Signaling pathways at the leading edge of chemotaxing cells

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1 Journal of Muscle Research and Cell Motility 23: , Ó 2003 Kluwer Academic Publishers. Printed in the Netherlands. Signaling pathways at the leading edge of chemotaxing cells CHANG Y. CHUNG 1 and RICHARD A. FIRTEL 2, * 1 Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN , USA; 2 Section of Cell and Developmental Biology, Division of Biological Sciences and Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA , USA Abstract Chemotaxis, or directed cell movement towards small molecule ligands, is a central function of many cell types and plays a key role in diverse biological processes. This review summarizes our present understanding of the signaling pathways that control the ability of cells to sense the chemoattractant gradient and respond by converting a shallow extracellular gradient into a steep intracellular gradient that leads to formation of a pseudopod in the direction of the chemoattractant gradient and contraction of the cell s posterior. The review focuses on the phosphatidylinositol 3-kinase pathway in Dictyostelium and our understanding of parallel pathways in leukocytes. Introduction *To whom correspondence should be addressed: Tel.: ; Fax: ; rafirtel@ucsd.edu The ability of cells to sense and respond to environmental stimuli is a key determinant of cell growth, development, differentiation, and oncogenesis. Many cells are capable of translating an extracellular chemical gradient into a guidance cue for directional migration, a process called chemotaxis. Chemotaxis, directed movement towards a chemoattractant agent, is involved in diverse biological responses. Chemotaxis is essential for the migration of polymorphonuclear leukocytes and macrophages to an inflammatory site (Devreotes and Zigmond, 1988; Downey, 1994). The directed movement of fibroblasts towards locally released PDGF is a critical event in wound healing (Heldin and Westermark, 1999). Axonal guidance in the developing nervous system appears to function via pathways that are analogous to chemotaxis of amoeboid cells (Wu et al., 1999; Rajagopalan et al., 2000). Higher concentrations of chemoattractant near target destinations of migratory neuroblasts appear to be involved in the regulation of the migration of neuroblasts. Diffusible repulsive agents from tissue may exclude axonal growth and help regulate the direction of the leading edge. Chemotaxis in leukocytes and Dictyostelium is regulated by ligands that interact with serpentine receptors (Devreotes and Zigmond, 1988; Chen et al., 1996). In leukocytes, chemokine receptors are coupled to heterotrimeric G proteins containing the Gai subunit pathways and mediate chemotaxis through the release of Gbc (Neptune and Bourne, 1997). In Dictyostelium, camp, the chemoattractant that regulates the formation of the multicellular organism, mediates chemotaxis through serpentine camp receptors (cars) coupled to the G protein containing the Ga2 subunit, whereas folate mediates chemotaxis through a distinct receptor and G protein containing the Ga4 subunit (Parent and Devreotes, 1996; Aubry and Firtel, 1999). Our understanding of the integrated pathways that regulate chemotaxis has been significantly advanced through genetic and molecular genetic analyses using Dictyostelium (Firtel and Chung, 2000; Chung et al., 2001a). Dictyostelium provides an excellent biological system for identifying genes required for chemotaxis, as such genes would be defective in the ability to form aggregates and thus are easily identified in mutant screens. Among these genes, we will focus on signaling components that are vital for establishing polarity and conferring the cells ability to respond to a chemoattractant gradient in which the difference in the level of chemoattractant between the front and back of the cell can be as low as 2%. This review focuses on the signaling pathways controlling leading edge formation in Dictyostelium and the parallels this work has established for the understanding of directional sensing in human leukocytes. How do cells achieve a polarized response to the gradient? The ability of cells to move directionally in a shallow gradient raises the possibility that cells can augment their sensitivity to a chemoattractant in the front by the redistribution of chemoattractant receptors. However, evidence indicates that a polarized response in the direction of a chemoattractant source is not due to a difference in chemoattractant-elicited second messenger

2 774 responses that might be elicited by the concentration difference of chemoattractant sensed by the front or the back of a chemotaxing cell. Although cells are able to respond to differences of chemoattractant as low as 2 5% between the front and back of cells, there would be no detectable biochemical difference in second messenger respond if cells were stimulated with a 1.0 or 0.95 concentration of a ligand. Thus, under even minute differences of chemoattractant concentration, cells must be able to sense the spatial gradient and locally activate signaling events leading to cell polarity, the formation of the leading edge driven by F-actin polymerization, and finally directional cell movement. Cells must therefore have a mechanism to amplify the extracellular shallow chemoattractant gradient into a steep intracellular gradient that will provide the necessary differential in second messengers that causes localized F-actin assembly at the front of the cell. Several pieces of work have demonstrated that the initial asymmetry in the response is not due to a preferential localization of either receptors or the coupled heterotrimeric G proteins at the leading edge. car1, the major camp receptor that controls aggregation in Dictyostelium, is uniformly distributed around the periphery of the cell, and, more importantly, the receptors remain uniformly distributed when the cells chemotax or change direction, providing strong evidence that it is not receptor localization that controls polarity (Xiao et al., 1997). Similar observations have been made using the C5a chemotaxis receptor fused to GFP in neutrophils (Servant et al., 1999). Furthermore, it has been shown that membraneassociated Gbc is distributed in only a very shallow anterior posterior gradient in highly polarized Dictyostelium cells (Jin et al., 2000), similar to the extracellular chemoattractant gradient, and thus does not represent a mechanism of providing a spatial amplification of the extracellular signal. FRET analysis of Ga2 andgbc similarly does not exhibit a significant spatial difference between the front and the back of cells, suggesting that any significant asymmetry in amplifying the extracellular chemoattractant gradient is not the result of differential spatial activation at the level of the receptor or heterotrimeric G protein. A cartoon of the subcellular localization of signaling components in a chemotaxing cells is presented in Figure 1. A major breakthrough in our understanding of the establishment of asymmetry at the leading edge came from experiments demonstrating that a subclass of PH domain-containing proteins, including CRAC, Akt/ PKB, and PhdA, that preferentially bind the PI3K lipid products PI(3,4,5)P 3 /PI(3,4)P 2 rapidly and transiently translocate to the plasma member in response to a global stimulation of cells by a chemoattractant (Parent et al., 1998; Meili et al., 1999; Funamoto et al., 2001). Furthermore, these proteins localize to the leading edge of chemotaxing cells. PH domain localization is sensitive to the PI3K inhibitor LY and Fig. 1. Cartoon of a chemotaxing Dictyostelium cell: this cartoon illustrates the general organization of polarized chemotaxing cells with an actin-enriched leading edge. PI3K is preferentially activated at the leading edge of cells. Activation requires Ras. This leads to a localized production of the PI3K products PI(3,4,5)P 3 and PI(3,4)P 2, which function as binding sites for a specific subset of PH domain-containing proteins, including Akt/PKB. These result in actin assembly, which is mediated by Rac in association with the WASp family proteins WASp and SCAR/WAVE. F-actin is shown at the leading edge. F-actin is also found at the posterior of cells, where, in association with myosin II, it regulates contraction of the uropod or posterior of the cell. Analysis of F-actin assembly is described in other reviews in this volume. Myosin I and PAKc preferentially localize to the leading edge and are essential for proper chemotaxis. Myosin II disassembly at the leading edge is mediated through the localization of MHCKA. There is a preferential localization of a variety of actin binding proteins and cytoskeletal components at the leading edge of cells that are required for proper extension of the pseudopod or lamellipod (not shown in this figure). Myosin II assembly, which controls both cortical tension along the sides of cells and contractility at the posterior, is mediated by chemoattractant stimulation. The preferential localization of assembled myosin II is shown. Myosin II assembly in the rear is controlled, in part, by PAKa, a p21-activated Ser/Thr protein kinase, which preferentially localizes to the posterior of the cells. PAKa is directly phosphorylated by Akt/PKB, leading to its activation, and is thus downstream from PI3K. PAKa mediates myosin assembly and contraction of the posterior or uropod. PTEN, a negative regulator of the PI3K pathway, is not shown in this figure. does not take place in pi3k null cells, indicating that the localization is dependent on PI3K activation. Neutrophils and fibroblasts were subsequently found to exhibit the same response, indicating these pathways are evolutionarily conserved (Haugh et al., 2000; Servant et al., 2000). These studies suggest that the localized activation of PI3K and the recruitment of PH domain-containing proteins at the leading edge may be one of first initial events that establishes a spatial asymmetry in signaling pathways leading to chemotaxis (Parent and Devreotes, 1999; Firtel and Chung, 2000; Rickert et al., 2000; Chung et al., 2001a; Iijima et al., 2002).

3 775 Localized activation Phosphatidylinositol 3-kinase (PI3K) plays a central role in local activation In Dictyostelium cells and mammalian neutrophils, a growing body of experimental evidence suggests that cells locally activate and amplify distinct sets of signaling pathways in the front of the cell that create a new leading edge, the establishment of cell polarity, and directional cell movement. This spatially restricted activation and amplification creates an internal signaling asymmetry and enables cells to move directionally in a shallow chemoattractant gradient. The findings described above with PH domain-containing proteins suggest that PI3K plays an essential part in the amplification of internal signaling asymmetry (Parent and Devreotes, 1999; Rickert et al., 2000; Chung et al., 2001a; Katanaev, 2001; Stephens et al., 2002). In mammals, the product of PI3K, phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P 3 ], is produced by two classes of PI3Ks, class I A (PI3Ka, PI3Kb, and PI3Kd) and class I B (PI3Kc) (Vanhaesebroeck et al., 1999). Dictyostelium contains three PI3Ks related to mammalian type I PI3Ks (Zhou et al., 1995) that are most closely related to mammalian Class I A PI3KS that include p110a. Cells lacking the two Dictyostelium class I PI3Ks PI3K1 and PI3K2 (pi3k1/2 null cells) or wild-type cells treated with the PI3K inhibitor LY are unable to properly polarize, are very defective in the temporal, spatial, and quantitative regulation of chemoattractant-mediated filamentous (F)- actin polymerization, and chemotax very slowly (Funamoto et al., 2001, 2002). This suggests that PI3K is important for chemotaxis and is consistent with the models derived from PH domain localization postulating that PI(3,4,5)P 3 production might be a key step in amplifying internal asymmetry. Dictyostelium PI3K1 and PI3K2 had been thought to be genetically redundant (Zhou et al., 1998). However, a recent study demonstrated that PI3K2 might play a more important role in controlling chemotaxis (Funamoto et al., 2002). Both single knockout strains (pi3k1 and pi3k2) exhibit chemotaxis defects as measured by using a micropipette emitting camp. pi3k2 null cells exhibited greater deficiencies than pi3k1, but less than the double knockout strain pi3k1/2. Akt/PKB activation, an assay of PI3K activation, is 60% of the wild-type level in pi3k1 null cells, but only 14% in pi3k2 null cells. These results suggest that PI3K2 is a major determinant of the level of PI(3,4,5)P 3 and local activation of signaling pathways at the leading edge, and that there is cooperation between PI3K1 and PI3K2 in regulating downstream effector pathways. As pi3k1/2 null cells still exhibit a low level of Akt/PKB activation, we assume that the third Class I PI3K, PI3K3, also has a minor function in controlling PI3K pathways. Parallel studies in mice have similarly established that PI3Kc, which lies downstream from chemokine receptors, is required for directional sensing in these cells, suggesting that the role of PI3K in directional sensing of chemotaxing cells may be universal (Hirsch et al., 2000; Li et al., 2000; Sasaki et al., 2000; Hannigan et al., 2002; Stephens et al., 2002). Regulation of PI3K activity and its localization Given the fact that PI3K activity is essential for local activation of signaling, dynamic regulation of the activity and localization of PI3Ks would be an important step in establishing the local activation. Both PI3K1 and PI3K2 were found to rapidly and transiently localize to the plasma membrane in response to global stimulation with camp and to the leading edge of chemotaxing cells (Funamoto et al., 2002). One can imagine that the simplest mechanism for positive feedback regulation of PI3K signaling would be that PI3K is recruited to the membrane by binding to PI(3,4,5)P 3. However, this is not the case, as PI3K localizes to the plasma membrane in response to chemoattractant stimulation with similar kinetics in the presence of a PI3K inhibitor, LY In addition to C-terminal lipid kinase and lipid-kinase-accessory domains, PI3Ks contain a Ras binding domain, a C2 domain, and a long N- terminal domain with little homology to other proteins. Surprisingly, the domain that is necessary and sufficient for chemoattractant-mediated localization resides in the N-terminal domain of PI3Ks. Translocation of this domain occurs normally in pi3k1/2 null cells, a result that is consistent with PI3K function not being required for activation. In mammalian cells, Rac family small GTPases and F-actin have been proposed to mediate amplification of the PI3K pathway (Wang et al., 2002; Weiner et al., 2002). The Ras binding domains (RBD) of PI3K1 and PI3K2 bind strongly to constitutively active RasG and human H-Ras in a yeast two-hybrid assay. When these RBDs were mutated so that the interactions with activated Ras were lost, both PI3K1 and PI3K2 were still able to translocate to the plasma membrane despite these mutations, but PI3K was not activated (Funamoto et al., 2002). These results are consistent with the structure/function analysis on the PI3K localization domain indicating that the interaction of Ras with RBD is not required for the translocation. However, Ras appears to be an essential upstream regulator of PI3K activation, as cells expressing myr-pi3k1 carrying the RBD mutation preventing Ras GTP binding, which is targeted to the membrane constitutively and cannot bind to Ras, exhibit only a minimal Akt/PKB activation. These findings indicate that PI3K activation is controlled in parallel by membrane localization and Ras. A cartoon illustrating the subcellular localization of PI3K and PH domain-containing proteins in resting and chemotaxing cells is depicted in Figure 2. Local activation: recruitment of PH domain-containing proteins The genetic defects of the three characterized PH domain-containing proteins that localize to the plasma

4 776 Fig. 2. Localization of PI3K, PH domain-containing proteins, and PTEN in cells placed in a chemoattractant gradient. Left panel: an unstimulated (not in a chemoattractant gradient), polarized Dictyostelium cell with actin localization at the front (not shown) and myosin II at the lateral sides and posterior (see Figure 1). In these cells, PTEN is uniformly localized around the plasma membrane. PI3K and the PI3K PH domain-containing effectors CRAC, Akt/PKB, and PhdA are cytosolic. In response to a directional signal, PI3K and the PH domain-containing proteins preferentially localize to the leading edge in response to the production of PI(3,4,5)P 3. Concomitant with this localization is a delocalization of PTEN from the leading edge. PTEN remains on the lateral sides and posterior as described in (Funamoto et al., 2002). Results on PTEN distribution in work by Iijima and Devreotes (2002) shows PTEN localization to be significantly less lateral and more restricted to the posterior half of the cell. The reason for the difference between PTEN localizations observed by the Firtel and Devroetes group is not known and may depend upon strain differences, extent of polarity of the cells, expression level of the construct, or the construct. However, the results of both groups are consistent with the conclusion that PTEN is essential for restricting the PI3K localization signal to the very anterior of the chemotaxing cell. PI3K, Akt/PKB, CRAC, and PhdA localization to the leading edge mediates preferential F-actin assembly and directional movement. membrane in response to chemoattractant stimulation have been analyzed. CRAC is a cytosolic activator of adenylyl cyclase and is essential for chemoattractantmediated camp production (Insall et al., 1994; Lilly and Devreotes, 1995). Presently, there is no published data indicating that CRAC is directly involved in controlling chemotaxis; however, this is not excluded by previous results. The Dictyostelium protein Akt/PKB is a structural homologue of mammalian Akt/PKB, having an N-terminal PH domain, a conserved kinase domain, and a C-terminal tail with conserved phosphorylation sites for activation by upstream kinases. Cells lacking Akt/ PKB are unable to properly polarize when placed in a chemotactic gradient, and the cells move slowly. More detailed analysis demonstrated that the cells produce multiple lateral pseudopodia as well as a pseudopod at the leading edge. Analysis of mutants suggests that the cells tumble rather than move smoothly in the direction of the chemoattractant source, similar to pi3k1/2 null cells. This tumbling leads to defective chemotaxis and the inability to form multicellular aggregates when plated at a low density, conditions in which cells do not have direct cell cell contacts. The downstream functions of Akt/PKB are described below. Another PH domain-containing protein, PhdA, appears to function as a docking site for cellular proteins that must assemble at the leading edge in response to chemoattractant signals. phda null cells have a reduced polarity, and move significantly more slowly than wild-type cells (Funamoto et al., 2001). In addition, phda null cells exhibit a defect in spatially localized F-actin assembly at the leading edge. In response to moving the position of the micropipette containing a chemoattractant, these cells show a slower response to depolymerized F-actin at the old leading edge and to polymerize new F-actin at the new leading edge. These cells also show a small (30%) reduction in the level of F-actin accumulation in response to global stimulation, while pi3k1/2 null cells show a >50% reduction (Funamoto et al., 2001). A flow diagram of the activation of PI3K and downstream Fig. 3. Flow diagram of the PI3K pathways controlling chemotaxis. PI3K is activated by a chemoattractant through a G protein-coupled receptor and the coupled heterotrimeric G protein Ga2bc. This leads to the localization of PI3K to the plasma membrane, as illustrated in previous figures, and a delocalization of PTEN. Three PH domaincontaining proteins have been described that are downstream effectors of PI3K. CRAC, PhdA, and Akt/PKB all localize to the leading edge in a PI3K-dependent manner. Akt/PKB is a direct activator of PAKa, which mediates myosin II assembly, probably by negatively regulating MHCK. It may have a direct function in causing myosin II assembly. PhdA is required for effective localized actin assembly at the leading edge. Akt/PKB is thought to have additional effectors, as the phenotypes of paka null cells represent only a subset of the phenotypes of the akt/pkb null cells.

5 777 effectors is presented in Figure 3. Ras activation, which is required for PI3K activation, is not shown. The translocation of GFP-fused PH domains indicates there is a spatially localized activation of pathways and may represent the underlying mechanism by which cells generally produce a pseudopod only in the direction of the chemoattractant source. This conclusion is highlighted by two sets of experiments. First, point mutations in the PH domain of Akt/PKB and PhdA that abrogate the ability of the proteins to bind the PI3K products PI(3,4,5)P 3 /PI(3,4)P 2 block the ability of these proteins to localize to the plasma membrane and Akt/ PKB is no longer activated in response to chemoattractant signaling (Meili et al., 1999, 2000; Funamoto et al., 2001). Furthermore, PhdA and Akt/PKB carrying these point mutations are unable to complement the respective null mutations. If these proteins are constitutively localized to the plasma membrane via an N-terminal lipid modification (myristoylation), the proteins are unable to complement the null mutations, indicating that the spatial localization of the proteins at the leading edge is essential for them to function properly to control chemotaxis. Global inhibition PTEN: setting a threshold for the activation of PI3K? The tumor suppressor PTEN is a phosphatidylinositide 3-phosphatase that removes the 3-phosphate from the PI3K products PI(3,4,5)P 3 and PI(3,4)P 2 (Maehama and Dixon, 1998; Maehama et al., 2001). Many studies suggest that PTEN is a potential negative regulator of the PI3K pathways during chemotaxis (Maehama et al., 2001), and recent analyses of Dictyostelium cells lacking PTEN provide a clue for understanding the mechanism of global inhibition (Funamoto et al., 2002; Iijima and Devreotes, 2002). In Dictyostelium cells and lymphocytes lacking PTEN or cells expressing a very low level of PTEN, the PI3K signaling and chemoattractant responses are augmented (Fox et al., 2002; Funamoto et al., 2002; Iijima and Devreotes, 2002). Compared to the lower frequency of pseudopod extension in Dictyostelium pi3k1/2 null cells, pten null cells extended three or more pseudopodia pointing generally but not directly up the gradient, indicating a lack of localized activation. Targeting of myristoylated PI3K to the membrane elicits the same phenotype, demonstrating that the balance between PI3K and PTEN activities is required to establish polarity under the gradient. Translocation of a GFP-labeled PH domain to the plasma membrane was dramatically enhanced and prolonged compared to wild-type cells, with a much broader leading edge, indicating accumulation of more PI(3,4,5)P 3. More importantly, significantly lower doses of camp are required to elicit an equivalent response in pten null cells compared to wild-type cells, suggesting PTEN is a component of global inhibition. Cells expressing higher levels of exogenous PTEN-CFP exhibit a significantly reduced level of YFP-PKB-PH translocation to the plasma membrane. Cells overexpressing PTEN exhibited a decrease in the rate of chemotaxis, presumably due to the inability to extend pseudopodia. These results suggest that the level of PTEN activity plays an important role in controlling the responsiveness of cells to the stimulation of a chemoattractant, which is consistent with PTEN being a component of global inhibition. The localization and translocation of Dictyostelium PTEN and PI3K to the membrane is reciprocal (Funamoto et al., 2002; Iijima and Devreotes, 2002). PTEN is found uniformly on the plasma membrane in unstimulated cells. On camp stimulation, there is a rapid and transient release of PTEN from the plasma membrane. PTEN delocalizes from the membrane in pi3k1/2 null cells, suggesting that PI3K activity is not required for PTEN translocation. In chemotaxing cells, PTEN is on the plasma membrane along the lateral sides and posterior of the cell but absent at the leading edge, consistent with the possibility of PTEN being a component of a global inhibition mechanism. This pattern of localization is the opposite of that observed for PH domain-containing proteins. Removal of the putative PI(4,5)P 2 binding domain of PTEN resulted in a PTEN- GFP protein that no longer associated with the membrane. The cytosolic version of PTEN-GFP was unable to rescue the pten null cells, suggesting that membrane localization is required for the function of PTEN in chemotaxis. Production of PI(4,5)P 2 by dephosphorylation of PI(3,4,5)P 3 could recruit more PTEN to the membrane, which might function as a positive feedback mechanism and could be a key component of the global inhibition and polarized response. The results on the phenotypes of pten null cells and cells expressing myr- PI3K posit that PTEN down-regulates the PI3K pathway and restricts and sharpens the PI3K pathway at the leading edge. The absence of PTEN at the leading edge of chemotaxing cells and its presence along the sides and backs of the cells may help restrict the PI3K lipid products to the narrow region at the cell s anterior. Myosin II: mechanical barrier for restricting pseudopod formation In a number of cell types including leukocytes, lymphocytes, and Dictyostelium, myosin II filaments are assembled at the rear cell body (uropod) and along the lateral sides of the cell where they are important in defining axial polarity, retracting the posterior of the cell during chemotaxis, and biasing the direction of movement by repressing extensions of lateral pseudopodia through cortical tension (Clow and McNally, 1999). Myosin II assembly and disassembly in response to extracellular signals is controlled by phosphorylation of threonine residues on the myosin II tail by myosin heavy chain kinases (MHCKs) in Dictyostelium and some mammalian cell types (van Leeuwen et al., 1999). Phosphorylation

6 778 of these residues leads to myosin II filament disassembly, whereas dephosphorylation leads to the formation of myosin II filaments. PAKa, a structural homologue of mammalian PAKs (p21-activated kinase), is essential for proper cell polarity, chemotaxis, and cytokinesis in Dictyostelium (Chung and Firtel, 1999). PAKa controls these processes, in part, by regulating myosin II assembly. paka null cells do not exhibit chemoattractant-mediated myosin II assembly, and expression of constitutively active PAKa results in hyper-assembly of myosin II and F-actin. Consistent with these findings, paka and myosin II (myoii) null strains exhibit similar chemotaxis and cytokinesis defects. PAKa does not directly phosphorylate myosin II and probably promotes myosin II assembly by negatively regulating MHCKs (Egelhoff et al., 1993; Chung and Firtel, 1999). PAKa co-localizes with assembled myosin II in the posterior of chemotaxing cells and cleavage furrows of dividing cells. In chemotaxing cells, chemoattractants control PAKa s kinase activity (and the downstream regulation of myosin II assembly) and subcellular localization. Stimulation of cells with the chemoattractant camp results in a rapid, transient increase in PAKa kinase activity and a transient association of PAKa with the cytoskeleton. The localization of PAKa at the posterior of the cell and its association with the cytoskeleton therefore spatially restrict PAKa s site of action, leading to an inhibition of the activity of one or more MHCKs in this subcellular domain and localized myosin II assembly to this site. This localized myosin II assembly is vital for maintaining cell polarity and cortical tension and retracting the posterior cell body. PAKa is a key downstream effector of the PI3K and PKB signaling pathway that controls chemotaxis (Chung et al., 2001b), suggesting PAKa is a key regulator for the anterior-to-posterior coordination during chemotaxis. PAKa is phosphorylated by PKB in vitro and 2D gel analysis indicates that phosphorylation of PAKa at the PKB phosphorylation site does not occur in Akt/PKB (pkba) null cells. Furthermore, phosphorylation at this site is essential for the in vivo regulation of PAKa kinase activity and its subcellular localization. In vivo, PAKa is not activated and exhibits an abnormal subcellular localization in either pkba or pi3k1/2 null cells, suggesting that phosphorylation of PAKa by PKB through a chemoattractant receptor and PI3K-dependent pathway controls cellular polarity and cell movement during chemotaxis. Consistent with this finding, addition of the PI3K inhibitor LY to chemotaxing cells results in a rapid loss of cell polarity and a disruption of the actin/myosin cytoskeletons and the posterior localization of PAKa. Disassembly of myosin II filaments at the leading edge is required to reduce cortical tension, allowing protrusion of pseudopodia. Translocation of myosin heavy chain kinase A (MHCKA) to the leading edge appears to control this process during chemotaxis (Steimle et al., 2002). MHCKA was recently reported to display dynamic recruitment to the leading edge. Localized MHCKA accumulation may drive disassembly of myosin II filaments, preventing myosin II filament accumulation at sites of F-actin-based protrusive activity. Future directions Present evidence, arising mostly from studies in Dictyostelium, suggests that localized activation of the PI3K pathway at the leading edge is essential for directional sensing of chemoattractant gradients in cell types ranging from human leukocytes to Dictyostelium cells. Key issues to be understood are the mechanisms by which PI3K translocates to the site of the new leading edge and how PTEN is preferentially delocalized from this same region. 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