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RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation

2001; Springer Nature; Volume: 20; Issue: 16 Linguagem: Inglês

10.1093/emboj/20.16.4490

1460-2075

Chinten James Lim,

Microtubule and mitosis dynamics

Article15 August 2001free access RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation Chinten James Lim Chinten James Lim Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3 Canada Search for more papers by this author George B. Spiegelman George B. Spiegelman Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3 Canada Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, V6T 1Z3 Canada Search for more papers by this author Gerald Weeks Corresponding Author Gerald Weeks Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3 Canada Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, V6T 1Z3 Canada Search for more papers by this author Chinten James Lim Chinten James Lim Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3 Canada Search for more papers by this author George B. Spiegelman George B. Spiegelman Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3 Canada Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, V6T 1Z3 Canada Search for more papers by this author Gerald Weeks Corresponding Author Gerald Weeks Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3 Canada Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, V6T 1Z3 Canada Search for more papers by this author Author Information Chinten James Lim1, George B. Spiegelman1,2 and Gerald Weeks 1,2 1Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3 Canada 2Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, V6T 1Z3 Canada *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:4490-4499https://doi.org/10.1093/emboj/20.16.4490 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Disruption of Dictyostelium rasC, encoding a Ras subfamily protein, generated cells incapable of aggregation. While rasC expression is enriched in a cell type-specific manner during post-aggregative development, the defect in rasC− cells is restricted to aggregation and fully corrected by application of exogenous cAMP pulses. cAMP is not produced in rasC− cells stimulated by 2′-deoxy-cAMP, but is produced in response to GTPγS in cell lysates, indicating that G-protein-coupled cAMP receptor activation of adenylyl cyclase is regulated by RasC. However, cAMP-induced ERK2 phosphorylation is unaffected in rasC− cells, indicating that RasC is not an upstream activator of the mitogen-activated protein kinase required for cAMP relay. rasC− cells also exhibit reduced chemotaxis to cAMP during early development and delayed response to periodic cAMP stimuli produced by wild-type cells in chimeric mixtures. Furthermore, cAMP-induced Akt/PKB phosphorylation through a phosphatidylinositide 3-kinase (PI3K)-dependent pathway is dramatically reduced in rasC− cells, suggesting that G-protein-coupled serpentine receptor activation of PI3K is regulated by RasC. Cells lacking the RasGEF, AleA, exhibit similar defects as rasC− cells, suggesting that AleA may activate RasC. Introduction The ras subfamily genes encode monomeric GTPases that function as molecular switches in cellular signal transduction by cycling between an active GTP-bound or an inactive GDP-bound state (Bourne et al., 1991). A variety of extracellular stimuli potentiate the activation of Ras by exchanging GDP for GTP (Campbell et al., 1998; Gutkind, 1998), a process catalyzed by guanine nucleotide exchange factors (GEFs) (Boguski and McCormick, 1993). GTPase activating proteins increase the intrinsic GTPase activity of Ras, hydrolyzing the bound GTP to GDP (Boguski and McCormick, 1993). In the active state, Ras proteins activate multiple cellular signaling pathways including mitogen-activated protein kinase (MAPK) cascades, the phosphatidylinositide 3-kinase (PI3K)-regulated pathways and RalGDS-dependent activation of Ral (Campbell et al., 1998). These Ras-mediated responses to membrane receptor stimuli regulate a wide range of cellular processes, including proliferation, cytoskeletal functions and differentiation. The discovery of a large number of Ras subfamily homologs in mammals and in the model organisms Drosophila melanogaster, Caenorhabditis elegans and Dictyostelium discoideum (Reuther and Der, 2000; Wilkins and Insall, 2001) has raised important questions regarding the specific functions of individual Ras proteins that cannot be readily resolved by biochemical analysis. To understand Ras function in a multicellular context, it is necessary to analyze organisms that are genetically disrupted in the ras gene of interest. The tractability of the Dictyostelium haploid genome facilitates the functional characterization of strains with specifically targeted gene disruptions and, furthermore, its unique biology allows studies of growth and differentiation as distinct processes (Parent and Devreotes, 1996; Aubry and Firtel, 1999). Nutrient deprivation triggers the developmental program whereby the secretion and chemotactic response to the chemoattractant cAMP result in the aggregation of up to 105 cells. The aggregate then elongates to become a phototactic and thermotactic migrating slug. Cells within the slug differentiate and sort into prestalk or prespore cells that segregate into spatially separated populations, which, upon culmination, form a stalk consisting of dead vacuolated cells supporting a sorus of spores. Underlying this deceptively simple developmental program is a complex of cellular signaling pathways that are highly conserved relative to mammalian systems. Six Dictyostelium Ras subfamily proteins with at least 50% amino acid identity to the mammalian H-, N- and K-Ras proteins have been described (Reymond et al., 1984; Daniel et al., 1995). Several lines of evidence have indicated that a Ras signaling pathway is involved in the regulation of the cAMP relay and in the chemotactic response to cAMP during aggregation. Disruption of aleA, the gene encoding a putative Ras GEF (Insall et al., 1996), and rip3, encoding a Ras interacting protein (Lee et al., 1999), resulted in cells that cannot aggregate due to defects in both the cAMP relay and chemotaxis. Furthermore, disruption of genes homologous to components of metazoan Ras-activated effector pathways also resulted in cells incapable of aggregation, suggesting a possible upstream role for a Ras protein. For example, the MAPK homolog ERK2 was shown to be essential for cAMP relay, but not necessary for chemotaxis (Segall et al., 1995), while cells lacking both PI3K1 and PI3K2, and cells lacking Akt/PKB, a downstream effector of PI3K activity, exhibit defects in chemotaxis but not in cAMP relay (Zhou et al., 1998; Meili et al., 1999). Three of the Dictyostelium ras genes, rasS, rasG and rasD, have been disrupted, but these disruptions were found to have negligible effects on the aggregation process (Tuxworth et al., 1997; Chubb et al., 2000; Wilkins et al., 2000). In the studies described here, we present genetic and biochemical evidence that RasC is the Ras protein that had been previously implicated in the aggregation processes. Dictyostelium cells in which the rasC gene had been disrupted by targeted gene replacement failed to aggregate. RasC appears to be a central regulatory molecule acting downstream of serpentine receptor stimulation by cAMP that is required for two distinct effector pathways: the activation of Akt/PKB through PI3K and the activation of adenylyl cyclase. The discovery that the RasC protein is essential for aggregation provides evidence for a novel role for a Ras subfamily protein in the sensing of and response to chemotactic signals. Results Growth and developmental expression of rasC In order to analyze the spatial expression of rasC during development, the 0.55 kb DNA fragment upstream of the 5′ rasC coding region (Daniel et al., 1994) was isolated by PCR and ligated to the hislacZ gene, a variant of lacZ that encodes a short half-life β-galactosidase (Detterbeck et al., 1994). The resulting rasC::hislacZ fusion construct was transformed into the wild-type AX2 strain. A northern blot of RNA isolated from different developmental stages of this transformant showed a lacZ expression pattern that was almost identical to that for the rasC transcript (Figure 1A), indicating that the 0.55 kb DNA fragment encodes sufficient promoter sequence to reproduce the normal regulation of the rasC gene. Figure 1.(A) rasC and lacZ gene expression during development. rasC::hislacZ-transformed AX2 cells were plated for development on nitrocellulose filters and total RNA harvested at the times indicated for northern blot analysis. Duplicate blots were probed with either rasC or lacZ cDNA. (B) Spatial expression of lacZ driven from the rasC promoter during development. rasC::hislacZ-transformed AX2 cells were plated for development on nitrocellulose filters, fixed at various developmental stages and stained in situ for β-galactosidase activity. Shown are representative stained structures from the following developmental stages: first finger (a); slug (b); early to mid culmination (c–e); late culmination (f); and terminal fruiting body (g). All were stained for 2 h (a–f), except (g), which was stained overnight. Bar = 0.5 mm. Download figure Download PowerPoint When the rasC::hislacZ transformant was developed on nitrocellulose filters, staining for β-galactosidase activity was observed predominantly in the tip during the transition from tipped mound to slug, and by the slug stage there was also some staining in the posterior (Figure 1B). These results indicate that rasC expression is enriched in the prestalk cell population, and this pattern of expression was maintained through early and mid culmination, as demonstrated by staining in the tip, stalk tube and basal disk. The whole population became equally stained during late culmination; however, in terminally differentiated fruiting bodies, staining was predominantly in the stalk tube, the upper cup and the lower cup, indicating a reversion to the enriched prestalk pattern of expression (Figure 1B). The transient change in rasC spatial expression during late culmination correlated with the peak of mRNA at 20 h of development (Figure 1A). This spatial and temporal expression pattern is suggestive of functional roles for RasC during slug formation and culmination. Generation of a rasC null strain In order to generate a rasC− strain, a rasC disruption vector was transformed into AX2 cells, and blasticidin S-resistant colonies were screened by western blotting using the RasC antibody. Out of 70 independently isolated clones analyzed, three exhibited no detectable RasC protein (one of the three is shown in Figure 2A). Southern blotting, using a rasC cDNA probe, verified that all three transformants contained a simple targeted disruption at the rasC locus (Figure 2B). Subsequent rehybridization with the bsr gene probe revealed a single insertion of bsr into the genome for all three isolates (data not shown), indicating that the null strains were the result of rasC disruption and not attributable to secondary effects resulting from random integration. All three rasC− cell lines exhibited identical phenotypes under all assay conditions and, therefore, all subsequent data presented are for one of the three transformants. Figure 2.Disruption of the rasC gene. (A) Western blot of cell lysates from the parental AX2, rasC− and rasC−/RasC, a rasC− transformant expressing RasC from the rasC promoter, probed with a highly specific RasC antibody. The higher molecular weight of the RasC protein in the rasC−/RasC strain is due to the fact that the expression construct encoded a RasC protein with an additional 17 amino acids at the N-terminus. Molecular weight markers in kilodaltons are as indicated. (B) Southern blot of genomic DNA from parental AX2 and three independent rasC− isolates. Genomic DNA was digested with DdeI, separated in 0.7% agarose gel, blotted onto nylon and probed with rasC cDNA. DdeI restriction sites flank the entire rasC genomic locus and are not present within the disruption construct used for homologous recombination. Approximate sizes in kilobases are as indicated. Download figure Download PowerPoint Phenotype of the rasC− cells When plated on a lawn of bacteria, Dictyostelium cells grow by ingesting the bacteria, resulting in plaque formation. Cells at the plaque periphery continue to feed, expanding plaque size, while starving cells toward the center initiate multicellular development and eventually form fruiting bodies. When rasC− cells were clonally grown on a bacterial lawn, initial plaque formation was indistinguishable from that of the parental AX2, indicating normal growth and phagocytosis. However, rasC− cells failed to aggregate, appearing as clear plaques 5 days after initial plating (Figure 3A). At that time, all phases of multicellular development were observed in the AX2 plaques. Ectopic expression of the RasC protein from the rasC promoter restored multicellular development in the null strain (Figures 2A and 3A), confirming that the observed phenotype was a functional consequence of rasC gene disruption. Figure 3.Developmental phenotypes of parental AX2 and rasC− strains. (A) Clonal plaques of parental AX2, rasC− and rasC−/RasC strains after 5 days growth on a bacterial lawn. Bar = 5 mm. (B) AX2 and rasC− cells were seeded as monolayers at a density of 5 × 105 cells/cm2 in Nunc tissue culture dishes and submerged under Bonner's salts solution. Images were taken at the times indicated following plating. Bars = 100 μm. Download figure Download PowerPoint The aggregation process can also be observed by incubating cell monolayers on plastic surfaces submerged under non-nutrient buffer. Under these conditions, aggregation streams were observed for AX2 after 8 h, and distinct aggregates formed by 12 h (Figure 3B, top panels). rasC− cells did not form aggregation streams or centers, even after prolonged starvation for 36 h (Figure 3B, bottom panels). The aggregation defects observed in the rasC− cells were similar to those reported for the aleA− strain (Insall et al., 1996), suggesting the possibility that AleA is the GEF responsible for regulating RasC activation. Exogenous cAMP pulses circumvent the block in aggregation of rasC− cells Dictyostelium cells are unable to aggregate if they are deficient in the cAMP relay, a process whereby cells synthesize and secrete cAMP in response to an extracellular cAMP stimulus. When cells in suspension were pulsed every 6 min for 5 h with 50 nM cAMP (herein referred to as 'cAMP-pulsed cells') and then plated on nitrocellulose filters, development of rasC− cells was indistinguishable from that of AX2 cells (data not shown). cAMP-pulsed rasC− cells produced spores in equal numbers to cAMP-pulsed AX2 cells, and these spores exhibited the same viability (data not shown). Furthermore, when developed in chimeric mixtures with equal numbers of AX2 cells, rasC− cells completed development, producing equal numbers of spores (data not shown). The rasC− spores germinated to form aggregation-negative plaques when plated on bacterial lawns (data not shown). Contrary to the spatial expression results suggesting functional roles for RasC during mid to late development, the development of rasC− cells is fully restored if the block in aggregation is circumvented by exogenous application of cAMP pulses, an indication that RasC function is necessary for the cAMP relay. cAMP receptor and heterotrimeric G-protein-dependent activation of adenylyl cyclase is mediated by RasC To confirm that the rasC− cells were defective in the cAMP relay, cAMP-pulsed cells were stimulated in vivo with 2′-deoxy-cAMP, and cell lysates assayed for cAMP accumulation at various time points following stimulation. There was negligible accumulation of cAMP in the rasC− cells relative to that observed for AX2 cells (Figure 4A), indicating that RasC is required for cAMP-stimulated activation of adenylyl cyclase A (ACA), the predominant adenylyl cyclase present during early Dictyostelium development (Parent and Devreotes, 1996). A northern blot of mRNA isolated from cAMP-pulsed cells showed that the rasC− cells overexpress the cAMP receptor carA and the heterotrimeric G-protein gα2, relative to AX2 cells (Figure 4B). While the significance of this increased expression is not apparent, it is clear that the inability of the rasC− cells to activate ACA is not due to insufficient expression of required signaling components such as cAR1 and Gα2 (Parent and Devreotes, 1996). Figure 4.Adenylyl cyclase assays and expression of aggregation phase genes. (A) cAMP-pulsed AX2 (triangles) and rasC− (circles) cells were stimulated with 10 μM 2′-deoxy-cAMP, and cell lysates assayed for total cAMP accumulation at the indicated times. Plotted values are the means ± SD for three independent experiments. (B) AX2 and rasC− cells were shaken in suspension in KK2, with or without the application of 50 nM cAMP pulses every 6 min. Total RNA was prepared from cells harvested at the times indicated and duplicate northern blots probed with either carA or gα2 cDNA. (C) cAMP-pulsed AX2, rasC− and aleA− cell-free lysates were assayed for adenylyl cyclase activity in the presence of either 5 mM MnSO4 (hatched bar), 40 μM GTPγS (black bar) or no additional component (white bar) (see Materials and methods). Plotted values are normalized relative to the unstimulated activity obtained in the absence of MnSO4 or GTPγS. Values for AX2 and rasC− cell lysates are the means ± SD for three independent experiments. Values for aleA− cell lysates are from a single experiment. Download figure Download PowerPoint To further assess the nature of the signaling defect, GTPγS-mediated activation of ACA was assayed in vitro using extracts prepared from cAMP-pulsed cells. Since aleA− cells also exhibit negligible cAMP synthesis when stimulated in vivo with cAMP (Insall et al., 1996), we included aleA− cell-free extracts in this assay for comparison. GTPγS is thought to stimulate ACA activity by uncoupling the Gβγ subunit from the heterotrimeric G-protein, thus bypassing the need for receptor activation in cell-free lysates (Parent and Devreotes, 1996). The addition of GTPγS stimulated the ACA activity of AX2 lysates ∼24-fold over the basal level, and the ACA activity of rasC− and aleA− cell lysates ∼12-fold (Figure 4C). Thus, cell lysates lacking RasC or AleA were capable of generating appreciable amounts of cAMP in the presence of GTPγS, although the levels were not as high as those obtained with wild-type cells. The lower levels of in vitro cAMP synthesis in the rasC− and aleA− cell lysates were not due to reduced expression of ACA, since the Mn2+-activated levels were similar for AX2, rasC− and aleA− cell lysates (Figure 4C). RasC is not necessary for activation of ERK2 Dictyostelium ERK2 is transiently activated in response to a cAMP stimulus (Maeda et al., 1996) and cells lacking ERK2 are unable to aggregate due to a defective cAMP relay (Segall et al., 1995). Since ERK proteins are important downstream effectors for Ras in metazoan signaling, the possibility that RasC is an upstream activator of ERK2 was tested. cAMP-pulsed cells were stimulated with cAMP and cell lysates were analyzed for ERK2 phosphorylation in western blots using a phospho-MAPK-specific antibody. Following cAMP stimulation, a 42 kDa protein corresponding to the predicted molecular weight of ERK2 was transiently phosphorylated in wild-type cells (Figure 5) with kinetics similar to that reported previously for ERK2 activation (Maeda et al., 1996). An erkB− strain assayed similarly showed only a very faint phosphorylation of the 42 kDa component (Figure 5). Since this particular erkB− strain (HS174) expresses low levels of ERK2 due to insertional disruption of a plasmid in the 3′ untranslated region of the gene (Segall et al., 1995), our results verify that the phosphorylated protein detected in the immunoblots is indeed ERK2. When rasC− cells were stimulated with cAMP, ERK2 was phosphorylated to similar levels and with similar kinetics to those seen for wild-type cells. This demonstrates that the activation of ERK2 is not downstream of RasC in the classical Ras–MAPK signaling cascade observed in metazoan systems. Figure 5.cAMP-induced stimulation of ERK2 phosphorylation. cAMP-pulsed AX2, rasC−, aleA− and erkB− cells were stimulated with cAMP to a final concentration of 100 nM, and cells lysed directly in SDS gel loading buffer at the indicated times. Protein samples of 10 μg were fractionated by SDS–PAGE, and western blots probed with a phospho-MAPK-specific antibody to assay for ERK2 phosphorylation. Results shown are representative of at least three independent experiments for each strain. Download figure Download PowerPoint Since there had been a correlation between the phenotypes of the rasC− and aleA− cells, we repeated the ERK2 activation assay with the aleA− cells. Phosphorylation of ERK2 in aleA− cells reached a higher level, peaked at a later time point and was more persistent compared with wild-type cells (Figure 5), confirming the results of an earlier experiment using an in-gel kinase assay to measure ERK2 activity (Aubry et al., 1997). The results indicate that AleA may activate another Ras (Aubry et al., 1997; Kosaka et al., 1998), in addition to RasC. rasC− cells exhibit an altered chemotactic response to cAMP Dictyostelium aggregation is also dependent on the cell's ability to sense and respond chemotactically to cAMP. To examine the chemotactic behavior of cells lacking RasC, green fluorescent protein (GFP)-labeled rasC− cells were mixed with unlabeled AX2 cells and allowed to aggregate on plastic submerged under buffer. The fluorescent label allowed tracking of individual rasC− cells in response to the natural cAMP oscillations produced by the wild-type cells. At 8 h of development, there were very few labeled rasC− cells in the small aggregates that had formed, while, in contrast, there were numerous labeled rasC− cells in the aggregates and aggregation streams by 12 h (Figure 6A, left panels). When GFP-labeled AX2 cells were mixed with unlabeled AX2 cells as a control, numerous labeled cells were observed both within the small aggregates at 8 h and within the aggregation streams by 12 h (Figure 6A, right panels), indicating that the GFP label had no deleterious effect on chemotactic behavior. Further analysis of the mixed population at 6 h following starvation by time-lapse microscopy revealed that synchronous, pulsatile movements of AX2 cells could be observed, whereas GFP-labeled rasC− cells exhibited no such response (data not shown). In contrast, by 12 h, the rasC− cells within the aggregation streams exhibited the same pulsatile responses as the AX2 cells, while the rasC− cells that remained outside the streams remained unresponsive and non-polarized. Figure 6.cAMP-mediated chemotaxis. (A) Aggregation of rasC− cells in chimeric mixtures with AX2 cells. GFP-labeled rasC− or GFP-labeled AX2 cells were mixed with unlabeled AX2 cells in a ratio of 1:4 and seeded at 5 × 105 cells/cm2 in Nunc tissue culture dishes submerged under Bonner's salts solution. Shown are the images of GFP-labeled fluorescent cells (in green) that have been overlayed on phase-contrast images of the total aggregating population, at 8 and 12 h of development. (B) AX2 and rasC− cells seeded on Nunc dishes at 105 cells/cm2 submerged under Bonner's salts were starved for 6 h and subjected to a micropipet filled with 100 μM cAMP at T = 0. Shown are phase-contrast images of the cells in the vicinity of the micropipet tip at the indicated times. (C) AX2 and rasC− cells that had been pulsed with cAMP for 5 h were seeded onto Nunc dishes and subjected to a cAMP filled micropipet as described in (B). Bars = 100 μm. Download figure Download PowerPoint AX2 cells that had been starved for 6 h without exogenous cAMP pulsing responded within 20 min to an artificial cAMP gradient released from a micropipet (Figure 6B, top panels). In contrast, under these conditions, rasC− cells underwent chemotaxis poorly and did not exhibit the characteristic clustering of cells around the micropipet tip even after 40 min (Figure 6B, bottom panels). AX2 cells that had been pulsed for 5 h with cAMP polarized and underwent chemotaxis toward the cAMP source within 20 min of micropipet tip application (Figure 6C, top panels). However, equivalently treated rasC− cells polarized and underwent chemotaxis toward the tip within 6 min of application of the micropipet (Figure 6C, bottom panels). This enhanced chemotaxis of the rasC− cells might be the result of overexpression of carA, gα2 (Figure 4B) and possibly other components involved in mediating chemotaxis. Thus, rasC− cells are initially slow in undergoing chemotaxis to cAMP, but underwent chemotaxis rapidly after being pulsed. These results are consistent with the behavior of the rasC− cells in mixtures with AX2 described above. Akt/PKB phosphorylation through a PI3K-dependent pathway requires RasC pi3k1−/pi3k2− and pkbA− cells exhibit aggregation defects despite having normal cAMP relays, suggesting that the chemotactic response to cAMP requires a PI3K-mediated signaling pathway (Zhou et al., 1998; Meili et al., 1999). Consistent with this model, cAMP-stimulated activation of Akt/PKB does not occur in pi3k1−/pi3k2− cells, but is fully restored in pi3k1−/pi3k2− cells constitutively expressing PI3K1 (Meili et al., 1999). Dictyostelium Akt/PKB activation requires phosphorylation at conserved threonine residues in the kinase domain and at the C-terminus (Meili et al., 1999). When cell lysates of cAMP-stimulated AX2 cells were analyzed by western blotting using a phospho-threonine-specific antibody, a protein with the predicted molecular weight of Dictyostelium Akt/PKB (51 kDa) was transiently phosphorylated following cAMP stimulation (Figure 7A, left panels). When the blot was stripped and re-probed with an Akt/PKB-specific antibody, a 51 kDa protein was also detected (Figure 7A, right panels), consistent with the idea that the phospho-threonine-specific antibody had detected the transient phosphorylation of Akt/PKB. Pre-treatment of AX2 cells with 12.5 μM LY294002, a PI3K inhibitor (Vlahos et al., 1995), caused an ∼90% reduction in threonine phosphorylation of the 51 kDa protein, indicating that the observed phosphorylation was PI3K dependent (Figure 7A). Finally, the phosphorylation kinetics of the 51 kDa protein observed here was consistent with the activation of Akt/PKB reported previously (Meili et al., 1999). We are therefore confident that the transiently phosphorylated 51 kDa component is the Akt/PKB protein encoded by pkbA. Figure 7.cAMP-induced stimulation of Akt/PKB phosphorylation. (A) cAMP-pulsed AX2 cells were either not treated (Control) or pre-treated with 12.5 μM LY294002 for 1 min, before stimulation with 100 nM cAMP. At the indicated times, cells were lysed directly in SDS gel loading buffer, 10 μg of protein fractionated by SDS–PAGE and western blots probed with a phospho-threonine-specific antibody (left panels). The blots were subsequently stripped of bound antibodies and re-probed with an Akt/PKB-specific antibody (right panels). Molecular weight markers in kilodaltons are as indicated beside each blot. (B) cAMP-pulsed AX2, rasC− and aleA− cells were stimulated with 100 nM cAMP and analyzed as described in (A). Results shown are representative of at least three independent experiments for each strain. (C) Ten micrograms of protein from cAMP-pulsed AX2, rasC− and aleA− cells were analyzed by western blotting with the Akt/PKB specific-antibody to demonstrate equal Akt/PKB expression levels in all three strains. Closed arrowheads indicate phosphorylated Akt/PKB and open arrowheads indicate total Akt/PKB protein. Download figure Download PowerPoint cAMP-pulsed rasC− cells exhibit dramatically reduced levels of phosphorylated Akt/PKB at 10 s following cAMP stimulation (Figure 7B), indicating that RasC is a major upstream effector of Akt/PKB phosphorylation. cAMP-stimulated Akt/PKB phosphorylation in aleA− cells was also reduced to levels similar to those observed for rasC− cells (Figure 7B), indicating that AleA also functions upstream of Akt/PKB, consistent with the possibility that it is a GEF for RasC. The trace of Akt/PKB phosphorylation at 10 s observed in the rasC− and aleA− cells suggests that low levels of activation not involving RasC or AleA can occur. The lower levels of Akt/PKB phosphorylation were not due to reduced expression of Akt/PKB in rasC− and aleA− cells, since western blots probed with an Akt/PKB-specific antibody revealed identical levels to those in AX2 (Figure 7C). Discussion A role for a Ras protein in aggregation has been suggested by several lines of evidence (Segall et al., 1995; Insall et al., 1996; Aubry et al., 1997; Lee et al., 1999; Meili et al., 1999; Firtel and Chung, 2000). Gene disruption of rasG produced cells that exhibited reduced motil