The role of historical contingency in evolution has been much debated, but rarely tested. Twelve initially identical populations of Escherichia coli were founded in 1988 to investigate this issue. They have since evolved in a glucose-limited medium that also contains citrate, which E. coli cannot use as a carbon source under oxic conditions. No population evolved the capacity to exploit citrate for >30,000 generations, although each population tested billions of mutations. A citrate-using (Cit ؉ ) variant finally evolved in one population by 31,500 generations, causing an increase in population size and diversity. The long-delayed and unique evolution of this function might indicate the involvement of some extremely rare mutation. Alternately, it may involve an ordinary mutation, but one whose physical occurrence or phenotypic expression is contingent on prior mutations in that population. We tested these hypotheses in experiments that ''replayed'' evolution from different points in that population's history. We observed no Cit ؉ mutants among 8.4 ؋ 10 12 ancestral cells, nor among 9 ؋ 10 12 cells from 60 clones sampled in the first 15,000 generations. However, we observed a significantly greater tendency for later clones to evolve Cit ؉ , indicating that some potentiating mutation arose by 20,000 generations. This potentiating change increased the mutation rate to Cit ؉ but did not cause generalized hypermutability. Thus, the evolution of this phenotype was contingent on the particular history of that population. More generally, we suggest that historical contingency is especially important when it facilitates the evolution of key innovations that are not easily evolved by gradual, cumulative selection.adaptation ͉ experimental evolution ͉ mutation ͉ selection

Inactivation of the tumor suppressor PTEN gene is found in a variety of human cancers and in cancer predisposition syndromes. Recently, PTEN protein has been shown to possess phosphatase activity on phosphatidylinositol 3,4,5-trisphosphate, a product of phosphatidylinositol 3-kinase. We have identified a homolog of PTEN in Caenorhabditis elegans and have found that it corresponds to the daf-18 gene, which had been defined by a single, phenotypically weak allele, daf-18(e1375). By analyzing an allele, daf-18(nr2037), which bears a deletion of the catalytic portion of CePTEN͞DAF-18, we have shown that mutation in daf-18 can completely suppress the dauer-constitutive phenotype caused by inactivation of daf-2 or age-1, which encode an insulin receptor-like molecule and the catalytic subunit of phosphatidylinositol 3-kinase, respectively. In addition, daf-18(nr2037) dramatically shortens lifespan, both in a wild-type background and in a daf-2 mutant background that normally prolongs lifespan. The lifespan in a daf-18(nr2037) mutant can be restored to essentially that of wild type when combined with a daf-2 mutation. Our studies provide genetic evidence that, in C. elegans, the PTEN homolog DAF-18 functions as a negative regulator of the DAF-2 and AGE-1 signaling pathway, consistent with the notion that DAF-18 acts a phosphatidylinositol 3,4,5-trisphosphate phosphatase in vivo. Furthermore, our studies have uncovered a longevity-promoting activity of the PTEN homolog in C. elegans.

Receptor tyrosine phosphatases have been implicated in playing important roles in cell signaling events by their ability to regulate the level of protein tyrosine phosphorylation. Although the catalytic activity of their phosphatase domains has been well established, the biological roles of these molecules are, for the most part, not well understood. Here we show that the Caenorhabditis elegans protein CLR-1 (CLeaR) is a receptor tyrosine phosphatase (RTP) with a complex extracellular region and two intracellular phosphatase domains. Mutations in clr-1 result in a dramatic Clr phenotype that we have used to study the physiological requirements for the CLR-1 RTP. We show that the phosphatase activity of the membrane-proximal domain is essential for the in vivo function of CLR-1. By contrast, we present evidence that the membrane-distal domain is not required to prevent the Clr phenotype in vivo. The Clr phenotype of clr-1 mutants is mimicked by activation of the EGL-15 fibroblast growth factor receptor (FGFR) and is suppressed by mutations that reduce or eliminate the activity of egl-15. Our data strongly indicate that CLR-1 attenuates the action of an FGFR-mediated signaling pathway by dephosphorylation.

Activation of fibroblast growth factor (FGF) receptors elicits diverse cellular responses including growth, mitogenesis, migration, and differentiation. The intracellular signaling pathways that mediate these important processes are not well understood. In Caenorhabditis elegans, suppressors of clr-1 identify genes, termed soc genes, that potentially mediate or activate signaling through the EGL-15 FGF receptor. We demonstrate that three soc genes, soc-1, soc-2, and sem-5, suppress the activity of an activated form of the EGL-15 FGF receptor, consistent with the soc genes functioning downstream of EGL-15. We show that soc-2 encodes a protein composed almost entirely of leucine-rich repeats, a domain implicated in protein-protein interactions. We identified a putative human homolog, SHOC-2, which is 54% identical to SOC-2. We find that shoc-2 maps to 10q25, shoc-2 mRNA is expressed in all tissues assayed, and SHOC-2 protein is cytoplasmically localized. Within the leucine-rich repeats of both SOC-2 and SHOC-2 are two YXNX motifs that are potential tyrosine-phosphorylated docking sites for the SEM-5͞GRB2 Src homology 2 domain. However, phosphorylation of these residues is not required for SOC-2 function in vivo, and SHOC-2 is not observed to be tyrosine phosphorylated in response to FGF stimulation. We conclude that this genetic system has allowed for the identification of a conserved gene implicated in mediating FGF receptor signaling in C. elegans.Fibroblast growth factor (FGF) receptors comprise a family of transmembrane receptor tyrosine kinases (RTKs) that mediate diverse cellular responses including growth, mitogenesis, migration, and differentiation. FGF receptors are activated by the concerted action of secreted polypeptide growth factor ligands and heparin sulfate proteoglycans (reviewed in refs. 1 and 2). Like other RTKs, activation is associated with receptor dimerization and autophosphorylation on specific intracellular tyrosine residues (3). Autophosphorylation leads to activation of receptor kinase activity and generates potential binding sites for Src homology 2 or phosphotyrosine binding domain proteins (4).Six major autophosphorylation sites (Y463, Y583, Y585, Y653, Y654, and Y730) on human FGF receptor 1 have been identified. Two of these sites, Y653 and Y654, are required for the stimulation of receptor kinase activity (5). Phospholipase C-␥ has been shown to associate with human FGF receptor 1 requiring Y766 (6); however, the significance of this interaction is unclear because mutation of Y766 abolishes phosphatidylinositol hydrolysis without affecting mitogenic or differentiation responses to FGF in several cell lines (7-9). In fact, FGF receptors lacking all autophosphorylation sites except those required for kinase activity are capable of inducing proliferation and differentiation in a variety of cell lines (5), suggesting that, unlike certain other RTKs, target recruitment to autophosphorylation sites plays a limited role in FGF receptor signaling.Members of the Ras͞mitogen-activate...

EGL-15 is a fibroblast growth factor receptor in the nematode Caenorhabditis elegans. Components that mediate EGL-15 signaling have been identified via mutations that confer a Clear (Clr) phenotype, indicative of hyperactivity of this pathway, or a suppressor-of-Clr (Soc) phenotype, indicative of reduced pathway activity. We have isolated a gain-of-function allele of let-60 ras that confers a Clr phenotype and implicated both let-60 ras and components of a mitogen-activated protein kinase cascade in EGL-15 signaling by their Soc phenotype. Epistasis analysis indicates that the gene soc-1 functions in EGL-15 signaling by acting either upstream of or independently of LET-60 RAS. soc-1 encodes a multisubstrate adaptor protein with an amino-terminal pleckstrin homology domain that is structurally similar to the DOS protein in Drosophila and mammalian GAB1. DOS is known to act with the cytoplasmic tyrosine phosphatase Corkscrew (CSW) in signaling pathways in Drosophila. Similarly, the C. elegans CSW ortholog PTP-2 was found to be involved in EGL-15 signaling. Structure-function analysis of SOC-1 and phenotypic analysis of single and double mutants are consistent with a model in which SOC-1 and PTP-2 act together in a pathway downstream of EGL-15 and the Src homology domain 2 (SH2)/SH3-adaptor protein SEM-5/GRB2 contributes to SOC-1-independent activities of EGL-15.Receptor tyrosine kinases (RTKs) play critical roles in translating cues gathered from the extracellular environment into biological responses such as cellular differentiation, proliferation, and migration events. RTKs such as the platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF) receptors define a family of single-pass transmembrane proteins with an intracellular tyrosine kinase domain (51). Binding of growth factor to the extracellular portions of these receptors promotes receptor dimerization and activation of the intracellular tyrosine kinase domain (43). Receptor activation leads to receptor autophosphorylation as well as phosphorylation of cytoplasmic signaling proteins. Specific phosphotyrosine sites on the receptor can serve to propagate signaling by recruiting Src homology domain 2 (SH2) and PTB domain-containing signaling proteins directly to the activated receptor (37, 38). For example, many RTKs can signal to the RAS/mitogen-activated protein kinase (MAPK) cascade via direct recruitment of the SH2/SH3 domain-containing adaptor protein GRB2 in complex with SOS, the guanine nucleotide exchange factor for RAS (31). EGF and PDGF RTKs can utilize direct recruitment of the GRB2/ SOS complex to achieve RAS activation. This canonical pathway is well-conserved in Caenorhabditis elegans, Drosophila, and mammalian systems (37).There is compelling evidence that the RAS/MAPK signaling pathway plays an important role in signaling via FGF receptors (FGFRs) as well (32). However, unlike the EGF and PDGF receptors, FGFRs do not appear to recruit GRB2/SOS directly. Instead, mammalian FGFR-1 makes use of the m...