The C. elegans G-protein-coupled receptor SRA-13 inhibits RAS/MAPK signalling during olfaction and vulval development

In C. elegans, the RAS/MAPK pathway is used in different tissues to regulate various cell fate decisions. Several positive and negative regulators tightly control the activity of the RAS/MAPK pathway at different steps. We demonstrate a link between a G-protein-coupled receptor signalling pathway and the RAS/MAPK cascade. SRA-13, a member of the SRA family of chemosensory receptors, negatively regulates RAS/MAPK signalling during vulval induction and the olfaction of volatile attractants. Epistasis analysis indicates that SRA-13 inhibits the RAS/MAPK pathway at the level or upstream of MAPK. In both tissues, the vulval precursor cells and the chemosensory neurones, SRA-13 acts through the GPA-5 Galpha protein subunit, suggesting a common mechanism of crosstalk. Moreover, we find that vulval induction is repressed by food withdrawal during larval development and that SRA-13 activity is required for the suppression of vulval induction in response to food starvation. Thus, SRA-13 may serve to adapt the activity of the RAS/MAPK pathway to environmental conditions.     

Distinct roles of the Pumilio and FBF translational repressors during C. elegans vulval development

The C. elegans PUF and FBF proteins regulate various aspects of germline development by selectively binding to the 3' untranslated region of their target mRNAs and repressing translation. Here, we show that puf-8, fbf-1 and fbf-2 also act in the soma where they negatively regulate vulvaI development. Loss-of-function mutations in puf-8 cause ectopic vulval differentiation when combined with mutations in negative regulators of the EGFR/RAS/MAPK pathway and suppress the vulvaless phenotype caused by mutations that reduce EGFR/RAS/MAPK signalling. PUF-8 acts cell-autonomously in the vulval cells to limit their temporal competence to respond to the extrinsic patterning signals. fbf-1 and fbf-2, however, redundantly inhibit primary vulval cell fate specification in two distinct pathways acting in the soma and in the germline. The FBFs thereby ensure that the inductive signal selects only one vulval precursor cell for the primary cell fate. Thus, translational repressors regulate various aspects of vulval cell fate specification, and they may play a conserved role in modulating signal transduction during animal development.     

A conserved PTEN/FOXO pathway regulates neuronal morphology during C. elegans development

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is a conserved signal transduction cascade that is fundamental for the correct development of the nervous system. The major negative regulator of PI3K signaling is the lipid phosphatase DAF-18/PTEN, which can modulate PI3K pathway activity during neurodevelopment. Here, we identify a novel role for DAF-18 in promoting neurite outgrowth during development in Caenorhabditis elegans. We find that DAF-18 modulates the PI3K signaling pathway to activate DAF-16/FOXO and promote developmental neurite outgrowth. This activity of DAF-16 in promoting outgrowth is isoform-specific, being effected by the daf-16b isoform but not the daf-16a or daf-16d/f isoform. We also demonstrate that the capacity of DAF-16/FOXO in regulating neuron morphology is conserved in mammalian neurons. These data provide a novel mechanism by which the conserved PI3K signaling pathway regulates neuronal cell morphology during development through FOXO.     

C. elegans DAF-18/PTEN mediates nutrient-dependent arrest of cell cycle and growth in the germline

The molecular pathways that link nutritional cues to developmental programs are poorly understood. Caenorhabditis elegans hatchlings arrest in a dormant state termed "L1 diapause" until food is supplied. However, little is known about what signal transduction pathways mediate nutritional status to control arrest and initiation of postembryonic development. We report that C. elegans embryonic germline precursors undergo G2 arrest with condensed chromosomes and remain arrested throughout L1 diapause. Loss of the DAF-18/PTEN tumor suppressor bypasses this arrest, resulting in inappropriate germline growth dependent on the AGE-1/PI-3 and AKT-1/PKB kinases. DAF-18 also regulates an insulin/IGF-like pathway essential for longevity and dauer larva formation. However, DAF-16/FoxO, which is repressed by this pathway, is not required for germline arrest in L1 diapause. Thus, these findings indicate that quiescence of germline development during L1 diapause is not a passive consequence of nutrient deprivation, but rather is actively maintained by DAF-18 through a pathway distinct from that which regulates longevity and dauer formation.     

The cross-talk of NOTCH and GSK-3 signaling in colon and other cancers

The GSK-3 kinases, GSK-3α and GSK-3β, have a central role in regulating multiple cellular processes such as glycogen synthesis, insulin signaling, cell proliferation and apoptosis. GSK-3β is the most well studied, and was originally described for its role in regulating glycogen synthase. GSK-3β has been studied as a participant in the oncogenic process in a variety of cancers due to its intersection with the PTEN/PI3K/AKT and RAS/RAF/MEK/ERK pathways. Dysregulated signaling through the Notch family of receptors can also promote oncogenesis. Normal Notch receptor signaling regulates cell fate determination in stem cell pools. GSK-3β and Notch share similar targets such β-catenin and the WNT pathway. WNT and β-catenin are involved in several oncogenic processes including those of the colon. In addition, GSK-3β may directly regulate aspects of Notch signaling. This review describes how crosstalk between GSK-3β and Notch can promote oncogenesis, using colon cancer as the primary example.     

Tissue-specific functions of the Caenorhabditis elegans p120 Ras GTPase activating protein GAP-3

All metazoan genomes encode multiple RAS GTPase activating proteins (RasGAPs) that negatively regulate the conserved RAS/MAPK signaling pathway. In mammals, several RasGAPs exhibit tumor suppressor activity by preventing excess RAS signal transduction. We have identified gap-3 as the to date missing Caenorhabditiselegans member of the p120 RasGAP family. By studying the genetic interaction of gap-3 with the two previously identified RasGAPs gap-1 and gap-2, we find that different combinations of RasGAPs are used to repress LET-60 RAS signaling depending on the cellular context. GAP-3 is the predominant negative regulator of RAS during meiotic progression of the germ cells, while GAP-1 is the key inhibitor of RAS during vulval induction. In other tissues such as the sex myoblasts or the chemosensory neurons, all three RasGAPs act in concert. The C. elegans RasGAPs have thus undergone partial specialization after gene duplication to allow the differential regulation of the RAS/MAPK signaling pathway in different cell types. A similar tissue specialization of the human tumor suppressor genes may explain the strong bias in the type of cancer they promote when mutated.     

Alphabet, a Ser/Thr phosphatase of the protein phosphatase 2C family, negatively regulates RAS/MAPK signaling in Drosophila

Signal transduction through the RAS/mitogen-activated protein kinase (MAPK) pathway depends on a diverse collection of proteins regulating positively and negatively signaling flow. We previously conducted a genetic screen in Drosophila to identify novel components of this signaling pathway. Here, we present the identification and characterization of a new gene, alphabet (alph), whose activity negatively regulates RAS/MAPK-dependent developmental processes in Drosophila and this, at a step downstream or in parallel to RAS. alph encodes a protein phosphatase 2C (PP2C) family member closely related to the mammalian PP2C alpha and beta isoforms. Interestingly, although alph gene product does not appear to be essential for viability, its elimination leads to weak but significant developmental defects reminiscent of an overactivated RAS/MAPK pathway. Consistent with this interpretation, strong genetic interactions are observed between alph alleles and mutations in bona fide components of the pathway. Together, this work identifies a PP2C of the alpha/beta subfamily as a novel negative regulator of the RAS/MAPK pathway and suggests that these evolutionarily conserved enzymes play a similar role in other metazoans. Finally, despite the relatively large size of the PP2C gene family in metazoans, this study represents only the second genetic characterization of a PP2C in these organisms.     

Cell fate-specific regulation of EGF receptor trafficking during Caenorhabditis elegans vulval development

By controlling the subcellular localization of growth factor receptors, cells can modulate the activity of intracellular signal transduction pathways. During Caenorhabditis elegans vulval development, a ternary complex consisting of the LIN-7, LIN-2 and LIN-10 PDZ domain proteins localizes the epidermal growth factor receptor (EGFR) to the basolateral compartment of the vulval precursor cells (VPCs) to allow efficient receptor activation by the inductive EGF signal from the anchor cell. We have identified EGFR substrate protein-8 (EPS-8) as a novel component of the EGFR localization complex that links receptor trafficking to cell fate specification. EPS-8 expression is upregulated in the primary VPCs, where it creates a positive feedback loop in the EGFR/RAS/MAPK pathway. The membrane-associated guanylate kinase LIN-2 recruits EPS-8 into the receptor localization complex to retain the EGFR on the basolateral plasma membrane, and thus allow maximal receptor activation in the primary cell lineage. Low levels of EPS-8 in the neighboring secondary VPCs result in the rapid degradation of the EGFR, allowing these cells to adopt the secondary cell fate. Extracellular signals thus regulate EGFR trafficking in a cell type-specific manner to control pattern formation during organogenesis.     

bFGF-mediated pluripotency maintenance in human induced pluripotent stem cells is associated with NRAS-MAPK signaling

Background

Human pluripotent stem cells (PSCs) open new windows for basic research and regenerative medicine due to their remarkable properties, i.e. their ability to self-renew indefinitely and being pluripotent. There are different, conflicting data related to the role of basic fibroblast growth factor (bFGF) in intracellular signal transduction and the regulation of pluripotency of PSCs. Here, we investigated the effect of bFGF and its downstream pathways in pluripotent vs. differentiated human induced (hi) PSCs.

Methods

bFGF downstream signaling pathways were investigated in long-term culture of hiPSCs from pluripotent to differentiated state (withdrawing bFGF) using immunoblotting, immunocytochemistry and qPCR. Subcellular distribution of signaling components were investigated by simple fractionation and immunoblotting upon bFGF stimulation. Finally, RAS activity and RAS isoforms were studied using RAS assays both after short- and long-term culture in response to bFGF stimulation.

Results

Our results revealed that hiPSCs were differentiated into the ectoderm lineage upon withdrawing bFGF as an essential pluripotency mediator. Pluripotency markers OCT4, SOX2 and NANOG were downregulated, following a drastic decrease in MAPK pathway activity levels. Notably, a remarkable increase in phosphorylation levels of p38 and JAK/STAT3 was observed in differentiated hiPSCs, while the PI3K/AKT and JNK pathways remained active during differentiation. Our data further indicate that among the RAS paralogs, NRAS predominantly activates the MAPK pathway in hiPSCs.

Conclusion

Collectively, the MAPK pathway appears to be the prime signaling pathway downstream of bFGF for maintaining pluripotency in hiPSCs and among the MAPK pathways, the activity of NRAS-RAF-MEK-ERK is decreased during differentiation, whereas p38 is activated and JNK remains constant.

     

The Caenorhabditis elegans homologue of the proto-oncogene ect-2 positively regulates RAS signalling during vulval development

Guanine nucleotide exchange factors (GEFs) regulate the activity of small GTP-binding proteins in a variety of biological processes. We have identified a gain-of-function mutation in the Caenorhabditis elegans GEF ect-2, the homologue of the mammalian ect2 proto-oncogene that has an essential role during cytokinesis. Here, we report that, in addition to its known function during mitosis, ECT-2 promotes the specification of the primary vulval cell fate by activating RAS/mitogen-activated protein kinase (MAPK) signalling before the end of the S-phase. Epistasis analysis indicates that ECT-2 crosstalks to the canonical RAS/MAPK cascade upstream of the RAS GEF SOS-1 by means of a RHO-1 signalling pathway. Our results raise the possibility that the transforming activity of the mammalian ect-2 oncogene could be due to hyperactivation of the RAS/MAPK pathway.