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Vol. 17, Issue 1, 357-366, January 2006
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* Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4256;
Pharmacology Research Associate Training Fellowship Program, National Institute of General Medical Sciences, National Institutes of Health, Bethesda, MD 20892-6200
Submitted August 19, 2005;
Accepted October 21, 2005
Monitoring Editor: John York
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Iijima et al., 2002; Weiner, 2002; Parent, 2004). It has been determined that these cells are extremely sensitive and can detect spatial gradients in which the chemoattractant concentration differs by as little as 1% between the front and the back of the cell (Mato et al., 1975; Zigmond, 1977). Furthermore, these cells are responsive over a wide dynamic range of chemoattractant concentrations, which can vary over at least 4 orders of magnitude. This dynamic responsiveness requires a tightly regulated balance between stimulatory and inhibitory responses, which are both initiated upon chemoattractant receptor activation (Zigmond and Sullivan, 1979; Dinauer et al., 1980a,b). Temporal modulation of stimulatory and inhibitory inputs give rise to transient responses composed of an initial peak of activation followed by a period of adaptation, during which the system is refractory to further signals, and finally deadaptation, which resets the system and allows cells to respond to additional chemotactic signals (Parent and Devreotes, 1999; Devreotes and Janetopoulos, 2003). Intriguingly, although the mechanisms that control the activation process have been extensively characterized, most of the adaptive mechanisms remain poorly understood.
For neutrophils and Dictyostelium, the binding of chemoattractants to G protein-coupled receptors stimulates a variety of responses, including an increase in the levels of cAMP (Harvath et al., 1991; Dai et al., 1994; Saran et al., 2002). In Dictyostelium, the signaling pathways that lead to the activation of the adenylyl cyclase expressed during aggregation (ACA), which converts ATP into cAMP, have been extensively studied (Kriebel and Parent, 2004). In this organism, cAMP acts as a chemoattractant by binding to specific seven-transmembrane cAMP receptors, cARs. Stimulation of the receptors leads to the activation of ACA and the synthesis of more cAMP, a fraction of which is secreted to initiate a signal relay loop that propagates the signal to neighboring cells. As chemotaxis proceeds, Dictyostelium cells begin to migrate in a head-to-tail manner, generating long streams of cells (Pitt et al., 1992; Kriebel et al., 2003). We have previously shown that this streaming process not only depends on the presence of ACA itself but also on its cellular distribution to the rear of cells (Kriebel et al., 2003). We propose that the spatial restriction of ACA provides a compartment from which cAMP is secreted, thereby locally attracting neighboring cells.
The activation of ACA shows an absolute requirement for the pleckstrin homology (PH) domain-containing protein cytosolic regulator of adenylyl cyclase (CRAC) (Insall et al., 1994; Lilly and Devreotes, 1995). On receptor stimulation, G
subunits activate phosphoinositide 3-kinase (PI3K) in a Ras-dependent manner, leading to the production of 3-phosphoinositides (3-PI) to which CRAC binds via its PH domain (Funamoto et al., 2002; Comer et al., 2005). After the addition of a uniform dose of chemoattractant, CRAC is rapidly and transiently recruited to the plasma membrane around the entire periphery of the cell. In contrast, when cells are exposed to a gradient of chemoattractant, the membrane recruitment is restricted to the side of the cell facing the highest concentration of attractant (Parent et al., 1998). This dynamic distribution is part of an elegant mechanism that cells use to compartmentalize signal transduction events. In both neutrophils and Dictyostelium, PI3K signaling has been shown to localize to the leading edge of chemotaxing cells, whereas the enzyme that degrades the products of PI3K, the tumor suppressor PTEN, localizes to the back and sides (Servant et al., 2000; Funamoto et al., 2002; Iijima and Devreotes, 2002; Li et al., 2005). Disruption of the balance between these two enzyme activities, either by pharmacological inhibition or targeted gene disruption, results in defects in cell polarity, spatial actin organization, gradient sensing, and efficiency of chemotaxis (Chung et al., 2001; Iijima et al., 2002; Stephens et al., 2002). Although the mechanism by which CRAC activates ACA remains to be determined, recent findings have shown that the PI3K-mediated activation of CRAC independently regulates ACA activation and chemotaxis, suggesting that these two processes are subject to coordinated regulation (Comer et al., 2005).
Interestingly, the activation of ACA requires another cytosolic regulator called Pianissimo (Pia) (Chen et al., 1997). In contrast to CRAC, Pia harbors no known protein motifs, but several Pia homologues in organisms that range from yeast to mammals have been identified. The mammalian Pia homologue, called Rictor or mAVO3, is part of the target of rapamycin (TOR) complex 2 (TORC2), along with TOR and LST8, and has been shown to regulate cytoskeletal rearrangements (Sarbassov et al., 2004). We have recently shown that in Dictyostelium the components of TORC2, which also include the AVO1 homologue ras interacting protein 3 (RIP3), function together in a preformed complex to regulate ACA activity (Lee et al., 1999, 2005). Moreover, reconstitution experiments established that TORC2 can form in the absence of CRAC. Given previous data showing that ACA activity can be reconstituted in lysates derived from cells lacking both Pia and CRAC only when both proteins are added back (Chen et al., 1997), these findings suggest that ACA activation requires an input from both TORC2 and CRAC.
In this article, we show that PI3K is central to both activation and adaptation of ACA in response to chemoattractant stimulation. Our results are consistent with a model in which PI3K coordinately regulates pathways that control chemotaxis, ACA activation, and ACA adaptation. Control of all three pathways by PI3K provides an efficient mechanism for a strict regulation and ensures the proper balance of these counteracting inputs on ACA activation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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-32P]ATP (800 Ci/mmol) was purchased from MP Biomedicals (Irvine, CA). Precast NuPage Bis-Tris protein electrophoresis gradient gels were purchased from Invitrogen (Carlsbad, CA).
Cell Lines
The wild-type Ax3 and pten-/pi3k1-/2- cell lines (Janetopoulos et al., 2005) were a kind gift from Peter Devreotes (Johns Hopkins University School of Medicine, Baltimore, MD). The pi3k1-/2- (Zhou et al., 1995; Buczynski et al., 1997) and PI3K2/pi3k1-/2- (Funamoto et al., 2002) cell lines were a kind gift from Rick Firtel (University of California, San Diego, San Diego, CA).
Cell Growth and Differentiation
Cells were cultured in shaking suspension in HL5 medium and differentiated to the chemotaxis competent aggregation stage essentially as described previously (Devreotes et al., 1987; Parent et al., 1998). In brief, cells from log phase suspension culture were harvested and differentiated for 4.5-7 h in suspension at 2 x 107 cells/ml in development buffer (DB; 5 mM sodium phosphate buffer, pH 6.2, 2 mM MgSO4, and 0.2 mM CaCl2) with exogenous pulses of 75 nM cAMP every 6 min. To verify that cells differentiated to the chemotaxis competent stage, we performed Western blot analysis of the early developmental markers, chemoattractant receptor (cAR1), and the aggregation stage adenylyl cyclase (ACA).
Microscopy
For the micropipette chemotaxis assay and CRAC translocation assay, cells were viewed with an inverted Zeiss Axiovert 200 microscope (Carl Zeiss, Thornwood, NY) equipped with automated filter wheels (Ludl Electronic Products, Hawthorne, NJ). Images were recorded with a CoolSnap HQ charge-coupled device camera (Roper Scientific, Trenton, NJ) operated by IPLab software (Scanalytics, Fairfax, VA). All images in a series were processed identically using IPLab and/or Adobe Photoshop. Fluorescent images were viewed with the appropriate filter set and nonfluorescent cells were viewed using either Varel or differential interference contrast optics.
Chemotaxis Assays
To analyze the migration of groups of individual cells, the micropipette chemotaxis assay was carried out essentially as described previously (Parent et al., 1998). In this assay, cells undergo starvation induced differentiation to the chemotaxis competent aggregation stage (4-7 h), and their directed migration is observed in a gradient supplied by a micropipette filled with the chemoattractant cAMP at a concentration of 10 µM. The chemotaxis was recorded with a 15-s interval between successive frames. Chemotaxis of a large population of cells was observed with the under agarose assay, as described previously (Nelson et al., 1975; Comer et al., 2005). Briefly, 3-4 ml of 0.5% agarose in DB containing 1 mM caffeine was poured into 35-mm Petri dishes. The agarose was allowed to cool completely, and then three equidistant wells were cut out of the agarose with a wide-bore Pasteur pipet. The middle well was filled with 1 µM cAMP in DB, and the gradient was allowed to establish for 30 min before plating cells. Differentiated cells were suspended at 2 x 107 cells/ml in DB containing 2 mM caffeine and plated into the outer wells. After the cells were allowed to chemotax for 
1 h, the wells were carefully overlaid with phosphate buffer and visualized on a Leica stereoscope. Each assay was performed in triplicate, and the results are representative of at least three independent experiments on different days, and wild-type cells were always assayed along with the tester cell line.
CRAC Translocation Assay
The chemoattractant-mediated recruitment of CRAC (CRAC-GFP) to the plasma membrane was observed on a fluorescence microscope as described previously (Parent et al., 1998). Briefly, CRAC-GFP-expressing cells were stimulated with 20 µM cAMP and fixed (1% formaldehyde, 0.125% glutaral-dehyde, and 0.01% Triton X-100 in phosphate buffer) after 5 s of stimulation. Cells were viewed as described above.
Adenylyl Cyclase and cAMP Accumulation Assays
Activation of adenylyl cyclase via chemoattractant receptor stimulation was measured essentially as described previously (Parent and Devreotes, 1995; Parent et al., 1998). Briefly, differentiated cells were stimulated with 50 µM cAMP and filter lysed at the indicated time points into Tris buffer containing [-32P]ATP diluted with unlabeled ATP to a final concentration of 150 Ci/mol. The reaction was allowed to proceed for 1 min, stopped with SDS/ATP, and the radiolabeled cAMP was purified by sequential Dowex-AG 50W X-4 and alumina column chromatography (Salomon, 1979). Adenylyl cyclase measurements by this assay exhibit considerable variation from experiment to experiment. Although the relative extent of activation of different cell lines or treatment conditions is highly reproducible between experiments, the absolute activity can vary significantly (2- to 3-fold at times) from day to day. Therefore we, and others in the field, have chosen to present adenylyl cyclase activation data as representative of results from at least three to five or more independent experiments, each performed in duplicate on a given day. Furthermore, comparisons between different cell lines or treatment conditions are made on samples assayed on the same day, using the same reagent mixture.
To measure activation of adenylyl cyclase by global stimulation of both heterotrimeric G proteins and small GTPases, lysates were incubated with 100 µM guanosine 5'-O-(3-thio)triphosphate (GTPS) and, after 5 min, aliquots of the lysate were assayed for adenylyl cyclase activity for 2 min. For the mixing experiments, cell lines were mixed, lysed together, and otherwise assayed exactly as described above. To prepare cytosolic and membrane extracts for the in vitro complementation experiment, cells were suspended at 8 x 107 cells/ml in simple lysis buffer (SLB; 10 mM Tris, pH 7.5, 0.2 mM EGTA, and 200 mM sucrose), filter lysed, and centrifuged at 9500 x g for 20 min. The supernatant fraction was denoted the cytosolic extract and was used without further dilution. The pellet fraction was resuspended in an equivalent volume of SLB and designated the membrane fraction. The in vitro complementation was performed in essentially the same manner as the direct GTPS-stimulated adenylyl cyclase activation assay, except that cells were lysed directly into a tube on ice containing the cytosolic or membrane extract, as indicated. After 5 min, the adenylyl cyclase activity of the mixture was assayed for 2 min, as described.
Chemoattractant-mediated cAMP production was measured in differentiated cells after the addition of 5 µM 2'deoxy-cAMP in the presence of 10 mM DTT and 200 µM 3-isobutyl-1-methylxanthine using the cAMP radioimmunoassay kit from GE Healthcare.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). To gain insight into this finding, we more carefully investigated the role of PI3K action in these processes. Although there are six PI3K isoforms in Dictyostelium (Janetopoulos et al., 2005), most of the chemoattractant-mediated PI3K activity arises from PI3K1 and PI3K2 (Huang et al., 2003). We therefore used cells in which the PI3K1 and PI3K2 isoforms have been disrupted (pi3k1-/2-) for our studies (Zhou et al., 1995; Buczynski et al., 1997). When we plated pi3k1-/2- cells on nonnutrient agar, we observed that these cells aggregate and differentiate, but they make misshapen terminal differentiation structures, as described previously (Zhou et al., 1995). To study this phenotype further, we recorded the differentiation process and found that the normal oscillatory pattern of periodic waves present in wild-type cells is disrupted in pi3k1-/2- cells. Although wild-type cells display regular wave patterns, which arise as cells respond to passing waves of synchronized chemoattractant release from cells in an aggregation center (Parent and Devreotes, 1996; Sawai et al., 2005), the wave patterns of the pi3k1-/2- cells are irregular and do not propagate far from the aggregation center (our unpublished data). To determine whether this defect is simply caused from aberrant expression of key signaling components, we assessed the expression of the chemoattractant receptor, cAR1, and ACA in pi3k1-/2- cells and found that they were comparable with wild-type cells (Figure 1A). We next investigated whether cAMP stimulation affects the extent or kinetics of cAR1 phosphorylation in pi3k1-/2- cells. On cAMP stimulation, cAR1 is rapidly phosphorylated on a cluster of C-terminal serine residues, giving rise to a 3- to 5-fold reduction in the cAMP binding affinity of cAR1 (Caterina et al., 1995). We found that cAMP-induced cAR1 phosphorylation is normal in pi3k1-/2- cells (Figure 1B). These results show that the wave propagation defect observed in pi3k1-/2- cells is not a consequence of defects at the level of cAMP detection by cAR1 or at alterations in the extent of cAMP-mediated cAR1 phosphorylation by downstream kinases.
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). Figure 1C shows images of wild-type and pi3k1-/2- cells migrating in a gradient of cAMP generated by a micropipette filled with 10 µM cAMP. Images taken just before and 18 min after the micropipette is activated are shown. Although wild-type cells polarize and migrate in long streams of cells (see Video 1), we found that pi3k1-/2- cells migrate exclusively as individual cells to the micropipette tip (see Video 2). As observed previously, we noted that the pi3k1-/2- cells display reduced polarity and do not migrate with the same directionality as wild-type cells (Funamoto et al., 2001), a behavior that is more pronounced in cells that are differentiated for 4-5 h (our unpublished data). To observe a larger population of cells, we examined the behavior of pi3k1-/2- cells in the under agarose chemotaxis assay (Nelson et al., 1975; Comer et al., 2005). Again, we found that, in contrast to wild-type cells, pi3k1-/2- cells do not align and migrate as a diffuse halo of individual chemotaxing cells (Figure 1D). To further test the role of PI3K in signal relay and stream formation, we subjected wild-type cells to the PI3K inhibitor LY294002 during the process of streaming (Figure 2 and Video 3). In this experiment, we allowed cells to migrate toward the micropipette and, after 18 min, added 30 µM LY294002. Again, we observed that inhibition of PI3K function leads to profound streaming defects. Remarkably, we found that LY294002 acts very rapidly, dissociating streams within 5 min. Together, these results show that PI3K activity is required for streaming during Dictyostelium chemotaxis.
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). Nevertheless, pi3k1-/2- cells and LY294002-treated wild-type Ax3 cells can still undergo chemotaxis, albeit with reduced polarity and directionality (Figures 1C and 2; Funamoto et al., 2001). Thus, we next examined whether chemoattractant-mediated translocation of CRAC to the plasma membrane is impaired in pi3k1-/2- cells or in LY294002-treated wild-type cells. We observed that, as for other PH domain-containing proteins (Meili et al., 1999; Servant et al., 2000; Funamoto et al., 2001), the recruitment of CRAC-GFP to the membrane is undetectable in pi3k1-/2- cells, whether observed microscopically (Figure 3A, left) or in a biochemical translocation assay (Figure 3A, right). Likewise, membrane translocation of CRAC-GFP was undetectable in LY294002-treated cells (Figure 3B). Thus, although the R42C-CRAC mutation and the reduction of PI3K activity both impair CRAC translocation beyond the detection limits of our assays, they confer distinct chemotaxis phenotypes. Although these results would seem to be in discrepancy, we submit that the R42C-CRAC mutant gives a more definitive chemotaxis phenotype because the mutation abolishes the interaction with PI3K products, which is tantamount to complete inhibition of PI3K activity. In contrast, it has been shown that in pi3k1-/2- cells, steady-state phosphatidylinositol-(3,4,5)-trisphosphate [PI(3,4,5)P3] levels are reduced to <50% (Zhou et al., 1998), whereas chemoattractant-induced PI(3,4,5)P3 production is reduced to 4% that of wild-type cells (Huang et al., 2003). Likewise, although Akt/PKB activation is greatly reduced in pi3k1-/2- cells, it is not abolished (Meili et al., 1999). Because chemotaxis is very sensitive, we propose that this residual PI3K response is sufficient to promote low levels of CRAC translocation, which are undetectable by our assay, but sufficient to drive the highly sensitive process of gradient sensing and chemotaxis. One consequence of this hypothesis is that there is likely to be signal amplification downstream of CRAC translocation. Thus, cells can recover from the low levels of PI3K-mediated CRAC translocation in pi3k1-/2- cells and upon LY294002 treatment because signal amplification renders cells resilient to significant perturbations in the chemotaxis signaling machinery.
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PI3K Regulates Adenylyl Cyclase Activation and Adaptation, but Not Its Cellular Distribution
Although ACA is properly expressed in pi3k1-/2- cells, we reasoned that the streaming defect of these cells may arise by a defect in regulating its activity or its cellular distribution. Indeed, we demonstrated previously that cells lacking ACA are unable to stream, although they can sense and migrate directionally toward a point source of cAMP (Pitt et al., 1992; Kriebel et al., 2003). We also established that ACA is highly enriched at the back of polarized, chemotaxing cells (Kriebel et al., 2003) and proposed that this provides a spatially restricted compartment from which cAMP is locally secreted to specifically attract neighboring cells, thus comprising a primary mechanism for signal relay and streaming. To determine whether the streaming defect of PI3K-deficient cells is caused by mislocalization of ACA, we examined its cellular distribution in pi3k1-/2- cells. For these experiments, we expressed ACA-YFP in pi3k1-/2- cells (ACA-YFP/pi3k1-/2-) or treated aca- cells expressing ACA-YFP (ACA-YFP/aca-) with LY294002. We found that, in both cases, ACA-YFP is properly enriched at the back of migrating cells (Figure 4, A and B). We conclude that the streaming defect caused by PI3K deficiency does not arise from disrupted cellular distribution of ACA.
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We next assessed the activity of ACA in PI3K-deficient cells. For these experiments, we used an activation trap assay, in which cells are stimulated with chemoattractant and at a specific time, rapidly lysed and assayed for adenylyl cyclase activity. When we examined receptor-mediated activation of ACA in pi3k1-/2- cells, we found that they exhibited a dramatic defect in adaptation of the response (Figure 4C). Wild-type cells exhibit a peak of activation at 1 min poststimulation, followed by a period of adaptation that reduces the activity to basal levels within 3-5 min. In contrast, pi3k1-/2- cells show a normal initial peak of activation, followed by a slight dip in activity and a dramatic loss of adaptation that results in a subsequent rise in activity, which often exceeds the initial peak, within 5-8 min poststimulation. The activity continues to rise for at least 12 min, and eventually begins to subside, although we have measured significant activity even 30 min after stimulation (our unpublished data). We further found that the reexpression of PI3K2 in pi3k1-/2- cells rescues the ability to properly attenuate the ACA response (Figure 4D) and restores streaming during chemotaxis (our unpublished data), consistent with previous observations showing that reexpression of PI3K2 in pi3k1-/2- cells rescues PKB activation (Funamoto et al., 2002). These findings show that PI3K activity controls the adaptation phase of the chemoattractant-mediated activation of ACA.
To further study the balance between 3-PI production and degradation in chemoattractant signaling to ACA, we examined the ACA response in cells deficient in both PI3K and PTEN activity. Deletion of the 3-PI-degrading enzyme PTEN in the pi3k1-/2- background results in cells that have an elevated basal level of CRAC at the plasma membrane, but they do not recruit more CRAC upon chemoattractant stimulation (Janetopoulos et al., 2005). These data suggest that, despite elevated basal levels of 3-PIs and membrane-associated CRAC, these cells do not exhibit proper temporal and spatial regulation of 3-PIs upon chemoattractant stimulation. We found that chemoattractant stimulation of these cells gives rise to a normal peak of ACA activation and a partial rescue of the adaptation defect observed in pi3k1-/2- cells (Figure 4D). This result has several implications. First, it shows that fluctuations in 3-PI production are not necessary for the ACA activation when there is an elevated basal level of CRAC at the plasma membrane. Second, it demonstrates that ACA activation must require a secondary input, such as TORC2, in addition to PI3K-mediated CRAC recruitment, because, in spite of the fact that there is a significant amount of CRAC at the plasma membrane in unstimulated cells, the basal ACA activity is not elevated. Finally, the partial rescue of the ACA adaptation response in these cells suggests that the balance of PI3K-mediated activation and adaptation pathways is partially restored by slowing the degradation of the small amount of 3-PIs that are synthesized in the pi3k1-/2- background. We therefore wanted to test whether inhibition of all PI3K isoforms with LY294002 would give the same ACA adaptation defect. Surprisingly, in contrast to the pi3k1-/2- cells but in agreement with our results using R42C-CRAC/crac- cells, LY294002 treatment significantly inhibited ACA activation (Figure 4E), suggesting that PI3K activity is indeed separately required for both activation and adaptation of ACA signaling. Furthermore, LY294002 treatment of pi3k1-/2- cells also ablated ACA activation (our unpublished data). Although we cannot rule out the possibility that treatment with LY294002 inhibits ACA activity via a non-PI3K pathway, such as through inhibition of TOR signaling (Brunn et al., 1996), our previous observation that crac- cells expressing a point mutation in the 3-PI binding motif within the CRAC PH domain are strongly impaired in their ability to activate ACA supports a direct role for PI3K. From these results, we infer that the loss of streaming during chemotaxis of wild-type cells treated with LY294002 is likely the result of too little ACA activity, as in the streaming defect reported previously for aca- cells (Kriebel et al., 2003). We propose that the streaming defect in the pi3k1-/2- cells, in contrast, is because of misregulation of the adaptation response, resulting in locally high concentrations of chemoattractant, which neighboring cells are unable to integrate into a proper streaming response. This hypothesis is further supported by our finding that net cAMP production is dramatically increased in pi3k1-/2- cells and significantly reduced in LY294002-treated wild-type cells (Figure 4F).
Distinct G Protein-mediated Pathways Control Chemoattractant Signaling
Another means of activating ACA is by direct G protein stimulation of cell lysates with the nonhydrolyzable GTP analogue GTPS, which simultaneously activates the entire cellular complement of GTP binding proteins, both heterotrimeric G proteins and small GTPases. In wild-type cells, GTPS stimulation results in a net activation of ACA, as observed previously (Figure 5A). Surprisingly, however, we found that the addition of GTPS to pi3k1-/2- lysates does not increase ACA activity (Figure 5A). We infer from these findings that GTPS stimulation is not functionally equivalent to chemoattractant receptor stimulation. To test whether the GTPS defect of the pi3k1-/2- cells can be reconstituted, we mixed aca- cells with the pi3k1-/2- cells before lysis. In these experiments, the aca- cells, which are devoid of G protein-coupled adenylyl cyclase activity, are thought to supply the PI3Ks required to reconstitute the lost activity. We found that the mixed pi3k1-/2-/aca- lysate is fully capable of GTPS-mediated ACA activation (Figure 5A). Furthermore, we observed that wild-type cytosol, but not wild-type membranes, restores GTPS-mediated ACA activity to pi3k1-/2- lysates (Figure 5B), showing that the wild-type cytosol effectively restores the cytosolic PI3K activity, as observed previously for the reconstituted translocation of CRAC (Huang et al., 2003). Finally, we again found that reexpression of PI3K2 in the pi3k1-/2- cells restores GTPS-mediated ACA activation (Figure 5C). We conclude that the loss of PI3K2 impairs the ability of GTPS to activate ACA.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) and mammalian leukocytes (Zigmond and Sullivan, 1979). Selective adaptation seems to be important for neutrophils to integrate conflicting chemotactic signals in a complex environment of multiple chemat-tractants (Foxman et al., 1999) and is important for both chemotaxis and signal relay in Dictyostelium (Devreotes and Steck, 1979; Van Haastert, 1983). However, although G9 has recently been identified as a general modulator of inhibitory chemotactic responses (Brzostowski et al., 2004), the mechanisms that regulate adaptation remain largely unknown. Progress in this area has undoubtedly been hampered by the complexity of the chemoattractant signaling system. Moreover, the phenotype of Dictyostelium adaptation mutants may be subtle and difficult to identify in a mutagenesis screen. For example, the pi3k1-/2- cells, in spite of their profound ACA adaptation defect, develop relatively normal terminal differentiation structures (Zhou et al., 1995).
Two opposing receptor-mediated biochemical processes—an excitatory and an inhibitory response—have been proposed to mediate the transient nature of the ACA response (Parent and Devreotes, 1999). When both responses reach a steady state, ACA activity returns to basal levels. We present evidence that supports a role for PI3K in both the excitation and inhibition pathways that control ACA in response to chemoattractant stimulation. Analysis of the chemoattractant receptor-mediated ACA activation in pi3k1-/2- cells reveals a profound adaptation defect, implying a key role for PI3K in the mechanism of adaptation. Our finding that deletion of PTEN in the pi3k1-/2- background, which elevates basal 3-PI levels, partially suppresses the adaptation defect suggests a direct role for 3-PIs in this process. In contrast, treatment of wild-type cells with a PI3K inhibitor or expressing a mutant of CRAC that has lost the capacity to bind to 3-PIs in crac- cells drastically reduces activation of the ACA response, pointing to an essential role for 3-PIs in ACA activation. These results are consistent with a model in which PI3K controls both the excitation and the inhibition of the ACA response through independent pathways (see model, Figure 6).
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We envision four nonmutually exclusive mechanisms for coordinating the output from PI3K signaling to the excitation and inhibition pathways controlling ACA. The first possibility is that the excitation branch is more sensitive than the inhibition branch. Thus, because the inhibition branch would require a greater input from PI3K, inhibition would be more affected than excitation in conditions of reduced PI3K activity and a loss of the adaptation response would occur before the activation is affected. Indeed, the chemoattractant-induced PI(3,4,5)P3 production in pi3k1-/2- cells is greatly reduced compared with wild-type cells, yet pi3k1-/2- cells show a robust initial peak of ACA activation. The second possibility is that the two branches have distinct kinetics, and the temporal delay in the inhibition pathway is exaggerated in the pi3k1-/2- cells. Third, PI3K isoforms other than PI3K1 and PI3K2 could be responsible for the initial activation peak, whereas these two predominant isoforms would specifically control the inhibition branch. Finally, because PI3Ks are known to exhibit both lipid and protein kinase activities (Carpenter et al., 1993; Dhand et al., 1994; Naga Prasad et al., 2005), it is possible that the inhibition pathway leading to ACA adaptation depends on the protein kinase activity of PI3K, which could be regulated independently of the lipid kinase activity. At the moment, it is not known whether the Dictyostelium PI3Ks display protein kinase activity.
Our finding that stimulation of cell lysates with GTPS does not promote ACA activation in pi3k1-/2- cells provides further insight into the mechanisms controlling the adaptive response. Our data are consistent with at least two hypotheses. First, it is possible that pleiotropic defects in the pi3k1-/2- cells impair the GTPS-mediated activation of ACA, despite the ability of these cells to activate ACA through the G protein-coupled chemoattractant receptor. Alternatively, our results may suggest the presence of a small GTPase that functions downstream of PI3K to attenuate the ACA activation response. This putative GTPase would need to be kinetically slower and/or less sensitive than the receptor-coupled heterotrimeric G protein and the Ras GTPase that has been proposed to activate PI3K (Funamoto et al., 2002; Sasaki et al., 2004). Thus, in wild-type cells, activation of the upstream G and Ras would dominate with a net activation of ACA upon GTPS stimulation. In contrast, in the pi3k1-/2- cells, the efficiency of the G and Ras signals is reduced because of the low PI3K activity in the excitation branch, and thus, a GTPase functioning in the inhibition branch dominates, resulting in a shift of the balance between excitation and inhibition, thus yielding little or no net activation. The identification of the GTPase and its associated regulators as mediators of ACA inhibition presents a considerable challenge. In Dictyostelium, there are at least six Ras proteins, some with redundant functions, and greater than 20 Ras guanine nucleotide exchange factors (Wilkins and Insall, 2001; Kae et al., 2004; Wilkins et al., 2005). Nevertheless, as these challenges are met, elucidation of the mechanisms of adaptation will provide fundamental insight into chemotaxis and the integration of chemoattractant signal transduction responses.
Reduction of PI3K activity affects multiple chemoattractant-mediated processes, pointing to a central role for PI3Ks in the integration of chemoattractant signaling. Previous studies have shown that pi3k1-/2- Dictyostelium cells and neutrophils from PI3K-null mice have reduced directionality, polarity and speed, but chemotaxis can still occur (Hirsch et al., 2000; Li et al., 2000; Chung et al., 2001; Funamoto et al., 2001; Iijima et al., 2002; Stephens et al., 2002; Weiner, 2002). Similar effects on chemotaxis were obtained upon PI3K inhibition with LY294002 or wortmannin (Funamoto et al., 2001; Wang et al., 2002). These observations are consistent with our hypothesis that, in addition to the upstream actin-dependent signal amplification loops that lead to the polarization of PI3K activity (Weiner et al., 2002), further downstream amplification of the PI3K response is likely to occur, such that even small PI3K inputs can be amplified into functional responses. This exquisite regulation may help explain the wide dynamic range of chemotactic responsiveness of Dictyostelium. These cells not only migrate directionally in very shallow gradients supplied by a source of 10-9 M cAMP (Mato and Konijn, 1975), they can also chemotax in a gradient applied over a constant background concentration of 10-5 M cAMP (Van Haastert, 1983). It therefore seems that PI3Ks are involved in regulating multiple effectors of chemoattractant signaling and that these effectors display distinct sensitivities to PI3K products.
Our findings suggest a novel mechanism by which PI3K activity regulates the excitation and inhibition pathways that control ACA in response to chemoattractant stimulation. We propose that such a mechanism, in which activation and adaptation are controlled by the same input, allows for a stringent regulation of the excitation/inhibition cycle. By linking two opposing responses, inhibition and excitation responses would always be proportional, ensuring the proper balance that is required to maintain the oscillatory circuit of chemoresponsiveness. We envision that similar coordinated controls operate in higher eukaryotes.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Abbreviations used: ACA, adenylyl cyclase expressed during aggregation; CRAC, cytosolic regulator of adenylyl cyclase; GTPS, guanosine 5'-3-O-(thio)triphosphate.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Carole A. Parent (parentc{at}helix.nih.gov).
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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