Overexpression of an activated rasG gene during growth blocks the initiation of Dictyostelium development.

Transformants that expressed either the wild-type rasG gene, an activated rasG-G12T gene, or a dominant negative rasG-S17N gene, all under the control of the folate-repressible discoidin (dis1gamma) promoter, were isolated. All three transformants expressed high levels of Ras protein which were reduced by growth in the presence of folate. All three transformants grew slowly, and the reduction in growth rate correlated with the amount of RasG protein produced, suggesting that RasG is important in regulating cell growth. The pVEII-rasG transformant containing the wild-type rasG gene developed normally despite the presence of high levels of RasG throughout development. This result indicates that the down regulation of rasG that normally occurs during aggregation of wild-type strains is not essential for the differentiation process. Dictyostelium transformants expressing the dominant negative rasG-S17N gene also differentiated normally. Dictyostelium transformants that overexpressed the activated rasG-G12T gene did not aggregate. The defect occurred very early in development, since the expression of car1 and pde, genes that are normally induced soon after the initiation of development, was repressed. However, when the transformant cells were pulsed with cyclic AMP, expression of both genes returned to wild-type levels. The transformants exhibited chemotaxis to cyclic AMP, and development was synergized by mixing with wild-type cells. Furthermore, cells that were pulsed with cyclic AMP for 4 h before being induced to differentiate by plating on filters produced small, but otherwise normal, fruiting bodies. These results suggest that the rasG-G12T transformants are defective in cyclic AMP production and that RasG - GTP blocks development by interfering with the initial generation of cyclic AMP pulses.     

Rap1 activation in response to cAMP occurs downstream of ras activation during Dictyostelium aggregation

We have used a doubly disrupted rasC(-)/rasG(-) strain of Dictyostelium discoideum, which ectopically expresses the carA gene, to explore the relationship between the activation of RasC and RasG, the two proteins that are necessary for optimum cAMP signaling, and the activation of Rap1, a Ras subfamily protein, that is also activated by cAMP. The ectopic expression of carA restored early developmental gene expression to the rasC(-)/rasG(-) strain, rendering it suitable for an analysis of cAMP signal transduction. Because there was negligible signaling through both the cAMP chemotactic pathway and the adenylyl cyclase activation pathway in the rasC(-)/rasG(-)/[act15]:carA strain, it is clear that RasG and RasC are the only two Ras subfamily proteins that directly control these pathways. The position of Rap1 in the signal transduction cascade was clarified by the finding that Rap1 activation was totally abolished in rasC(-)/rasG(-)/[act15]:carA and rasG(-) cells but only slightly reduced in rasC(-) cells. Rap1 activation, therefore, occurs downstream of the Ras proteins and predominantly, if not exclusively, downstream of RasG. The finding that in vitro guanylyl cyclase activation is also abolished in the rasC(-)/rasG(-)/[act15]:carA strain identifies RasG/RasC as the presumptive monomeric GTPases required for this activation.     

Mediation of cell-substratum adhesion by RasG in Dictyostelium

Previous studies on the functions of the RasG gene in the cellular slime mold, Dictyostelium discoideum, have revealed that it is required for normal motility and cytokinesis. To further understand how the RasG gene regulates various cellular processes, we transformed an activated form of RasG, that is, RasG (G12T), a mutation from glycine to threonine at amino acid position 12 into wild type KAX-3 cells. This produced moderate but constitutive RasG(G12T) protein expression, which causes cells to become significantly more adherent to the substratum than are wild type cells. The RasG(G12T) transformants also grow slowly on bacterial plates, and engulf fewer bacteria on filter surfaces, indicating a defect in phagocytosis when cells are adhered. The expression of the activated RasG also dramatically reduces the number of filopodia on the cell surface. Tyrosine phosphorylation on a 43 kDa protein (most likely actin) of the RasG (G12T) transformants is highly elevated. Taken together, our observations suggest that RasG is crucial for Dictyostelium cell-substratum adhesion during growth and that RasG may play a role in adhesion-mediated phagocytosis. Our results also suggest that RasG is important in filopodial formation and that RasG is involved in the signal pathway that is regulated by tyrosine phosphorylation.     

The identification of Dictyostelium phosphoproteins altered in response to the activation of RasG

Dictyostelium RasG has been implicated in the regulation of a variety of cellular processes, including the initiation of development, cell movement, and cytokinesis, but the molecular components of the signaling pathways involved are largely unknown. We used a tetracycline-regulated protein expression system to study the effect of activated RasG, RasG(G12T), expression on the phosphorylation state of Dictyostelium proteins. Over 70 vegetative phosphoprotein components were resolved by two-dimensional (2-D) immunoblot analysis and of these 16 phosphothreonine and three phosphotyrosine protein components were found to reproducibly change upon RasG(G12T) expression. Thirteen of these were recovered from 2-D gels and identified by mass spectrometry of in-gel tryptic digestions. The proteins identified include the signaling proteins RasGEF-R and protein kinase B, the adhesion protein DdCAD-1, the cytoskeletal protein actin, the mitochondrial division protein FtsZA, and proteins involved in translation and metabolism. In addition to the direct demonstration of the phosphorylation of putative downstream targets of RasG activation, these findings reveal previously undetected phosphorylation of several proteins.     

Role of cAMP-dependent protein kinase during growth and early development of Dictyostelium discoideum

cAMP-dependent protein kinase (PKA) is an essential regulator of gene expression and cell differentiation during multicellular development of Dictyostelium discoideum. Here we show that PKA activity also regulates gene expression during the growth phase and at the transition from growth to development. Overexpression of PKA leads to overexpression of the discoidinIgamma promoter, while expression of the discoidinIgamma promoter is reduced when PKA activity is reduced, either by expression of a dominant negative mutant of the regulatory subunit or by disruption of the gene for the catalytic subunit (PKA-C). The discoidin phenotype of PKA-C null cells is cell autonomous. In particular, normal secretion of discoidin-inducing factors was demonstrated. In addition, PKA-C null cells are able to respond to media conditioned by PSF and CMF. We conclude that PKA is a major activator of discoidin expression. However, it is not required for production or transduction of the inducing extracellular signals. Therefore, PKA-dependent and PKA-independent pathways regulate the expression of the discoidin genes.     

The effects of expression of an activated rasG mutation on the differentiation of Dictyostelium.

Dictyostelium discoideum contains two ras genes, rasG and rasD, that are expressed during growth and differentiation, respectively. It was shown previously that Dictyostelium transformants expressing an activated rasD gene (a mutation producing a change in amino acid 12 from glycine to threonine) developed abnormally. When developed on filters these transformants formed multitipped aggregates, which did not go on to produce final fruiting bodies, but in a submerged culture assay on a plastic surface they either formed small aggregates or did not aggregate. In this study we transformed cells with the rasG gene, mutated to change amino acid 12 from glycine to threonine. The resulting transformants developed normally on filters, but aggregation under other conditions was impaired. In particular, in submerged culture on a plastic surface they either produced very small aggregates or did not aggregate, one of the phenotypes exhibited by the activated rasD transformants. Molecular analysis of the transformants revealed the presence of high copy numbers of the mutated rasG gene, but the level of expression of the mutant gene never exceeded the level of expression of the endogenous gene. These results indicate a powerful dominant effect of a relatively small amount of the activated RasG protein in Dictyostelium.     

Aberrant folate response and premature development in a mutant of Dictyostelium discoideum

Growth and development are mutually exclusive in Dictyostelium discoideum. The transition between the two stages of the life cycle is regulated by the relative abundance of nutrients and proteins secreted by the cells which reflect population density. At the transition from growth to development, the discoidin genes--developmental markers--are induced by the "quorum" protein PSF. The effect of PSF is counteracted by food bacteria and by folate [8]. We show that folate treatment during growth delays morphologic development. Furthermore, we demonstrate that in a mutant of Dictyostelium discoideum (V188, renamed HBW3), which expresses discoidinI during growth and which develops rapidly [46], discoidinI expression is less sensitive to folate than in wild type cells. Finally, we present evidence that fragments of the discoidinI gamma promoter which are unresponsive to PSF and CM are sufficient for misregulation in the mutant. The only known regulator of these promoter elements is folate. Changes in the expression of other early developmental genes are also shown. Taken together, these data suggest that the reduced sensitivity to folate might be the cause for the "rapid development" phenotype of the mutant and that folate regulates developmental timing.     

Folate responsiveness during growth and development of Dictyostelium: separate but related pathways control chemotaxis and gene regulation

Folate-controtled gene expression and chemotaxis have been examined in Dictyostelium wild-type and mutant strains. We show that regulation of the discoidin genes is sensitive to foiate in growing ceiis as weli as in suspension development. The signal is transferred via the N¹⁰-methylfoiate-sensitive folate receptor sites, which also appear to confer the chemotactic response. The strain HG5145 has previously been isolated as a mutant that does not display chemotactic movement towards folate. Nevertheless, these cells are fully functional in foiate-mediated downregulation of discoidin I expression. The strain ga 93 has been isolated as an overproducer mutant of the cyclic nucleotide phosphodiesterase inhibitor. Simultaneously, these cells fail to downregulate discoidin I in response to folate but are fully functional in folate chemotaxis. Therefore we conclude that the pathways for chemotaxis and for gene regulation diverge downstream of a common receptor type.     

A mutation that separates the RasG signals that regulate development and cytoskeletal function in Dictyostelium

The expression of an activated RasG, RasG-G12T, in vegetative cells of Dictyostelium discoideium produced an alteration in cell morphology. Cells underwent a transition between an extensively flattened form that exhibited lateral membrane ruffling to a less flattened form that exhibited prominent dorsal membrane ruffling. These rasG-G12T transformants exhibited a redistribution of F-actin at the cell periphery and did not undergo the rapid contraction upon refeeding that is characteristic of wild-type cells. These results suggest a role for RasG in regulating cytoskeletal rearrangement in D. discoideum. We had shown previously that expression of rasG-G12T inhibited starvation induced aggregation (M. Khosla et al., 1996, Mol. Cell. Biol. 16, 4156-4162). rasG-G12T genes containing secondary mutations were transformed into cells to test whether the effects of rasG-G12T were transmitted through a single downstream effector. Cells expressing rasG-G12T/T35S or rasG-G12T/Y40C (secondary mutations within the effector domain) exhibited normal morphology and underwent normal aggregation, suggesting that signaling through the effector domain was required for both the morphological and the development changes induced by rasG-G12T. In contrast, cells expressing rasG-G12T/T45Q (a secondary mutation in the effector distal flanking domain) exhibited normal aggregation but a morphology indistinguishable from that of rasG-G12T transformants. This result suggests that RasG regulates developmental and cytoskeletal functions by direct interaction with more than one downstream effector.     

gdt1, a new signal transduction component for negative regulation of the growth-differentiation transition in Dictyostelium discoideum

Discoidin I expression was used as a marker to screen for mutants affected in the growth-differentiation transition (GDT) of Dictyostelium. By REMI mutagenesis we have isolated mutant 2-9, an overexpressor of discoidin I. It displays normal morphogenesis but shows premature entry into the developmental cycle. The disrupted gene was denominated gdt1. The mutant phenotype was reconstructed by disruptions in different parts of the gene, suggesting that all had a complete loss of function. gdt1 was expressed in growing cells; the levels of protein and mRNA appear to increase with cell density and rapidly decrease with the onset of development. gdt1 encodes a 175-kDa protein with four putative transmembrane domains. In the C terminus, the derived amino acid sequence displays some similarity to the catalytic domain of protein kinases. Mixing experiments demonstrate that the gdt1(-) phenotype is cell autonomous. Prestarvation factor is secreted at wild-type levels. The response to folate, a negative regulator of discoidin expression, was not impaired in gdt1 mutants. Cells that lack the G protein alpha2 display a loss of discoidin expression and do not aggregate. gdt1(-)/Galpha2(-) double mutants show no aggregation but strong discoidin expression. This suggests that gdt1 is a negative regulator of the GDT downstream of or in a parallel pathway to Galpha2.     

Expression of activated Ras during Dictyostelium development alters cell localization and changes cell fate

There is now a body of evidence to indicate that Ras proteins play important roles in development. Dictyostelium expresses several ras genes and each appears to perform a distinct function. Previous data had indicated that the overexpression of an activated form of the major developmentally regulated gene, rasD, caused a major aberration in morphogenesis and cell type determination. We now show that the developmental expression of an activated rasG gene under the control of the rasD promoter causes a similar defect. Our results indicate that the expression of activated rasG in prespore cells results in their transdifferentiation into prestalk cells, whereas activated rasG expression in prestalk causes gross mislocalization of the prestalk cell populations.     

Delineation of the roles played by RasG and RasC in cAMP-dependent signal transduction during the early development of Dictyostelium discoideum

On starvation, the cellular slime mold Dictyostelium discoideum initiates a program of development leading to formation of multicellular structures. The initial cell aggregation requires chemotaxis to cyclic AMP (cAMP) and relay of the cAMP signal by the activation of adenylyl cyclase (ACA), and it has been shown previously that the Ras protein RasC is involved in both processes. Insertional inactivation of the rasG gene resulted in delayed aggregation and a partial inhibition of early gene expression, suggesting that RasG also has a role in early development. Both chemotaxis and ACA activation were reduced in the rasG- cells, but the effect on chemotaxis was more pronounced. When the responses of rasG- cells to cAMP were compared with the responses of rasC- and rasC- rasG- strains, generated in otherwise isogenic backgrounds, these studies revealed that signal transduction through RasG is more important in chemotaxis and early gene expression, but that signal transduction through RasC is more important in ACA activation. Because the loss of either of the two Ras proteins alone did not result in a total loss of signal output down either of the branches of the cAMP signal-response pathway, there appears to be some overlap of function.     

Regulation of the Discoidin I gamma gene in Dictyostelium discoideum: identification of individual promoter elements mediating induction of transcription and repression by cyclic AMP.

We dissected the promoter of the developmentally induced and cyclic AMP-repressed discoidin I gamma gene and identified a sequence element essential for developmental induction. Transfer of the element to an inactive heterologous promoter demonstrated that this sequence is sufficient to confer expression in axenically growing cells and to induce gene activity in development after growth on bacteria. A 16-base-pair sequence within this element was shown to be sufficient for induction in the discoidin promoter context and was used to reactivate different truncated promoter constructs. This led to the localization of an element necessary for down regulation of gene expression by extracellular cyclic AMP.     

Promoter methylation of O(6)-methylguanine-DNA-methyltransferase in lung cancer is regulated by p53

Methylation of the O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter is associated with G:C to A:T transitions in the p53 gene in various human cancers, including lung cancer. In tumors with p53 mutation, MGMT promoter methylation is more common in advanced tumors than in early tumors. However, in tumors with wild-type p53, MGMT promoter methylation is independent of tumor stage. To elucidate whether p53 participates in MGMT promoter methylation, we engineered three cell models: A549 cells with RNA interference (RNAi)-mediated knockdown of p53, and p53 null H1299 cells transfected with either wild-type p53 (WT-p53) or mutant-p53 (L194R, and R249S-p53). Knockdown of endogenous p53 increased MGMT promoter methylation in A549 cells, and transient expression of WT-p53 in p53 null H1299 cells diminished MGMT promoter methylation, whereas the MGMT promoter methylation status were unchanged by expression of mutant-p53. Previous work showed that p53 modulates DNA-methyltransferase 1 (DNMT1) expression; we additionally examined chromatin remodeling proteins expression levels of histone deacetylase 1 (HDAC1). We found that p53 knockdown elevated expression of both DNMT1 and HDAC1 in A549 cells. Conversely, expressing WT-p53 in p53 null H1299 cells reduced DNMT1 and HDAC1 expression, but the reduction of both proteins was not observed in expressing mutant-p53 H1299 cells. CHIP analysis further showed that DNMT1 and HDAC1 binding to the MGMT promoter was increased by MGMT promoter methylation and decreased by MGMT promoter demethylation. In conclusion, MGMT promoter methylation modulated by p53 status could partially promote p53 mutation occurrence in advanced lung tumors.     

Chemoattractant-induced Ras activation during Dictyostelium aggregation

Ras proteins are highly conserved molecular switches that regulate cellular response to external stimuli. Dictyostelium discoideum contains an extensive family of Ras proteins that function in regulation of mitosis, cytoskeletal function and motility, and the onset of development. Little is known about the events that lead to the activation of Ras proteins in Dictyostelium, primarily owing to a lack of a biochemical assay to measure the levels of activated Ras. We have adapted an assay, used successfully to measure activated Ras in mammalian cells, to monitor activation of two Dictyostelium Ras proteins, RasC and RasG. We have found that the Ras-binding domain (RBD) of mammalian Raf1 was capable of binding to the activated form of RasG, but not to the activated form of RasC; however, the RBD of Schizosaccharomyces pombe Byr2 was capable of binding preferentially to the activated forms of both RasC and RasG. Using this assay, we discovered that RasC and RasG showed a rapid and transient activation when aggregation-competent cells were stimulated with the chemoattractant cAMP, and this activation did not occur in a number of cAMP signalling mutants. These data provide further evidence of a role for both RasC and RasG in the early development of Dictyostelium.