic bone resorption, the clinical application of RANKL inhibition could be expected to have a major effect on metabolic bone disease therapy. In this study, we investigated whether or not YM529/ONO-5920, a nitrogen-containing bisphosphonate (a novel minodronic acid), inhibits RANKL expression in a bone marrow-derived stromal cell line (ST2 cells). Reverse transcription-polymerase chain reaction revealed that the administration of YM529/ONO-5920 to ST2 cells inhibited RANKL mRNA expression and reduced RANKL proteins as assessed by Western blot analysis. The inhibition of RANKL mRNA expression was reversed when geranylgeranyl pyrophosphate (GGPP), an intermediate in the mevalonate pathway, was used in combination. Furthermore, YM529/ONO-5920 reduced phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), and similarly, U0126, a mitogen-activated protein kinase kinase 1/2 inhibitor, inhibited RANKL expression. Pretreatment with GGPP reversed the YM529/ONO-5920-induced decrease in phosphorylation of ERK. Furthermore, YM529/ONO-5920 decreased TRAP-positive cells in co-culture of ST2 cells and an osteoclast cell line, C7 cells, and this decrease was inhibited by pretreatment with GGPP. This indicates that YM529/ONO-5920 inhibits GGPP biosynthesis in the mevalonate pathway and then signal transduction in the Ras–mitogen-activated protein kinase pathway, thereby inhibiting RANKL expression on ST2 cells. These results suggest a newly elucidated action of bisphosphonates in the inhibition of bone resorption.

Involvement of gangliosides in proliferation of immortalized neural progenitor cells

The CNS consists of neuronal and glial cells generated from common neural progenitor cells during development. Cellular events for neural progenitor cells, such as proliferation and differentiation, are regulated by multiple intrinsic and extrinsic cell signals. Although much is known on the importance of the proteinous factors in regulating the fate of neural progenitor cells, the involvement of other molecules such as gangliosides, sialic acid-containing glycosphingolipids, remains to be clarified. To elucidate the biological functions of gangliosides in neural progenitor cells, we transfected an immortalized neural progenitor cell line, C17.2, which does not express GD3 ganglioside, with a fusion protein of GD3-synthase (ST-II) and enhanced green fluorescent protein (ST-II-EGFP). Analysis of the ST-II transfectants revealed the ectopic expression of b- and c-series gangliosides. In the ST-II transfectants, proliferation induced by epidermal growth factor (EGF) was severely retarded. EGF-induced proliferation of C17.2 cells was dependent on the Ras-mitogen-activated protein kinase (Ras-MAPK) pathway, and the EGF-induced activation of this pathway was significantly repressed in the transfectants. Thus, ST-II overexpression retarded proliferation of C17.2 cells via repression of the Ras-MAPK pathway. The result supports the concept that gangliosides may play an important role in regulating the proliferation of neural progenitor cells.

A directed screen for genes involved in Drosophila blood cell activation.

An attack by a parasitic wasp activates a vigorous cellular immune response in Drosophila larvae. This response is manifested by an increased number of circulating cells, the hemocytes, and by the appearance of a specialized class of hemocyte, the lamellocytes, which participate in the encapsulation and killing of the parasite. To study the molecular mechanisms of this response, we have overexpressed different genes in the hemocytes, by using the GAL4-upstream activating sequence system and a hemocyte-specific Hemese-GAL4 driver. Multiple transgenes were tested, representing several important signaling pathways. We found that the proliferation response and the activation of lamellocyte formation are independent phenomena. A drastic increase in the number of circulating hemocytes is caused by receptor tyrosine kinases, such as Egfr, Pvr, and Alk, as well as by the downstream signaling components Ras85D and pointed, supporting the notion that the Ras-mitogen-activated protein kinase pathway regulates hemocyte numbers. In the case of Pvr and Alk, this phenotype also is accompanied by lamellocyte formation. By contrast, constitutively active hopscotch and hemipterous give massive activation of lamellocyte formation with little or no increase in total hemocyte numbers. This finding indicates that both the Jak/Stat and the Jun kinase pathways affect lamellocyte formation. Still other signals, mediated by aop(ACT), Toll(10b), and Rac1 expression, cause a simultaneous increase in lamellocyte and total cell numbers, and the same effect is seen when WNT signaling is suppressed. We conclude that the activation of a cellular response is complex and affected by multiple signaling pathways.

Regulation of Ras-MAPK pathway mitogenic activity by restricting nuclear entry of activated MAPK in endoderm differentiation of embryonic carcinoma and stem cells.

In response to retinoic acid, embryonic stem and carcinoma cells undergo differentiation to embryonic primitive endoderm cells, accompanied by a reduction in cell proliferation. Differentiation does not reduce the activation of cellular MAPK/Erk, but does uncouple mitogen-activated protein kinase (MAPK) activation from phosphorylation/activation of Elk-1 and results in inhibition of c-Fos expression, whereas phosphorylation of the cytoplasmic substrate p90RSK remains unaltered. Cell fractionation and confocal immunofluorescence microscopy demonstrated that activated MAPK is restricted to the cytoplasmic compartment after differentiation. An intact actin and microtubule cytoskeleton appears to be required for the restriction of MAPK nuclear entry induced by retinoic acid treatment because the cytoskeletal disrupting agents nocodazole, colchicine, and cytochalasin D are able to revert the suppression of c-Fos expression. Thus, suppression of cell proliferation after retinoic acid–induced endoderm differentiation of embryonic stem and carcinoma cells is achieved by restricting nuclear entry of activated MAPK, and an intact cytoskeleton is required for the restraint.

The Ras1–Mitogen-Activated Protein Kinase Signal Transduction Pathway Regulates Synaptic Plasticity through Fasciclin II-Mediated Cell Adhesion

Ras proteins are small GTPases with well known functions in cell proliferation and differentiation. In these processes, they play key roles as molecular switches that can trigger distinct signal transduction pathways, such as the mitogen-activated protein kinase (MAPK) pathway, the phosphoinositide-3 kinase pathway, and the Ral–guanine nucleotide dissociation stimulator pathway. Several studies have implicated Ras proteins in the development and function of synapses, but the molecular mechanisms for this regulation are poorly understood. Here, we demonstrate that the Ras–MAPK pathway is involved in synaptic plasticity at theDrosophilalarval neuromuscular junction. Both Ras1 and MAPK are expressed at the neuromuscular junction, and modification of their activity levels results in an altered number of synaptic boutons. Gain- or loss-of-function mutations in Ras1 and MAPK reveal that regulation of synapse structure by this signal transduction pathway is dependent on fasciclin II localization at synaptic boutons. These results provide evidence for a Ras-dependent signaling cascade that regulates fasciclin II-mediated cell adhesion at synaptic terminals during synapse growth.