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Report |
G protein–independent Ras/PI3K/F-actin circuit regulates basic cell motility
Correspondence to Richard A. Firtel: rafirtel{at}ucsd.edu
Phosphoinositide 3-kinase (PI3K)
and Dictyostelium PI3K are activated via G protein–coupled receptors through binding to the Gß subunit and Ras. However, the mechanistic role(s) of Gß and Ras in PI3K activation remains elusive. Furthermore, the dynamics and function of PI3K activation in the absence of extracellular stimuli have not been fully investigated. We report that gß null cells display PI3K and Ras activation, as well as the reciprocal localization of PI3K and PTEN, which lead to local accumulation of PI(3,4,5)P3. Simultaneous imaging analysis reveals that in the absence of extracellular stimuli, autonomous PI3K and Ras activation occur, concurrently, at the same sites where F-actin projection emerges. The loss of PI3K binding to Ras–guanosine triphosphate abolishes this PI3K activation, whereas prevention of PI3K activity suppresses autonomous Ras activation, suggesting that PI3K and Ras form a positive feedback circuit. This circuit is associated with both random cell migration and cytokinesis and may have initially evolved to control stochastic changes in the cytoskeleton.
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Introduction |
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 TopAbstract Introduction Results and discussionMaterials and methodsReferences |
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The Class IB PI3K, PI3K, is expressed most highly in neutrophils and activated by binding to Gß subunits and Ras upon G protein–coupled receptor activation (Stephens et al., 1994, 1997; Stoyanov et al., 1995; Suire et al., 2005). Overexpression of Gß subunits in HEK293 cells leads to PI3K activation via its interaction with the p101 catalytic subunit (Krugmann et al., 2002; Brock et al., 2003). Recently, p101 knockout mice and RBD-mutated PI3K knock-in mice have been generated. In the neutrophils from these mutant mice, chemoattractant-induced PI3K activation is significantly decreased (Suire et al., 2006). These in vitro and in vivo studies demonstrate a linkage between chemoattractant stimulation and PI3K activation. Interestingly, lipid PI3K assays demonstrated that cells have a basal level of PI3K activity in the absence of chemoattractants or serum (Huang et al., 2003; Suire et al., 2005). It is unclear whether this basal activity of PI3K is actively controlled or merely a passive property of this enzyme.
Dictyostelium Class I PI3Ks are considered to be the functional counterpart of PI3K on the basis of biochemical and structural characteristics (Janetopoulos et al., 2005; Sasaki and Firtel, 2006). As implicated in PI3K regulation in mammalian cells, Dictyostelium cells lacking Gß cannot activate PI3K in response to chemoattractant or GTPS stimulation (Meili et al., 1999; Huang et al., 2003). Dictyostelium cells, however, manage to divide and undergo random motility in the absence of functional heterotrimeric G proteins (Wu et al., 1995). It is known that these complex cell shape changes involve the polymerization of F-actin, but the upstream signals regulating these pathways have yet to be determined.
Cells have the intrinsic ability to produce pseudopodia and move in the absence of chemoattractants or nutrients (Wessels et al., 1988; Condeelis and Segall, 2003). This random cell migration allows cells to explore their environment and is associated with metastasis of tumor cells. Although much progress has been made in elucidating the molecular mechanisms of chemoattractant-mediated migration (chemotaxis), random cell migration has barely been investigated. In this study, we genetically separate the GPCR heterotrimeric G protein–dependent cellular events that regulate chemotaxis from the basal regulatory signaling loops that control random cell movement and cytokinesis. We demonstrate that both PI3K and Ras activation occur actively, at the same sites of new pseudopod formation in the absence of extracellular stimuli and without heterotrimeric G protein input. PI3K and Ras activation also occur at the poles of dividing cells in wild-type strains and in cells lacking functional heterotrimeric G proteins, implying that cell shape changes during cytokinesis are controlled by a similar mechanism. We suggest that this "unprompted" Ras activity and PI(3,4,5)P3 accumulation, which are independent of external stimuli, constitute a core regulatory pathway involved in a variety of physiological responses and provide the basis for many ligand- or substrate-mediated processes, such as chemotaxis and phagocytosis.
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Results and discussion |
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signaling is required for cells to become developmentally competent. We expressed the GFP-tagged PH domain of CRAC (PHcrac), a reporter for PI(3,4)P2 and PI(3,4,5)P3, in the KAx-3 wild-type strain and a null strain of Gß, the sole Gß subunit in Dictyostelium (Wu et al., 1995; Comer et al., 2005). Consistent with previous studies (Parent 1998; Meili et al., 1999), gß null (gß–) cells show no detectable folic acid–mediated translocation of PHcrac to the cell cortex (Fig. 1 A).
However, we noticed that, in the buffer containing only Na/KPO4, the PH domains spontaneously accumulated at both pseudopodial extensions and to macropinosomes (the structures responsible for fluid-phase uptake) of randomly moving KAx-3 cells (Lee and Knecht, 2002). The PHcrac accumulation in randomly moving cells is a consequence of PI3K activation because its membrane localization was abolished 
1 min after the addition of LY294002, a PI3K inhibitor (Fig. 1 A). Importantly, gß– cells also exhibited spontaneous PHcrac accumulation, which was sensitive to the PI3K inhibitor. This finding indicates that PI3K is spontaneously activated without heterotrimeric G protein signaling.
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As spontaneous PI(3,4,5)P3 accumulation appears to occur strictly at sites of F-actin protrusion, including both the sites of macropinosomes and pseudopodial extensions, we examined whether spontaneous PI3K activation requires F-actin synthesis. (In many instances, we could not resolve whether protrusions began as macropinosomes or pseudopodia, but consider them pseudopodia if they ultimately protruded from the cell and gave rise to a net movement.) After treatment with Latrunculin B (LatB), an inhibitor of F-actin polymerization, spontaneous PI(3,4,5)P3 accumulation (as visualized through PHcrac recruitment) was lost and cells rounded up. After washout of LatB, cells regained the spontaneous PI(3,4,5)P3 accumulation at the sites of new pseudopodial projections (Fig. 1 C). This finding suggests that Gß-independent, spontaneous PI3K activation requires and occurs at sites of F-actin polymerization. Furthermore, we found that GFP-PI3K2 labeled the sites of new F-actin projections in both wild-type and gß– cells (Fig. 1 D and Video 1, which is available at http://www.jcb.org/cgi/content/full/jcb.200611138/DC1), whereas PTEN-GFP detached from the membrane in pseudopodial extensions in both wild-type cells and gß– cells (unpublished data). The gß– cells did not exhibit PI3K translocalization in response to a chemoattractant, whereas wild-type cells did (unpublished data). Thus, although PI3K and PTEN localization is regulated by extracellular stimuli in the chemotaxing cells, the reciprocal localization of PI3K and PTEN occurs during random cell movement, even in the absence of chemoattractant, nutrients, and heterotrimeric G proteins.
Gß-independent, PI3K-dependent Ras activation in the absence of extracellular stimuli
As Ras is required for PI3K activation in response to extracellular stimuli in neutrophils and Dictyostelium, we monitored the localization of activated Ras during random movement using a GFP fusion of the human Raf1 RBD (GFP-RBD; Sasaki et al., 2004). As we demonstrated for PI(3,4,5)P3 accumulation, spontaneous localization of GFP-RBD occurs at the sites of F-actin projections in wild-type, gß–, and pten– cells. Surprisingly, the Ras activation was abrogated with LY294002 treatment and recovered after removal of the drug (Fig. 2 A and Video 2, which is available at http://www.jcb.org/cgi/content/full/jcb.200611138/DC1).
Biochemical assays confirmed a basal Ras activity in both wild-type and gß– vegetative cells in the absence of exogenous stimulation or nutrients (Fig. 2 B). The basal Ras and PI3K activation do not require cellular attachment to the substratum, as cells in suspension display the LY294002- sensitive spontaneous Ras and Akt activations (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200611138/DC1). The basal Ras activation is elevated in pten– cells (Fig. 2 C), supporting our notion of the requirement of PI3K. Furthermore, like PI3K activation, this unprompted Ras activation requires F-actin (Fig. 2 D and Video 3), although it is possible that a very low level of Ras and PI3K activity exists in these drug-treated cells. These drug-treated wild-type cells can activate Ras in response to chemoattractant stimulation (Fig. 2 E; Sasaki et al., 2004).
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It is worth noting that we previously observed spontaneous PI3K and Ras activation in developed PTEN-deficient Dictyostelium cells (Sasaki et al., 2004). The findings here differ because the developed pten– cells likely increase the level of the chemoattractant (cAMP) secretion (Iijima and Devreotes, 2002), which would evoke a GPCR/heterotrimeric G protein–mediated autocrine Ras activation (Comer et al., 2005).
Spontaneous activation of Ras and PI3K occurs simultaneously at the same sites
To compare the sites and timing of Ras activation to those of PI3K in randomly moving cells, we simultaneously imaged both GFP-RBD and RFP-PH by using a dual-wavelength beam splitter with high time resolution (200 msec). The RBD and PH domain appeared concurrently (within the limits of our resolution) at the same sites where pseudopodia start to form (Fig. 3 A, arrow, and Video 4, which is available at http://www.jcb.org/cgi/content/full/jcb.200611138/DC1).
The kinetics of the disappearance of the RBD and PHcrac probes were also closely correlated (Fig. 3 B). Interestingly, we observed this synchronized localization of RBD and PHcrac in the pten– cells. RBD and PHcrac also appear at micropinosome cups (clathrin-dependent small invaginating pits) with the same timing; however, PHcrac is retained considerably longer at these sites than RBD, suggesting differential regulation of Ras at later stages of micropinocytosis (Fig. S1 B). These data demonstrate that the timing of PI3K and Ras activation occurs in parallel and at the same sites during random movement.
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0.6 s faster than PHcrac to the plasma membrane (Fig. 3 C, cAMP, and Fig. S2 C, folic acid, which is available at http://www.jcb.org/cgi/content/full/jcb.200611138/DC1). Thus, Ras activation is not synchronized with PI3K activity in the initial phases (2 s) of chemoattractant signaling. Furthermore, in pten– cells stimulated with either folic acid or cAMP, PHcrac was retained at the plasma membrane for >1 min, whereas RBD returned to the cytosol in 20 s (Fig. 3 D). These results suggest that a chemoattractant/Gß-dependent pathway induces the signaling response that overrides or disrupts the intrinsic feedback activation of Ras/PI3K. In response to a chemoattractant gradient, cells activate a localized response at the site on the plasma membrane closest to the chemoattractant source while inhibiting the spontaneous activation of Ras/PI3K at the lateral sides of cells, thereby repressing random cell movement.
PI3K and Ras form a G protein–independent circuit and regulate random cell movement
We further investigated whether Gß-independent PI3K activation is involved in random motility. Fig. 4 A illustrates that random movement of two strains of wild-type cells (KAx-3 and NC-4) is rapidly blocked by 50 µM LY294002 (Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200611138/DC1; similar results are observed with 30 µM LY294002, unpublished data).
As NC-4 cells do not have macropinosomes, the cellular movement is driven by LY294002-sensitive pseudopodial extensions. This LY294002-sensitive random cell movement does not require heterotrimeric G proteins because the gß– cells exhibit random cell movement (Fig. 4 A). To clarify whether the random movement inhibition by LY294002 is due to the inhibition of PI3K signaling, we used pi3k1/2/3– cells (a strain lacking the three major PI3Ks responsible for PIP3 production in Dictyostelium [Takeda et al., 2007]). Strikingly, pi3k1/2/3– cells exhibit defects in random cell motility (Fig. 4 A and Video 6). LY294002-treated cells and pi3k1/2/3– cells still produce small pseudopodial projections, suggesting that a low level of PI3K-independent F-actin synthesis pathway is active. On the other hand, wild-type cells expressing membrane-bound PI3K2 (Myr-PI3K2; Funamoto et al., 2002), thus bypassing F-actin–induced PI3K translocalization, periodically exhibit sudden robust protrusions that are associated with rapid cell movement (Video 7). This finding suggests that F-actin–mediated PI3K translocation plays an important role in amplifying F-actin projection during random movement. Overall, these data demonstrate that PI3K regulates random cell movement and the extent of F-actin projections in the absence of extracellular stimuli.
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As Dd-target of rapamycin (Dd-TOR) may be another LY294002-sensitive kinase, we examined the role of TOR in random cell movement. We found that inhibition of TOR complex (TORC)1 by 1 µM rapamycin and disruption of TORC2 using pianissimo– cells, which lack the ortholog of mammalian Rictor/mAVO3 (Lee et al., 2005), did not result in random movement defects (unpublished data and Video 9, which is available at http://www.jcb.org/cgi/content/full/jcb.200611138/DC1). Akt is under the regulation of TORC2. We find that akt–cells, which have a defect in macropinocytosis, display normal RFP-PHcrac and GFP-RBD colocalization on F-actin projections linked to cellular movement, consistent with TORC2 not playing a major role in random cell movement (Rupper et al., 2001; Video 10).
The Ras/PI3K circuit regulates cell morphology during cytokinesis
We previously demonstrated that the regulation of PI(3,4,5)P3 plays a central role in cell shape changes during cytokinesis (Janetopoulos et al., 2005). We show here that Ras activation as examined by RBD cortical localization, like PI3K activation, was uniformly suppressed at the onset of cytokinesis as cells rounded up. As cells progressed through mitosis, the Ras activity reporter gradually localized to the polar ruffles during spindle assembly, cell elongation, and cytokinesis (Fig. 4 C). When the daughter cells separate from one another, cortically localized RBD and PI(3,4,5)P3 activity increase dramatically, resulting in high levels of random pseudopod extension. The gß– cells exhibit activation of Ras indistinguishable from that of wild-type cells during cytokinesis, suggesting that the Gß-independent Ras/PI3K circuit plays a fundamental role in cytokinesis.
Conclusions
Our studies reveal a critical role of a Gß-independent Ras/PI3K/PTEN/F-actin feedback loop in regulating random cell movement, a basic cellular function, and showed a linkage to cytokinesis. We dissect two distinct pathways that regulate Ras and PI3K activation and PTEN localization: (1) cells use Gß-dependent signaling to evoke Ras/PI3K activation and PTEN relocalization in response to chemoattractant stimulation; and (2) cells exploit Gß-independent machinery to induce stochastic Ras and PI3K activation and PTEN dissociation, for example, during random cell movement and cytokinesis. We assume that the regulators for Ras/PI3K/PTEN and F-actin polymerization/disassembly can influence the initiation and decay of the circuit (Fig. S2). As the process is stochastic, we hypothesize that an increase in the level of any of the responses over a threshold level may be sufficient to trigger the feedback loops and pseudopod formation, whereas components such as GAPs and phosphatases regulate the threshold and level/time of activation.
The pathways controlling random movement parallel those of the amplification step of chemotaxis that is controlled through a regulatory loop containing Ras, PI3K, PTEN, and F-actin (Sasaki and Firtel, 2006). We suggest that, in chemotaxis, the directed activation of the pathway by the chemoattractant/G proteins biases the localized activation of the intrinsic Ras/PI3K circuit and locally restricts the positive feedback loop that is the basis for random cell movement (Fig. S2). The output from the sensing mechanism activates Ras and generates PI3K binding sites and simultaneously results in the loss of PTEN binding sites. This reciprocal regulation, along with the activation of Ras, leads to the local production of PI(3,4,5)P3 and pseudopod extension.
RasG may be one of Ras isoforms that plays a role in the Ras/PI3K feedback circuit because the GFP-RBD recognizes RasG-GTP and random movement of rasG– cells is decreased (Tuxworth et al., 1997; Sasaki et al., 2004). There are likely other Ras isoforms that are integrated into the PI3K feedback loop, as rasG null cells show modest defects. The challenge for future investigation will be to elucidate the molecular mechanisms by which Ras, RasGEF, and RasGAP regulate basic cell motility in a Gß-independent fashion in the absence of extracellular stimuli.
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Materials and methods |
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We created an aimless null strain in the KAx-3 background using a targeting vector similar to that used to generate aimless null in a KAx2 background (Insall et al., 1996). The gß, pten, and pi3k1/2 null strains were described previously (Wu et al., 1995; Funamoto et al., 2001; Iijima and Devreotes, 2002).
Cell culture
All cell lines except for NC-4 were cultured axenically in HL5 medium at 22°C. NC-4 cells were grown on bacterial lawns. Transformants were maintained in 40 µg/ml G418, 50 µg/ml hygromycin, or both as required.
Biochemical assays
PKB activation and Ras activation were measured as described previously (Meili et al., 1999; Sasaki et al., 2004). Cells were treated or not treated with 50 µM LY294002 for 5 min. Endogenous Akt was immunoprecipitated and subjected to an in vitro kinase assay using H2B as a substrate. To monitor the Akt and Ras activation in vegetative cells, cells were harvested at 2–4 x 106 cells/ml, washed twice with 12 mM sodium/potassium phosphate buffer, pH 6.1, buffer A, and then resuspended in buffer A at 107 cells/ml. After 30 min of starvation, cells were subjected to the assay. For comparing Ras activation of wild-type cells to that of the mutant cells, washed cells were placed on the plate for 1 h and stimulated with 50 µM folic acid for the indicated duration, and then lysed on the plate.
Assays for random movement and cytokinesis
In a random movement assay, vegetative cells growing on plates were harvested and seeded onto a chambered coverglass in starvation buffer. Cells were rinsed three times with an excess amount of buffer A at 10 min after seeding, and then sat for 1 h. Images were collected on a microscope (model TE300; Nikon) with DIC and 40x/0.60 objectives. Initial images were captured using Metamorph software and analyzed with the DIAS program (Wessels et al., 1988). Speed refers to the speed of the cell's centroid movement along the total path. The cell movement during the 1 min between measured frames was measured to calculate speed so that genuine movement was measured rather than cytoplasmic rearrangement. Parallel experiments were performed with cells in HL5 axenic growth medium or cells starved for 2 h. No differences in the results were observed under these three conditions.
Cytokinesis was measured as described previously (Janetopoulos et al., 2005). Confocal images were obtained by using a CSU10 scanner unit (Yokogawa) on a Leica inverted DMIRE2 microscope with a 63x/1.4 objective using an ORCA-ER camera (Hamamatsu) or a Dual-View OI-11-EM–equipped EM-CCD camera (Hamamatsu) for simultaneous imaging. Imaging was described previously (Sasaki et al., 2004; Janetopoulos et al., 2005).
Online supplemental material
Fig. S1 A shows spontaneous Ras and PI3K activation in low density suspended cells, and Fig. S1 B shows the differences of RBD and PH accumulation kinetics in micropinosome formation from those in pseudopodial formation. Fig. S1 C shows translocation of GFP-RBD and PHcrac in vegetative wild-type cells by folic acid. Fig. S2 illustrates a model for the Ras/PI3K circuit during random movement and chemotaxis. Video 1 shows GFP-N-PI3K1 localization in gß null cells without extracellular stimuli. In Video 2, GFP-RBD localization in a gß null cell corresponds to Fig. 2 A in the text. In Video 3, GFP-RBD localization in a gß null cell corresponds to Fig. 2 D in the text. In Video 4, simultaneous imaging of GFP-RBD and RFP-PHcrac in the wild-type vegetative cells corresponds to Fig. 3 A in the text. In Video 5, random movement analysis of nonaxenic NC4 cells corresponds to Fig. 4 A in the text. In Video 6, random movement analysis of cells corresponds to Fig. 4 A in the text. Video 7 shows random movement analysis of myristoylated PI3K2-overexpressing wild-type cells. In Video 8, GFP-RBD and RFP-PHcrac in the wild-type cells correspond to Fig. 4 B in the text. Video 9 shows random movement analysis of pianissimo null cells. Video 10 shows GFP-RBD and RFP-PHcrac in the akt null cells. The online supplemental material can be found at http://www.jcb.org/cgi/content/full/jcb.200611138/DC1.
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Acknowledgments |
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A.T. Sasaki was supported, in part, by a Japanese Society for the Promotion of Science Research Fellowship for Research Abroad. This work was funded by research grants from the USPHS to P.N. Devreotes (GM28007, GM34933, and GM071920) and R.A. Firtel (GM037830, GM06847, and GM24279).
Submitted: 27 November 2006
Accepted: 14 June 2007
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References |
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TopAbstractIntroductionResults and discussionMaterials and methodsReferences |
|---|
Brock, C., M. Schaefer, H. Reusch, C. Czupalla, M. Michalke, K. Spicher, G. Schultz, and B. Nurnberg. 2003. Roles of Gß in membrane recruitment and activation of p110/p101 phosphoinositide 3-kinase . J. Cell Biol. 160:89–99.
Comer, F.I., C.K. Lippincott, J.J. Masbad, and C.A. Parent. 2005. The PI3K-mediated activation of CRAC independently regulates adenylyl cyclase activation and chemotaxis. Curr. Biol. 15:134–139.[CrossRef][Medline]
Condeelis, J., and J.E. Segall. 2003. Intravital imaging of cell movement in tumours. Nat. Rev. Cancer. 3:921–930.[CrossRef][Medline]
Engelman, J.A., J. Luo, and L.C. Cantley. 2006. The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat. Rev. Genet. 7:606–619.[CrossRef][Medline]
Funamoto, S., R. Meili, S. Lee, L. Parry, and R. Firtel. 2002. Spatial and temporal regulation of 3-phosphoinositides by PI 3-kinase and PTEN mediates chemotaxis. Cell. 109:611–623.[CrossRef][Medline]
Funamoto, S., K. Milan, R. Meili, and R. Firtel. 2001. Role of phosphatidylinositol 3' kinase and a downstream pleckstrin homology domain–containing protein in controlling chemotaxis in Dictyostelium. J. Cell Biol. 153:795–810.
Huang, Y., M. Iijima, C. Parent, S. Funamoto, R. Firtel, and P. Devreotes. 2003. Receptor-mediated regulation of PI3Ks confines PI(3,4,5)P3 to the leading edge of chemotaxing cells. Mol. Biol. Cell. 14:1913–1922.
Iijima, M., and P. Devreotes. 2002. Tumor suppressor PTEN mediates sensing of chemoattractant gradients. Cell. 109:599–610.[CrossRef][Medline]
Insall, R.H., J. Borleis, and P.N. Devreotes. 1996. The aimless RasGEF is required for processing of chemotactic signals through G-protein-coupled receptors in Dictyostelium. Curr. Biol. 6:719–729.[CrossRef][Medline]
Janetopoulos, C., J. Borleis, F. Vazquez, M. Iijima, and P. Devreotes. 2005. Temporal and spatial regulation of phosphoinositide signaling mediates cytokinesis. Dev. Cell. 8:467–477.[CrossRef][Medline]
Krugmann, S., M.A. Cooper, D.H. Williams, P.T. Hawkins, and L.R. Stephens. 2002. Mechanism of the regulation of type IB phosphoinositide 3OH-kinase by G-protein betagamma subunits. Biochem. J. 362:725–731.[CrossRef][Medline]
Lee, E., and D.A. Knecht. 2002. Visualization of actin dynamics during macropinocytosis and exocytosis. Traffic. 3:186–192.[CrossRef][Medline]
Lee, S., F.I. Comer, A. Sasaki, I.X. McLeod, Y. Duong, K. Okumura, J.R. Yates, C.A. Parent, and R.A. Firtel. 2005. TOR complex 2 integrates cell movement during chemotaxis and signal relay in Dictyostelium. Mol. Biol. Cell. 16:4572–4583.
Meili, R., C. Ellsworth, S. Lee, T. Reddy, H. Ma, and R. Firtel. 1999. Chemoattractant-mediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium. EMBO J. 18:2092–2105.[CrossRef][Medline]
Pacold, M., S. Suire, O. Perisic, S. Lara-Gonzalez, C. Davis, E. Walker, P. Hawkins, L. Stephens, J. Eccleston, and R. Williams. 2000. Crystal structure and functional analysis of Ras binding to its effector phosphoinositide 3-kinase gamma. Cell. 103:931–943.[CrossRef][Medline]
Parent, C.A., B.J. Blacklock, W.M. Froehlich, D.B. Murphy, and P.N. Devreotes. 1998. G protein signaling events are activated at the leading edge of chemotactic cells. Cell. 95:81–91.[CrossRef][Medline]
Rupper, A.C., J.M. Rodriguez-Paris, B.D. Grove, and J.A. Cardelli. 2001. p110-related PI 3-kinases regulate phagosome-phagosome fusion and phagosomal pH through a PKB/Akt dependent pathway in Dictyostelium. J. Cell Sci. 114:1283–1295.[Abstract]
Sasaki, A.T., C. Chun, K. Takeda, and R.A. Firtel. 2004. Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement. J. Cell Biol. 167:505–518.
Sasaki, A.T., and R.A. Firtel. 2006. Regulation of chemotaxis by the orchestrated activation of Ras, PI3K, and TOR. Eur. J. Cell Biol. 85:873–895.[CrossRef][Medline]
Stephens, L., A. Smrcka, F.T. Cooke, T.R. Jackson, P.C. Sternweis, and P.T. Hawkins. 1994. A novel phosphoinositide 3 kinase activity in myeloid-derived cells is activated by G protein beta gamma subunits. Cell. 77:83–93.[CrossRef][Medline]
Stephens, L.R., A. Eguinoa, H. Erdjument-Bromage, M. Lui, F. Cooke, J. Coadwell, A.S. Smrcka, M. Thelen, K. Cadwallader, P. Tempst, and P.T. Hawkins. 1997. The G beta gamma sensitivity of a PI3K is dependent upon a tightly associated adaptor, p101. Cell. 89:105–114.[CrossRef][Medline]
Stoyanov, B., S. Volinia, T. Hanck, I. Rubio, M. Loubtchenkov, D. Malek, S. Stoyanova, B. Vanhaesebroeck, R. Dhand, B. Nurnberg, et al. 1995. Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase. Science. 269:690–693.
Suire, S., J. Coadwell, G.J. Ferguson, K. Davidson, P. Hawkins, and L. Stephens. 2005. p84, a new Gbetagamma-activated regulatory subunit of the type IB phosphoinositide 3-kinase p110gamma. Curr. Biol. 15:566–570.[CrossRef][Medline]
Suire, S., A.M. Condliffe, G.J. Ferguson, C.D. Ellson, H. Guillou, K. Davidson, H. Welch, J. Coadwell, M. Turner, E.R. Chilvers, et al. 2006. Gbetagammas and the Ras binding domain of p110gamma are both important regulators of PI3Kgamma signalling in neutrophils. Nat. Cell Biol. 8:1303–1309.[CrossRef][Medline]
Takeda, K., A. Sasaki, H. Ha, H. Seung, and R.A. Firtel. 2007. Role of PI3 kinases in chemotaxis in Dictyostelium. J. Biol. Chem. 282:11874–11884.
Tuxworth, R.I., J.L. Cheetham, L.M. Machesky, G.B. Spiegelmann, G. Weeks, and R.H. Insall. 1997. Dictyostelium RasG is required for normal motility and cytokinesis, but not growth. J. Cell Biol. 138:605–614.
Vanhaesebroeck, B., K. Ali, A. Bilancio, B. Geering, and L.C. Foukas. 2005. Signalling by PI3K isoforms: insights from gene-targeted mice. Trends Biochem. Sci. 30:194–204.[CrossRef][Medline]
Wessels, D., D.R. Soll, D. Knecht, W.F. Loomis, A. De Lozanne, and J. Spudich. 1988. Cell motility and chemotaxis in Dictyostelium amebae lacking myosin heavy chain. Dev. Biol. 128:164–177.[CrossRef][Medline]
Wu, L., R. Valkema, P.J. Van Haastert, and P.N. Devreotes. 1995. The G protein beta subunit is essential for multiple responses to chemoattractants in Dictyostelium. J. Cell Biol. 129:1667–1675.
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