Neuronal KGB-1 JNK MAPK signaling regulates the dauer developmental decision in response to environmental stress in Caenorhabditis elegans

In response to stressful growth conditions of high population density, food scarcity, and elevated temperature, young larvae of nematode Caenorhabditis elegans can enter a developmentally arrested stage called dauer that is characterized by dramatic anatomic and metabolic remodeling. Genetic analysis of dauer formation of C. elegans has served as an experimental paradigm for the identification and characterization of conserved neuroendocrine signaling pathways. Here, we report the identification and characterization of a conserved c-Jun N-terminal Kinase-like mitogen-activated protein kinase (MAPK) pathway that is required for dauer formation in response to environmental stressors. We observed that loss-of-function mutations in the MLK-1-MEK-1-KGB-1 MAPK pathway suppress dauer entry. A loss-of-function mutation in the VHP-1 MAPK phosphatase, a negative regulator of KGB-1 signaling, results in constitutive dauer formation, which is dependent on the presence of dauer pheromone but independent of diminished food levels or elevated temperatures. Our data suggest that the KGB-1 pathway acts in the sensory neurons, in parallel to established insulin and TGF-β signaling pathways, to transduce the dauer-inducing environmental cues of diminished food levels and elevated temperature.     

Caenorhabditis elegans SDF-9 enhances insulin/insulin-like signaling through interaction with DAF-2

SDF-9 is a modulator of Caenorhabditis elegans insulin/IGF-1 signaling that may interact directly with the DAF-2 receptor. SDF-9 is a tyrosine phosphatase-like protein that, when mutated, enhances many partial loss-of-function mutants in the dauer pathway except for the temperature-sensitive mutant daf-2(m41). We propose that SDF-9 stabilizes the active phosphorylated state of DAF-2 or acts as an adaptor protein to enhance insulin-like signaling.     

C. elegans STAT cooperates with DAF-7/TGF-beta signaling to repress dauer formation

The DAF-7/TGF-beta pathway in C. elegans interprets environmental signals relayed through amphid neurons and actively inhibits dauer formation during reproductive developmental growth . In metazoans, the STAT pathway interprets external stimuli through regulated tyrosine phosphorylation, nuclear translocation, and gene expression , but its importance for developmental commitment, particularly in conjunction with TGF-beta, remains largely unknown. Here, we report that the nematode STAT ortholog STA-1 accumulated in the nuclei of five head neuron pairs, three of which are amphid neurons involved in dauer formation . Moreover, sta-1 mutants showed a synthetic dauer phenotype with selected TGF-beta mutations. sta-1 deficiency was complemented by reconstitution with wild-type protein, but not with a tyrosine mutant. Canonical TGF-beta signaling involves the DAF-7/TGF-beta ligand activating the DAF-1/DAF-4 receptor pair to regulate the DAF-8/DAF-14 Smads . Interestingly, STA-1 functioned in the absence of DAF-7, DAF-4, and DAF-14, but it required DAF-1 and DAF-8. Additionally, STA-1 expression was induced by TGF-beta in a DAF-3-dependent manner, demonstrating a homeostatic negative feedback loop. These results highlight a role for activated STAT proteins in repression of dauer formation. They also raise the possibility of an unexpected function for DAF-1 and DAF-8 that is independent of their normal upstream activator, DAF-7.     

Molecular cloning, chromosomal mapping, and developmental expression of a novel protein tyrosine phosphatase-like gene

Protein tyrosine phosphatases (PTPs) mediate the dephosphorylation of phosphotyrosine. PTPs are known to be involved in many signal transduction pathways leading to cell growth, differentiation, and oncogenic transformation. We have cloned a new family of novel protein tyrosine phosphatase-like genes, the Ptpl (protein tyrosine phosphatase-like; proline instead of catalytic arginine) gene family. This gene family is composed of at least three members, and we describe here the developmental expression pattern and chromosomal location for one of these genes, Ptpla. In situ hybridization studies revealed that Ptpla expression was first detected at embryonic day 8.5 in muscle progenitors and later in differentiated muscle types: in the developing heart, throughout the liver and lungs, and in a number of neural crest derivatives including the dorsal root and trigeminal ganglia. Postnatally Ptpla was expressed in a number of adult tissues including cardiac and skeletal muscle, liver, testis, and kidney. The early expression pattern of this gene and its persistent expression in adult tissues suggest that it may have an important role in the development, differentiation, and maintenance of a number of different tissue types. The human homologue of Ptpla (PTPLA) was cloned and shown to map to 10p13-p14.     

daf-1, a C. elegans gene controlling dauer larva development, encodes a novel receptor protein kinase

The dauer larva is a developmentally arrested, non-feeding dispersal stage normally formed in response to overcrowding and limited food. The daf-1 gene specifies an intermediate step in a hierarchy of genes thought to specify a pathway for neural transduction of environmental cues. Mutations in daf-1 result in constitutive formation of dauer larvae even in abundant food. This gene has been cloned by Tc1-transposon tagging, and it appears to encode a new class of serine/threonine kinase. A daf-1 probe detects a 2.5 kb mRNA of low abundance, and the DNA sequence indicates that the gene encodes a 669 amino acid protein, with a putative transmembrane domain and a C-terminal protein kinase domain most closely related to the cytosolic, raf proto-oncogene family. Hence, the daf-1 product appears to be a cell-surface receptor required for transduction of environmental signals into an appropriate developmental response.     

The C. elegans PH domain protein CED-12 regulates cytoskeletal reorganization via a Rho/Rac GTPase signaling pathway.

The C. elegans gene ced-12 functions in the engulfment of apoptotic cells and in cell migration, acting in a signaling pathway with ced-2 Crkll, ced-5 DOCK180, and ced-10 Rac GTPase and acting upstream of ced-10 Rac. ced-12 encodes a protein with a pleckstrin homology (PH) domain and an SH3 binding motif, both of which are important for ced-12 function. CED-12 acts in engulfing cells for cell corpse engulfment and interacts physically with CED-5, which contains an SH3 domain. CED-12 has Drosophila and human counterparts. Expression of CED-12 and its counterparts in murine Swiss 3T3 fibroblasts induced Rho GTPase-dependent formation of actin filament bundles. We propose that through interactions with membranes and with a CED-2/CED-5 protein complex, CED-12 regulates Rho/Rac GTPase signaling and leads to cytoskeletal reorganization by an evolutionarily conserved mechanism.     

UNC-16, a JNK-signaling scaffold protein, regulates vesicle transport in C. elegans.

Transport of synaptic components is a regulated process. Loss-of-function mutations in the C. elegans unc-16 gene result in the mislocalization of synaptic vesicle and glutamate receptor markers. unc-16 encodes a homolog of mouse JSAP1/JIP3 and Drosophila Sunday Driver. Like JSAP1/JIP3, UNC-16 physically interacts with JNK and JNK kinases. Deletion mutations in Caenorhabditis elegans JNK and JNK kinases result in similar mislocalization of synaptic vesicle markers and enhance weak unc-16 mutant phenotypes. unc-116 kinesin heavy chain mutants also mislocalize synaptic vesicle markers, as well as a functional UNC-16::GFP. Intriguingly, unc-16 mutations partially suppress the vesicle retention defect in unc-104 KIF1A kinesin mutants. Our results suggest that UNC-16 may regulate the localization of vesicular cargo by integrating JNK signaling and kinesin-1 transport.     

The cell junction protein VAB-9 regulates adhesion and epidermal morphology in C. elegans

Epithelial cell junctions are essential for cell polarity, adhesion and morphogenesis. We have analysed VAB-9, a cell junction protein in Caenorhabditis elegans. VAB-9 is a predicted four-pass integral membrane protein that has greatest similarity to BCMP1 (brain cell membrane protein 1, a member of the PMP22/EMP/Claudin family of cell junction proteins) and localizes to the adherens junction domain of C. elegans apical junctions. Here, we show that VAB-9 requires HMR-1/cadherin for localization to the cell membrane, and both HMP-1/alpha-catenin and HMP-2/beta-catenin for maintaining its distribution at the cell junction. In vab-9 mutants, morphological defects correlate with disorganization of F-actin at the adherens junction; however, localization of the cadherin-catenin complex and epithelial polarity is normal. These results suggest that VAB-9 regulates interactions between the cytoskeleton and the adherens junction downstream of or parallel to alpha-catenin and/or beta-catenin. Mutations in vab-9 enhance adhesion defects through functional loss of the cell junction genes apical junction molecule 1 (ajm-1) and discs large 1 (dlg-1), suggesting that VAB-9 is involved in cell adhesion. Thus, VAB-9 represents the first characterized tetraspan adherens junction protein in C. elegans and defines a new family of such proteins in higher eukaryotes.     

A hormonal signaling pathway influencing C. elegans metabolism, reproductive development, and life span.

During C. elegans development, animals must choose between reproductive growth or dauer diapause in response to sensory cues. Insulin/IGF-I and TGF-beta signaling converge on the orphan nuclear receptor daf-12 to mediate this choice. Here we show that daf-9 acts downstream of these inputs but upstream of daf-12. daf-9 and daf-12 mutants have similar larval defects and modulate insulin/IGF-I and gonadal signals that regulate adult life span. daf-9 encodes a cytochrome P450 related to vertebrate steroidogenic hydroxylases, suggesting that it could metabolize a DAF-12 ligand. Sterols may be the daf-9 substrate and daf-12 ligand because cholesterol deprivation phenocopies mutant defects. Sensory neurons, hypodermis, and somatic gonadal cells expressing daf-9 identify potential endocrine tissues. Evidently, lipophilic hormones influence nematode metabolism, diapause, and life span.     

NCR-1 and NCR-2, the C. elegans homologs of the human Niemann-Pick type C1 disease protein, function upstream of DAF-9 in the dauer formation pathways

Mutations in the human NPC1 gene cause most cases of Niemann-Pick type C (NP-C) disease, a fatal autosomal recessive neurodegenerative disorder. NPC1 is implicated in intracellular trafficking of cholesterol and glycolipids, but its exact function remains unclear. The C. elegans genome contains two homologs of NPC1, ncr-1 and ncr-2, and an ncr-2; ncr-1 double deletion mutant forms dauer larvae constitutively (Daf-c). We have analyzed the phenotypes of ncr single and double mutants in detail, and determined the ncr gene expression patterns. We find that the ncr genes function in a hormonal branch of the dauer formation pathway upstream of daf-9 and daf-12, which encode a cytochrome P450 enzyme and a nuclear hormone receptor, respectively. ncr-1 is expressed broadly in tissues with high levels of cholesterol, whereas expression of ncr-2 is restricted to a few cells. Both Ncr genes are expressed in the XXX cells, which are implicated in regulating dauer formation via the daf-9 pathway. Only the ncr-1 mutant is hypersensitive to cholesterol deprivation and to progesterone, an inhibitor of intracellular cholesterol trafficking. Our results support the hypothesis that ncr-1 and ncr-2 are involved in intracellular cholesterol processing in C. elegans, and that a sterol-signaling defect is responsible for the Daf-c phenotype of the ncr-2; ncr-1 mutant.     

Hormonal signals produced by DAF-9/cytochrome P450 regulate C. elegans dauer diapause in response to environmental cues

In response to the environment, the nematode C. elegans must choose between arrest at a long-lived alternate third larval stage, the dauer diapause, or reproductive development. This decision may ultimately be mediated by daf-9, a cytochrome P450 related to steroidogenic hydroxylases and its cognate nuclear receptor daf-12, implying organism-wide coordination by lipophilic hormones. Accordingly, here we show that daf-9(+) works cell non-autonomously to bypass diapause, and promote gonadal outgrowth. Among daf-9-expressing cells, the hypodermis is most visibly regulated by environmental inputs, including dietary cholesterol. On in reproductive growth, off in dauer, hypodermal daf-9 expression is strictly daf-12 dependent, suggesting feedback regulation. Expressing daf-9 constitutively in hypodermis rescues dauer phenotypes of daf-9, as well as insulin/IGF receptor and TGFbeta mutants, revealing that daf-9 is an important downstream point of control within the dauer circuits. This study illuminates how endocrine networks integrate environmental cues and transduce them into adaptive life history choices.     

Hyperactivation of the G12-mediated signaling pathway in Caenorhabditis elegans induces a developmental growth arrest via protein kinase C

The G(12) type of heterotrimeric G-proteins play an important role in development and behave as potent oncogenes in cultured cells. However, little is known about the molecular nature of the components that act in the G(12)-signaling pathway in an organism. We characterized a C. elegans Galpha subunit gene, gpa-12, which is a homolog of mammalian G(12)/G(13)alpha, and found that animals defective in gpa-12 are viable. Expression of activated GPA-12 (G(12)QL) results in a developmental growth arrest caused by a feeding behavior defect that is due to a dramatic reduction in pharyngeal pumping. To elucidate the molecular nature of the signaling pathways in which G(12) participates, we screened for suppressors of the G(12)QL phenotype. We isolated 50 suppressors that contain mutations in tpa-1, which encodes two protein kinase C isoforms, TPA-1A and TPA-1B, most similar to PKCtheta/delta. TPA-1 mediates the action of the tumor promoter PMA. Expression of G(12)QL and treatment of wild-type animals with PMA induce an identical growth arrest caused by inhibition of larval feeding, which is dependent on TPA-1A and TPA-1B function. These results suggest that TPA-1 is a downstream target of both G(12) signaling and PMA in modulating feeding and growth in C. elegans. Taken together, our findings provide a potential molecular mechanism for the transforming capability of G(12) proteins.     

Striatal‐enriched protein tyrosine phosphatase regulates the PTPα/Fyn signaling pathway

The tyrosine kinase Fyn has two regulatory tyrosine residues that when phosphorylated either activate (Tyr⁴²⁰) or inhibit (Tyr⁵³¹) Fyn activity. Within the central nervous system, two protein tyrosine phosphatases (PTPs) target these regulatory tyrosines in Fyn. PTPα dephosphorylates Tyr⁵³¹ and activates Fyn, while STEP (STriatal‐Enriched protein tyrosine Phosphatase) dephosphorylates Tyr⁴²⁰ and inactivates Fyn. Thus, PTPα and STEP have opposing functions in the regulation of Fyn; however, whether there is cross talk between these two PTPs remains unclear. Here, we used molecular techniques in primary neuronal cultures and in vivo to demonstrate that STEP negatively regulates PTPα by directly dephosphorylating PTPα at its regulatory Tyr⁷⁸⁹. Dephosphorylation of Tyr⁷⁸⁹ prevents the translocation of PTPα to synaptic membranes, blocking its ability to interact with and activate Fyn. Genetic or pharmacologic reduction in STEP₆₁ activity increased the phosphorylation of PTPα at Tyr⁷⁸⁹, as well as increased translocation of PTPα to synaptic membranes. Activation of PTPα and Fyn and trafficking of GluN2B to synaptic membranes are necessary for ethanol (EtOH) intake behaviors in rodents. We tested the functional significance of STEP₆₁ in this signaling pathway by EtOH administration to primary cultures as well as in vivo, and demonstrated that the inactivation of STEP₆₁ by EtOH leads to the activation of PTPα, its translocation to synaptic membranes, and the activation of Fyn. These findings indicate a novel mechanism by which STEP₆₁ regulates PTPα and suggest that STEP and PTPα coordinate the regulation of Fyn.