A PDK1 homolog is necessary and sufficient to transduce AGE-1 PI3 kinase signals that regulate diapause in Caenorhabditis elegans. (41/1944)

An insulin receptor-like signaling pathway regulates Caenorhabditis elegans metabolism, development, and longevity. Inactivation of the insulin receptor homolog DAF-2, the AGE-1 PI3K, or the AKT-1 and AKT-2 kinases causes a developmental arrest at the dauer stage. A null mutation in the daf-16 Fork head transcription factor alleviates the requirement for signaling through this pathway. We show here that a loss-of-function mutation in pdk-1, the C. elegans homolog of the mammalian Akt/PKB kinase PDK1, results in constitutive arrest at the dauer stage and increased life span; these phenotypes are suppressed by a loss of function mutation in daf-16. An activating mutation in pdk-1 or overexpression of wild-type pdk-1 relieves the requirement for AGE-1 PI3K signaling. Therefore, pdk-1 activity is both necessary and sufficient to propagate AGE-1 PI3K signals in the DAF-2 insulin receptor-like signaling pathway. The activating mutation in pdk-1 requires akt-1 and akt-2 gene activity in order to suppress the dauer arrest phenotype of age-1. This indicates that the major function of C. elegans PDK1 is to transduce signals from AGE-1 to AKT-1 and AKT-2. The activating pdk-1 mutation is located in a conserved region of the kinase domain; the equivalent amino acid substitution in human PDK1 activates its kinase activity toward mammalian Akt/PKB.  (+info)

Negative regulation of male development in Caenorhabditis elegans by a protein-protein interaction between TRA-2A and FEM-3. (42/1944)

The tra-2 gene of the nematode Caenorhabditis elegans encodes a predicted membrane protein, TRA-2A, that promotes XX hermaphrodite development. Genetic analysis suggests that tra-2 is a negative regulator of three genes that are required for male development: fem-1, fem-2, and fem-3. We report that the carboxy-terminal region of TRA-2A interacts specifically with FEM-3 in the yeast two-hybrid system and in vitro. Consistent with the idea that FEM-3 is a target of negative regulation, we find that excess FEM-3 can overcome the feminizing effect of tra-2 and cause widespread masculinization of XX somatic tissues. In turn, we show that the masculinizing effects of excess FEM-3 can be suppressed by overproduction of the carboxy-terminal domain of TRA-2A. A FEM-3 fragment that retains TRA-2A-binding activity can masculinize fem-3(+) animals, but not fem-3 mutants, suggesting that it is possible to release and to activate endogenous FEM-3 by titrating TRA-2A. We propose that TRA-2A prevents male development by interacting directly with FEM-3 and that a balance between the opposing activities of TRA-2A and FEM-3 determines sex-specific cell fates in somatic tissues. When the balance favors FEM-3, it acts through or with the other FEM proteins to promote male cell fates.  (+info)

Identification of an intramolecular interaction between the SH3 and guanylate kinase domains of PSD-95. (43/1944)

Postsynaptic density-95 (PSD-95/SAP-90) is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins that assemble protein complexes at synapses and other cell junctions. MAGUKs comprise multiple protein-protein interaction motifs including PDZ, SH3 and guanylate kinase (GK) domains, and these binding sites mediate the scaffolding function of MAGUK proteins. Synaptic binding partners for the PDZ and GK domains of PSD-95 have been identified, but the role of the SH3 domain remains elusive. We now report that the SH3 domain of PSD-95 mediates a specific interaction with the GK domain. The GK domain lacks a poly-proline motif that typically binds to SH3 domains; instead, SH3/GK binding is a bi-domain interaction that requires both intact motifs. Although isolated SH3 and GK domains can bind in trans, experiments with intact PSD-95 molecules indicate that intramolecular SH3/GK binding dominates and prevents intermolecular associations. SH3/GK binding is conserved in the related Drosophila MAGUK protein DLG but is not detectable for Caenorhabditis elegans LIN-2. Many previously identified genetic mutations of MAGUKs in invertebrates occur in the SH3 or GK domains, and all of these mutations disrupt intramolecular SH3/GK binding.  (+info)

Suppression of SHP-2 and ERK signalling promotes self-renewal of mouse embryonic stem cells. (44/1944)

The propagation of pluripotent mouse embryonic stem (ES) cells depends on signals transduced through the cytokine receptor subunit gp130. Signalling molecules activated downstream of gp130 in ES cells include STAT3, the protein tyrosine phosphatase SHP-2, and the mitogen-activated protein kinases, ERK1 and ERK2. A chimaeric receptor in which tyrosine 118 in the gp130 cytoplasmic domain was mutated did not engage SHP-2 and failed to activate ERKs. However, this receptor did support ES cell self-renewal. In fact, stem cell colonies formed at 100-fold lower concentrations of cytokine than the unmodified receptor. Moreover, altered ES cell morphology and growth were observed at high cytokine concentrations. These indications of deregulated signalling in the absence of tyrosine 118 were substantiated by sustained activation of STAT3. Confirmation that ERK activation is not required for self-renewal was obtained by propagation of pluripotent ES cells in the presence of the MEK inhibitor PD098059. In fact, the growth of undifferentiated ES cells was enhanced by culture in PD098059. Thus activation of ERKs appears actively to impair self-renewal. These data imply that the self-renewal signal from gp130 is a finely tuned balance of positive and negative effectors.  (+info)

The timing of lin-4 RNA accumulation controls the timing of postembryonic developmental events in Caenorhabditis elegans. (45/1944)

The lin-4 gene encodes a small RNA that is required to translationally repress lin-14 toward the end of the first larval stage of Caenorhabditis elegans development. To determine if the timing of LIN-14 protein down-regulation depends on the temporal profile of lin-4 RNA level, we analyzed the stage-specificity of lin-4 RNA expression during wild-type development and examined the phenotypes of transgenic worms that overexpress lin-4 RNA during the first larval stage. We found that lin-4 RNA first becomes detectable at approximately 12 h of wild-type larval development and rapidly accumulates to nearly maximum levels by 16 h. This profile of lin-4 RNA accumulation corresponded to the timing of LIN-14 protein down-regulation. Transgenic strains that express elevated levels of lin-4 RNA prior to 12 h of development display reduced levels of LIN-14 protein and precocious phenotypes consistent with abnormally early loss of lin-14 activity. These results indicate that the temporal profile of lin-4 RNA accumulation specifies the timing of LIN-14 down-regulation and thereby controls the timing of postembryonic developmental events.  (+info)

Dosage compensation proteins targeted to X chromosomes by a determinant of hermaphrodite fate. (46/1944)

In many organisms, master control genes coordinately regulate sex-specific aspects of development. SDC-2 was shown to induce hermaphrodite sexual differentiation and activate X chromosome dosage compensation in Caenorhabditis elegans. To control these distinct processes, SDC-2 acts as a strong gene-specific repressor and a weaker chromosome-wide repressor. To initiate hermaphrodite development, SDC-2 associates with the promoter of the male sex-determining gene her-1 to repress its transcription. To activate dosage compensation, SDC-2 triggers assembly of a specialized protein complex exclusively on hermaphrodite X chromosomes to reduce gene expression by half. SDC-2 can localize to X chromosomes without other components of the dosage compensation complex, suggesting that SDC-2 targets dosage compensation machinery to X chromosomes.  (+info)

p24 proteins and quality control of LIN-12 and GLP-1 trafficking in Caenorhabditis elegans. (47/1944)

Mutations in the Caenorhabditis elegans sel-9 gene elevate the activity of lin-12 and glp-1, which encode members of the LIN-12/NOTCH family of receptors. Sequence analysis indicates SEL-9 is one of several C. elegans p24 proteins. Allele-specific genetic interactions suggest that reducing sel-9 activity increases the activity of mutations altering the extracellular domains of LIN-12 or GLP-1. Reducing sel-9 activity restores the trafficking to the plasma membrane of a mutant GLP-1 protein that would otherwise accumulate within the cell. Our results suggest a role for SEL-9 and other p24 proteins in the negative regulation of transport of LIN-12 and GLP-1 to the cell surface, and favor a role for p24 proteins in a quality control mechanism for endoplasmic reticulum-Golgi transport.  (+info)

Regulation of the UNC-18-Caenorhabditis elegans syntaxin complex by UNC-13. (48/1944)

The Caenorhabditis elegans unc-13, unc-18, and unc-64 genes are required for normal synaptic transmission. The UNC-18 protein binds to the unc-64 gene product C. elegans syntaxin (Ce syntaxin). However, it is not clear how this protein complex is regulated. We show that UNC-13 transiently interacts with the UNC-18-Ce syntaxin complex, resulting in rapid displacement of UNC-18 from the complex. Genetic and biochemical evidence is presented that UNC-13 contributes to the modulation of the interaction between UNC-18 and Ce syntaxin.  (+info)