Leading the way: directional sensing through phosphatidylinositol 3-kinase and other signaling pathways

Sylvain Merlot and Richard A. Firtel*

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 92093-0634, USA

* Author for correspondence (e-mail: rafirtel{at}ucsd.edu)


    Summary
 Top
 Summary
 Introduction
 Directional sensing and cell...
 Role of class I...
 PTEN downregulates the...
 PI3K effectors and their...
 A PtdIns(3,4,5)P3 positive...
 Conclusion and perspectives
 References
 
Chemoattractant-responsive cells are able to translate a shallow extracellular chemical gradient into a steep intracellular gradient resulting in the localization of F-actin assembly at the front and an actomyosin network at the rear that moves the cell forward. Recent evidence suggests that one of the first asymmetric cellular responses is the localized accumulation of PtdIns(3,4,5)P3, the product of class I phosphoinositide 3-kinase (PI3K) at the site of the new leading edge. The strong accumulation of PtdIns(3,4,5)P3 results from the localized activation of PI3K and also from feedback loops that amplify PtdIns(3,4,5)P3 synthesis at the front and control its degradation at the side and back of cells. These different pathways are temporally and spatially regulated and integrate with other signaling pathways during directional sensing and chemotaxis.

Key words: Chemotaxis, Dictyostelium, PI 3-kinase, Fibroblasts, Neutrophils, Actin


    Introduction
 Top
 Summary
 Introduction
 Directional sensing and cell...
 Role of class I...
 PTEN downregulates the...
 PI3K effectors and their...
 A PtdIns(3,4,5)P3 positive...
 Conclusion and perspectives
 References
 
Chemotaxis, movement of a cell towards a chemoattractant signal, is a fundamental process involved in a wide range of cellular responses, including morphogenesis during development, wound healing, innate immune responses, and metastasis of tumor cells (Firtel and Chung, 2000Go). Much of our understanding of chemotaxis is derived from studies of leukocytes and Dictyostelium amoebae, which are able to move rapidly towards a variety of chemoattractants. When cells sense a chemoattractant gradient, they dramatically change their shape, polarizing in the direction of the gradient. The side of the cell facing up the chemoattractant gradient forms a leading edge or pseudopod that protrudes forward. This response is mediated by F-actin assembly, with the pointed ends of the F-actin filaments being directed outward. The process is regulated by Rac/Cdc42 small GTPases, acting through WASp/SCAR/WAVE proteins, which activate the Arp2/3 complex (Etienne-Manneville and Hall, 2002Go; Machesky and Insall, 1999Go; Pollard and Borisy, 2003Go). The posterior of the cell or uropod is enriched in an actomyosin network containing myosin II that provides the contractile force that helps release the cell from the substratum and allows the posterior to move forward. Myosin II is also found along the lateral sides of cells, where it increases the cortical tension in these regions, providing a physical barrier to restrict lateral pseudopod formation. Cells lacking myosin II or components required for myosin II assembly produce lateral pseudopodia, cannot properly retract the uropod, and consequently exhibit chemotaxis defects (Chung et al., 2001aGo; Wessels et al., 1988Go).

Remarkably, cells respond to chemoattractant gradients as shallow as a 2-5% difference across the anterior and posterior of the cell and convert this shallow external gradient into a steep intracellular gradient of signaling components (Parent and Devreotes, 1999Go). This is used to produce directional movement through the differential, localized reorganization of the cytoskeleton at the front and posterior of the cell. During the past four years, growing evidence has indicated that preferential activation of phosphoinositide 3-kinase (PI3K) at the side of the cell facing the chemoattractant gradient is important for establishing a new leading edge, cell polarization, and directional movement in a wide range of motile cell types (Bourne and Weiner, 2002Go; Firtel and Chung, 2000Go; Parent and Devreotes, 1999Go).

Here, we review recent studies demonstrating that the generation of a PtdIns(3,4,5)P3/PtdIns(3,4)P2 gradient is a conserved mechanism for chemoattractant directional sensing and cell polarization. These recent results explain how the temporal and spatial regulation of PI3K and the lipid phosphatase PTEN generate and maintain a steep PtdIns(3,4,5)P3/PtdIns(3,4)P2 gradient at the leading edge of chemotaxing cells. We also discuss several unanswered questions about how the signaling pathways involved are regulated.


    Directional sensing and cell polarization require PtdIns(3,4,5)P3 accumulation in the leading edge
 Top
 Summary
 Introduction
 Directional sensing and cell...
 Role of class I...
 PTEN downregulates the...
 PI3K effectors and their...
 A PtdIns(3,4,5)P3 positive...
 Conclusion and perspectives
 References
 
In leukocytes and Dictyostelium, the chemoattractant signal is perceived by G-protein-coupled receptors (GPCRs). Examination of GFP-tagged GPCRs in both Dictyostelium and leukocytes demonstrated that they remain evenly distributed along the plasma membrane in highly polarized chemotaxing cells (Servant et al., 1999Go; Xiao et al., 1997Go), indicating that differential activation of signaling pathways in the front and back of cells does not depend on differential distribution of the receptor. Similar studies revealed that the G protein ß subunit (Gß) exhibits a very shallow anterior-posterior gradient, similar to that of the extracellular chemoattractant gradient in chemotaxing Dictyostelium cells (Jin et al., 2000Go). This weak gradient may reflect receptor occupancy but cannot account for the polarization of the cell (Ueda et al., 2001Go). FRET analyses of the dissociation kinetics of the G{alpha} subunit from Gß{gamma} in response to cAMP also suggest that activation of G proteins only reflects the shallow receptor occupancy gradient and could not explain the generation of a steep signaling gradient in the leading edge (Janetopoulos et al., 2001Go). However, the spatial activation of G proteins during chemotaxis was not directly imaged in this study.

Studies in Dictyostelium with GFP fusions of PH-domain-containing proteins, including CRAC (cytosolic activator of adenylyl cyclase) and the PI3K effector Akt/PKB first demonstrated that proteins carrying PH domains that preferentially bind to the Class I PI3K products PtdIns(3,4,5)P3/PtdIns(3,4)P2 rapidly (peak at 5-10 seconds) and transiently translocate to the plasma membrane in response to a uniform stimulation with the chemoattractant cAMP (global stimulation) (Parent et al., 1998Go; Meili et al., 1999Go). More importantly, in chemotaxing cells, these proteins localize to the leading edge (Fig. 1). This was similarly shown for three other Dictyostelium PH-domain-containing proteins: PhdA (Funamoto et al., 2001Go), PhdB (S.M. and R.A.F., unpublished), and PDK1 (R. Meili and R. A. Firtel, unpublished). These responses do not occur in the presence of the PI3K inhibitors wortmannin and LY294002 or in pi3k1/pi3k2 null cells, in which the two partially redundant Class I PI3Ks (PI3K1 and PI3K2) are deleted, or if mutant PH domains that cannot bind PtdIns(3,4,5)P3/PtdIns(3,4)P2 are used (Funamoto et al., 2001Go) (R. Meili and R. A. Firtel, unpublished). The PH domains of the mammalian Akt/PKB exhibit similar responses in neutrophils after stimulation by the chemokine N-formyl-Met-Leu-Phe (fMLP) and in fibroblasts in response to the growth factor PDGF (Haugh et al., 2000Go; Servant et al., 2000Go). Studies using the F-actin inhibitor latrunculin A demonstrate that new F-actin synthesis is not required for the translocation of the PH domain proteins.



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Fig. 1. PH domain and PI3K localization. The upper panel illustrates the temporal localization of the PH-domain-containing protein PhdA to the plasma membrane in response to a directional signal. In the first panel (left), the micropipette (asterisk) is in the upper right-hand corner and there is a localization of the protein in the part of the cell closest to the micropipette. In the second panel, the micropipette has moved, leading to a delocalization of PhdA from the original site and localization to the new position of the micropipette (panel 3). The fourth panel (right) indicates that when the micropipette is moved again, there is once again a delocalization of PhdA from the old site and a relocalization to the new site of the micropipette. The lower panels illustrate a similar dynamic localization of PI3K to the position on the plasma membrane closest to the chemoattractant source. The first panel shows the localization on the right side of the cell. When the micropipette is moved, there is a loss of this localization and a relocalization to the new position on the membrane. This localization is not sensitive to latrunculin A (data not shown), indicating that it is not dependent on the actin cytoskeleton.

 

The subfamily of PH-domain-containing proteins described above are considered to be reporters for sites of PtdIns(3,4,5)P3/PtdIns(3,4)P2 accumulation. Spatio-temporal localization studies suggest that a directional stimulation through chemoattractant receptors (GPCRs in Dictyostelium and neutrophils, and receptor tyrosine kinases in fibroblasts) leads to the localized synthesis and accumulation of PtdIns(3,4,5)P3/PtdIns(3,4)P2 at the site of the membrane closest to the chemoattractant source. Use of antibodies specific for PtdIns(3,4,5)P3 has confirmed the presence of PtdIns(3,4,5)P3 in the leading edge of polarized neutrophils (Weiner et al., 2002Go). However, the presence and the role of the PtdIns(3,4,5)P3 5'-dephosphorylated product PtdIns(3,4)P2 in the leading edge remains unclear. Therefore, hereafter we only mention PtdIns(3,4,5)P3.

The accumulation of PtdIns(3,4,5)P3 leads to the rapid localization and activation of PH-domain-containing proteins, such as Akt/PKB, at the leading edge. These results provided the first evidence that a steep PtdIns(3,4,5)P3 gradient is generated at the leading edge of chemotaxing cells in response to a shallow chemoattractant gradient and provided a conserved mechanism that accounts for directional sensing and cell polarization. The finding that membrane-permeant PtdIns(3,4,5)P3 added to cells is sufficient to induce cell polarization and motility in different cell types supported this model (Derman et al., 1997Go; Niggli, 2000Go; Weiner et al., 2002Go).

Analysis of Dictyostelium PI3K1 and PI3K2 localization unexpectedly revealed that in vivo these two proteins rapidly and transiently translocate to the plasma membrane in response to a global stimulation by cAMP, with kinetics slightly faster than those of the GFP fusion proteins containing PH domains (Funamoto et al., 2002Go). Both PI3Ks localize to the leading edge during chemotaxis (Fig. 1). The domain responsible is near the N-terminus of the protein and does not require the putative lipid-interacting C2 domain, the Ras-binding domain (RBD) or the lipid kinase domain (Funamoto et al., 2002Go) [for additional information on PI3K classes and structure see the recent review by Foster et al. (Foster et al., 2003Go)]. Further, the translocation occurs in pi3k1/pi3k2 null cells or wild-type cells treated with LY294002, which suggests that PI3K activity is not required for translocation. The gradient and the kinetics of PI3K translocation are very similar to those observed for the translocation of PH domain proteins. The localization and subsequent activation of PI3K at the leading edge could thus account for the generation of a steep PtdIns(3,4,5)P3 gradient. However, other mechanisms must amplify and maintain this gradient.

The mechanism underlying the localization of PI3K to the leading edge is presently unknown, except that it lies downstream of the heterotrimeric G protein [translocation does not occur in cells lacking the G{alpha}2 or the Gß subunits (S. Funamoto and R. A. Firtel, unpublished) (Lilly and Devreotes, 1995Go)]. We expect that, as in the case of PH domain localization, PI3K localization requires the de novo formation of binding sites or the uncovering of pre-existing binding sites (e.g. release of free Gß{gamma} subunits from G{alpha}{gamma}). Recent studies (Brock et al., 2003Go) have demonstrated that the p101 regulatory subunit of mammalian PI3K{gamma} interacts with Gß{gamma} in vivo and that this interaction is important for PI3K activation in response to a chemoattractant. Therefore, in the case of mammalian PI3K{gamma}, once G protein is activated, Gß{gamma} might recruit PI3K by interacting with the p101 subunit. In such a model, the specific recruitment of PI3K to the leading edge would directly depend on strong G protein activation in this region or alternatively require an additional unidentified pathway. What specifies the leading edge as the site of localization is not known. Ras-GTP has been proposed to be a general activator of PI3K. In mammalian cells, PI3K{gamma} is directly activated both in vitro and in vivo by Ras (Pacold et al., 2000Go; Suire et al., 2002Go). Furthermore, in Dictyostelium, a functional RBD is necessary for PI3K activation following chemoattractant stimulation (Funamoto et al., 2002Go).


    Role of class I PI3Ks in directional sensing and cell polarization
 Top
 Summary
 Introduction
 Directional sensing and cell...
 Role of class I...
 PTEN downregulates the...
 PI3K effectors and their...
 A PtdIns(3,4,5)P3 positive...
 Conclusion and perspectives
 References
 
The results discussed above implicate Class I PI3Ks in generating a PtdIns(3,4,5)P3 gradient involved in directional sensing and cell polarization (Fig. 2). Direct genetic evidence that PI3K is important for directional sensing and chemotaxis derives from studies of PI3K-knockout mutations in mice and Dictyostelium. Neutrophils isolated from knockout mice lacking the gene coding for PI3K{gamma}, a Class Ib PI3K activated by signaling through the heterotrimeric G protein Gß{gamma} subunit and Ras-GTP, exhibit significantly impaired chemoattractant-induced PtdIns(3,4,5)P3 synthesis, PKB activation, and motility in response to the chemoattractant peptides fMLP, C5a, and interleukin-8 (Il-8) (Hirsch et al., 2000Go; Li et al., 2000Go; Sasaki et al., 2000Go). The analysis of these neutrophils during chemotaxis revealed that they exhibit significantly reduced maintenance of a persistent leading edge and project pseudopodia along their entire surface rather than predominantly at the side of the cell facing the chemoattractant source (Hannigan et al., 2002Go). Parallel studies using isoform-specific antibodies against class IA PI3Kß and PI3K{delta} in macrophages and PI3K{delta}-specific inhibitors in neutrophils indicate that these PI3Ks also play an essential role in directional sensing (Sadhu et al., 2003Go; Vanhaesebroeck et al., 1999Go). Dictyostelium pi3k1/pi3k2 null cells show a >90% reduction in Akt/PKB activation in response to the chemoattractant cAMP as well as significantly reduced cell polarity, chemotaxis speed, and directionality, although they still move up a chemoattractant gradient (Funamoto et al., 2001Go; Funamoto et al., 2002Go; Meili et al., 1999Go). Dictyostelium cells also chemotax towards folate, a chemoattractant released by bacteria, using a distinct GPCR and G{alpha} subunit but the same {gamma} subunit. Chemotaxis in response to folate is even further impaired in pi3k1/pi3k2 null cells compared to chemotaxis to cAMP (Funamoto et al., 2001Go). This suggests that in this system the relative importance of the pathways downstream from the heterotrimeric G protein in controlling chemotaxis can vary, depending on the chemoattractant signal. The conclusion from these genetic studies that Class I PI3Ks are required for chemotaxis in Dictyostelium cells, neutrophils and macrophages is supported by a large number of studies using the general PI3K inhibitors wortmannin and LY294002 [for a detailed discussion and references, see Chung et al. (Chung et al., 2001aGo)]. Lastly, in addition to this work, studies of other Dictyostelium PI3K pathway components also demonstrate that they are required for the establishment and maintenance of cell polarization. PAKa, a p21-activated kinase, is a direct Akt/PKB effector and thus lies downstream from PI3K. Mutation of the Akt/PKB phosphorylation site on PAKa prevents chemoattractant-stimulated PAKa activation and produces a dominant negative molecule in wild-type cells that causes loss of chemoattractant-induced polarity.



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Fig. 2. Model for activation of PI3K during chemotaxis. Chemoattractant binding on a G-protein-coupled-receptor (GPCR) leads to the translocation and activation of Class I PI3Ks at the plasma membrane. This activation mechanism that requires both G protein and Ras GTPase remains poorly understood at the molecular level. Activated PI3K catalyzes the phosphorylation of PtdIns(4,5)P2 at the 3' position of the inositol ring to produce PtdIns(3,4,5)P3. In response, PtdIns(3,4,5)P3 acts as a binding site for a subclass of PH domain proteins. These localize to the plasma membrane and are activated. In neutrophils, Rho family GEFs such as P-Rex are PI3K effectors, which lead to the accumulation of activated Rac (Rac-GTP). The feedback loop required for the amplification of the pathway may involve actin polymerization. In Dictyostelium, the delocalization of PTEN from the plasma membrane at the leading edge may function as an amplification pathway for PI3K signaling by preferentially extending the half-life of PtdIns(3,4,5)P3 at this site of the membrane.

 

Although studies with PI3K inhibitors and null strains indicate that PI3K is important for directional sensing in at least some cell types, they do not provide evidence that PI3K plays an instructional role. Such evidence is supplied by experiments in Dictyostelium (Funamoto et al., 2002Go). Myristoylated (myr) PI3K is constitutively associated with the plasma membrane. Cells expressing myr-PI3K do not exhibit a basal in vivo PI3K activity, as determined by the absence of a PH domain localized to the plasma membrane or a high basal activity of Akt/PKB. In response to global chemoattractant stimulation, the cells have a wild-type response. When placed in a chemoattractant gradient, these cells produce multiple, large pseudopodia along the sides and back of the cells. Analysis of PH domain localization shows that PI3K is activated at these sites (localization of GFP-PhdA and GFPPH-Akt/PKB). Thus, mis-localization and activation of PI3K along the sides of cells leads to pseudopodum formation.

As mention above, PI3K inhibitors (wortmannin and LY294002) inhibit cell polarization and chemotaxis in response to chemoattractant stimulation in a wide variety of cell types, although the extent of the effect varies significantly between cell types (Chung et al., 2001aGo; Funamoto et al., 2001Go; Wang et al., 2002Go). We cannot exclude the possibility that there is only partial inhibition by wortmannin and LY294002, and the remaining PI3K activity, albeit small, might be sufficient for the partial effect observed. The variability of results with PI3K inhibitors could indicate that PI3K plays only a partial role in directional sensing and that other parallel regulatory mechanisms exist. The relative importance of the PI3K versus other pathways is probably cell-type dependent; other pathways are also essential for directional sensing. For example, a dominant negative form of the Rho family GTPase Cdc42 impairs directional sensing in macrophages, which also require PI3K for proper directional movement (Allen et al., 1998Go). Furthermore, in cells chemotaxing towards growth factors, the actin-severing protein cofilin plays a key role in cell polarity (Bailly and Jones, 2003Go; Dawe et al., 2003Go). Cofilin may also be important for responding to directional signals by locally stimulating F-actin polymerization at the leading edge through the formation of new barbed ends, which would be the sites of new F-actin polymerization (Chan et al., 2000Go; Condeelis et al., 2001Go; Ichetovkin et al., 2002Go; Zebda et al., 2000Go). Because cofilin is negatively regulated by PtdIns(4,5)P2, signaling pathways that affect PtdIns(4,5)P2 levels might differentially activate or inhibit cofilin function. In addition, cofilin is negatively regulated by LIM kinase, a PAK1 effector, providing another mechanism for controlling the activity of cofilin and F-actin assembly.


    PTEN downregulates the PtdIns(3,4,5)P3 pathway at the back of the cell
 Top
 Summary
 Introduction
 Directional sensing and cell...
 Role of class I...
 PTEN downregulates the...
 PI3K effectors and their...
 A PtdIns(3,4,5)P3 positive...
 Conclusion and perspectives
 References
 
The persistence of the steep PtdIns(3,4,5)P3 gradient indicates that a mechanism must restrict the PtdIns(3,4,5)P3 to the leading edge. One possibility is that PtdIns(3,4,5)P3 is degraded along the lateral sides of the cell. The tumor suppressor PTEN is a phosphoinositide 3'-specific phosphatase that dephosphorylates PtdIns(3,4,5)P3 and PtdIns(3,4)P2 to PtdIns(4,5)P2 and PtdIns(4)P, respectively (Lee et al., 1999Go; Maehama and Dixon, 1998Go; Yamada and Araki, 2003). Mammalian pten-null cells exhibit increased motility in a wound healing assay; however, the effect of this mutation on chemotaxis has not been directly studied (Liliental et al., 2000Go).

Genetic studies in Dictyostelium have provided more evidence for a direct role of PTEN in regulating chemotaxis (Iijima and Devreotes, 2002Go; Funamoto et al., 2002Go). These studies revealed that, in resting cells, PTEN is localized to the plasma membrane and uniformly distributed around the cell. In response to global stimulation, PTEN transiently delocalizes from the plasma membrane with kinetics that are similar to those of PI3K relocalization. In chemotaxing cells, PTEN is excluded from the leading edge but persists at the sides and the back of the cell. Thus, PI3K and PTEN exhibit reciprocal patterns of spatial localization (Fig. 3). PTEN is therefore spatially restricted to prevent the accumulation of PtdIns(3,4,5)P3 at the sides and the back of the cell. According to this model, the inactivation of PTEN should lead to the appearance of PtdIns(3,4,5)P3 at the side of the cell and the subsequent loss of cell polarity.



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Fig. 3. Spatial-temporal regulation of PTEN and PI3K induces cell polarization in response to a chemoattractant signal. (A) In unstimulated cells, Class I PI3K is mainly cytoplasmic, whereas PTEN is localized at the plasma membrane, resulting in a homogenous distribution of PtdIns(4,5)P2 in the plasma membrane. (B) When cells sense the chemoattractant signal, a signaling pathway yet to be identified promotes the rapid PI3K translocation to the leading edge facing the higher chemoattractant concentration and the delocalization of PTEN from the leading edge. Therefore, PtdIns(3,4,5)P3 is synthesized from PtdIns(4,5)P2 at the leading edge and prevented from accumulating on the sides and at the back of the cell by PTEN, causing a very steep anterior/posterior PtdIns(3,4,5)P3 gradient. (C) PtdIns(3,4,5)P3 recruits and activates at the leading edge RhoGEF proteins and other PH domain-containing proteins. The activity of these proteins is important to stimulate the actin polymerization necessary for cell motility. In coordination with these events in the leading edge, signaling by one or more pathways that remain to be identified restricts certain proteins to the back of the cell. These proteins are important for inhibiting pseudopod protrusion from the sides of the cell and for retracting the back of the cell while the leading edge is moving forward.

 

Iijima and Devreotes (Iijima and Devreotes, 2002Go) have identified a PtdIns(4,5)P2-binding site in the N-terminus of PTEN (conserved in the mammalian PTEN) and shown that this is required for PTEN localization to the plasma membrane. They proposed that the phosphorylation of PtdIns(4,5)P2 to form PtdIns(3,4,5)P3 by PI3Ks in the leading edge might be responsible for the delocalization of PTEN (Iijima and Devreotes, 2002Go). This would explain why the kinetics of PI3K localization to the plasma membrane in response to cAMP mimic those of PTEN delocalization. However, Funamoto et al. have observed that PTEN similarly delocalizes from the plasma membrane in response to cAMP in pi3k1/pi3k2 null cells, which produce very little PtdIns(3,4,5)P3 at the plasma membrane, suggesting that the conversion of PtdIns(4,5)P2 to PtdIns(3,4,5)P3 by PI3K is not fully responsible for the delocalization of PTEN (Funamoto et al., 2002Go). Dictyostelium cells in which the single PTEN gene has been disrupted move slower and are subject to more directional changes than are wild-type cells in a cAMP gradient (Iijima and Devreotes, 2002Go) (R. Meili and R. A. Firtel, unpublished). Close examination of the mutant cells during chemotaxis reveals that the leading edge is poorly defined and pseudopodia often emerge from the sides and even from the back. The introduction of a PtdIns(3,4,5)P3 probe into these cells revealed that the distribution of PtdIns(3,4,5)P3 is extended to the sides and the back of the cell, which is similar to cells expressing myr-PI3K (Iijima and Devreotes, 2002Go; Funamoto et al., 2002Go) (R. Meili and R. A. Firtel, unpublished). The phenotype of the pten null strain demonstrates that PTEN is important to restrict the localization of PtdIns(3,4,5)P3 to the leading edge, and this function is required to maintain a strong cell polarity during chemotaxis. Further analysis of these cells demonstrated that chemoattractant-induced F-actin polymerization is more elevated and prolonged, providing direct evidence that the PI3K pathway can regulate F-actin polymerization in these cells.


    PI3K effectors and their roles in chemotaxis
 Top
 Summary
 Introduction
 Directional sensing and cell...
 Role of class I...
 PTEN downregulates the...
 PI3K effectors and their...
 A PtdIns(3,4,5)P3 positive...
 Conclusion and perspectives
 References
 
Disruption of the Akt/PKB and PhdA genes in Dictyostelium strongly impairs the generation of cell polarity and chemotaxis (Funamoto et al., 2001Go; Meili et al., 1999Go). Akt/PKB is activated in response to chemoattractants in leukocytes and Dictyostelium cells, and this does not occur in pi3k1/pi3k2 null cells. Moreover, akt/pkb null cells exhibit reduced cell polarization and chemotax poorly. The localization of the PH domain of Akt/PKB during chemotaxis suggested that this kinase phosphorylates and regulates proteins involved in remodeling the leading edge (Meili et al., 1999Go). Unexpectedly, one role of Akt/PKB is the phosphorylation and activation of PAKa, which is essential for myosin II assembly the back of the cell (Chung and Firtel, 1999Go; Chung et al., 2001bGo). The phenotypes of the paka and akt/pkb null strains suggest that Akt/PKB has other substrates, possibly in the leading edge. Although, we do not know how Akt/PKB, which is thought to be activated at the leading edge, phosphorylates PAKa at the back, this example might provide a partial explanation of how Akt/PKB regulates cell polarity.

A second PI3K effector in Dictyostelium is PhdA (Funamoto et al., 2001Go). phdA null cells exhibit a 30% reduction in chemoattractant-mediated F-actin assembly, which suggests that PhdA is involved in this process. Although the role of PhdA has not been elucidated, it might be an adaptor protein that must be properly localized to the leading edge to function. A PhdA construct carrying two point mutations in the non-PH domain, C-terminal tail does not complement phdA null cells and has no phenotype when expressed in wild-type cells. Expression of a construct carrying a point mutation in the PH domain that abrogates PtdIns(3,4,5)P3 binding also fails to complement the null mutation but exhibits strong dominant negative effects when expressed in wild-type cells. However, expression of a PhdA construct that carries both the N-terminal and C-terminal mutations has no effect. This suggests that PhdA functions as an adaptor protein that localizes cytosolic components to the leading edge through its C-terminal domain. Further evidence that leading edge localization is important is derived from studies showing that myr-PhdA, which constitutively and uniformly localizes along the plasma membrane, does not complement the null mutation. Although PhdA does not have a clear homologue in mammals, a similar docking protein might play the same role.

The analysis of PI3K and PTEN mutants suggests that the PI3K pathway regulates F-actin assembly. F-actin assembly during chemotaxis is thought to be mediated by at least two pathways: activation of Arp2/3 by WASp/SCAR(WAVE); and activation of cofilin, which, through its F-actin-severing activity, produces new barbed ends that can function as sites of actin polymerization (Condeelis et al., 2001Go; Pollard and Borisy, 2003Go). Members of the WASp family of proteins are activated by Rho family GTPases Rac and Cdc42 (Etienne-Manneville and Hall, 2002Go; Machesky and Insall, 1999Go; Pollard and Borisy, 2003Go). Chemoattractant stimulation leads to the production of activated Rac-GTP and Cdc42-GTP, presumably resulting from the activation of Rac/Cdc42 guanine-nucleotide-exchange factors (GEFs), RhoGEFs. Activated Rac localizes to sites of membrane protrusion, where it is thought to activate effectors that mediate lamellipodial protrusion (Gardiner et al., 2002Go; Kraynov et al., 2000Go). Canonical RhoGEFs contain a catalytic Dbl-homology domain that is invariably linked to a PH domain that has a regulatory function and is therefore a potential target of the PtdIns(3,4,5)P3 signaling pathway in the leading edge (Schmidt and Hall, 2002Go). PtdIns(3,4,5)P3 is able to activate, directly or indirectly through phosphorylation events, RhoGEFs such as Vav1, Pix, SWAP-70, and P-Rex1 (Han et al., 1998Go; Shinohara et al., 2002Go; Welch et al., 2002Go; Yoshii et al., 1999Go). P-Rex1 is strongly and directly activated by PtdIns(3,4,5)P3 both in vitro and in vivo in neutrophils, but its involvement in cell polarization and chemotaxis has not yet been established (Welch et al., 2002Go). It is unclear whether RhoGEFs such as Vav are directly activated by PI(3,4,5)P3. It is important to mention that a new class of unconventional RhoGEF proteins, called CDMs (for Ced15/Dock/Myoblastcity), has emerged recently. This family includes DOCK180, DOCK2, and ZIZIMIN1 (Brugnera et al., 2002Go; Cote and Vuori, 2002Go; Fukui et al., 2001Go; Meller et al., 2002Go). DOCK180 is a specific GEF for Rac (Brugnera et al., 2002Go; Cote and Vuori, 2002Go), whereas ZIZIMIN1 is a GEF for Cdc42 (Cote and Vuori, 2002Go; Meller et al., 2002Go). Interestingly, DOCK180 has a basic domain that binds to PtdIns(3,4,5)P3 (Kobayashi et al., 2001Go) and ZIZIMIN1 has an N-terminal PH domain (Meller et al., 2002Go). The presence of these domains suggests that these GEFs might be regulated directly by PtdIns(3,4,5)P3. Furthermore, the disruption of the mouse DOCK2 gene, which is closely related to DOCK180, affects lymphocyte migration (Fukui et al., 2001Go). This result suggests that members of the DOCK/ZIZIMIN1 GEF family might also be regulated by PtdIns(3,4,5)P3 at the leading edge and play a role in cell polarization.


    A PtdIns(3,4,5)P3 positive feedback loop that involves Rho family GTPases
 Top
 Summary
 Introduction
 Directional sensing and cell...
 Role of class I...
 PTEN downregulates the...
 PI3K effectors and their...
 A PtdIns(3,4,5)P3 positive...
 Conclusion and perspectives
 References
 
As mentioned earlier, addition of PtdIns(3,4,5)P3 to neutrophils is sufficient to induce cell polarity and motility in neutrophils and neutrophil-differentiated HL-60 cells. When these cells are treated with membrane-permeant PtdIns(3,4,5)P3, the lipid first distributes homogenously in the inner leaflet of the plasma membrane and then accumulates at a newly formed leading edge and induces cell motility. Interestingly, prior treatment of cells with the PI3K inhibitors wortmannin and LY294002 abrogates these effects. Therefore, the observed accumulation of PtdIns(3,4,5)P3 in the leading edge does not result solely from the redistribution of the exogenously added PI(3,4,5)P3 but requires de novo PtdIns(3,4,5)P3 synthesis stimulated by PtdIns(3,4,5)P3. These data support the existence of a PtdIns(3,4,5)P3 positive feedback loop activated in the leading edge that is required to create a steep PtdIns(3,4,5)P3 gradient and induce cell polarization (Niggli, 2000Go; Wang et al., 2002Go; Weiner et al., 2002Go) (Fig. 2). In Dictyostelium, the preferential delocalization of PTEN from the leading edge but not the sides and back of chemotaxing cells may lead to a similar amplification of the gradient by reducing the rate of PtdIns(3,4,5)P3 hydrolysis at the leading edge but not the sides or back of the cell.

Bourne's laboratory has observed that treatment of neutrophil-like HL-60 cells with toxin B, a toxin from Clostridium difficile, which inhibits Rho family GTPases, prevents the formation of a steep PtdIns(3,4,5)P3 gradient at the leading edge after both chemoattractant and membrane-permeant PtdIns(3,4,5)P3 stimulations (Servant et al., 2000Go; Weiner et al., 2002Go). This finding indicates that, in HL-60 cells, at least one Rho family GTPase is involved in the positive feedback loop. Studies using the toxin from Clostridium sordelli, which specifically inhibits the Rac and Cdc42 GTPases, combined with the expression of dominant interfering forms of these GTPases, revealed that Rac but not Cdc42 or RhoA is probably involved in this loop (Srinivasan et al., 2003Go).

The proposed PtdIns(3,4,5)P3 positive feedback loop has two branches (Fig. 2). The first branch leads from the synthesis of PtdIns(3,4,5)P3 to the activation of the Rac GTPase. Obvious candidates for this role are the RacGEFs activated by the PtdIns(3,4,5)P3 pathway. The efficiency of this feedback loop implicates a strong and localized activation of the RacGEF at the leading edge upon chemoattractant stimulation. Interestingly, the RacGEF P-Rex1 isolated from neutrophils is synergistically activated by both PtdIns(3,4,5)P3 and Gß{gamma}. Therefore, P-Rex1 appears to be particularly well-suited to play a part in this feedback loop (Weiner, 2002Go; Welch et al., 2002Go). Alternatively, a strong and localized activation of RacGEF by the PI3K pathway could be achieved if PI3K and RacGEF are part of a same signaling complex. The existence of such a signaling complex is supported by the recent finding that a Class I PI3K is found in a complex containing the RacGEF Sos-1, the SH3-containing protein Eps8, and the scaffold protein Abi-1. The formation of this complex is important for stimulation of the RacGEF activity of Sos-1 (Innocenti et al., 2003Go). Such a complex could be produced at the leading edge in response to chemoattractant stimulation.

The second branch of the loop leading from the activated Rho GTPase to PI3K may require the actin cytoskeleton. Treatment of Dictyostelium and HL-60 cells with latrunculin B, a drug that inhibits F-actin polymerization, strongly destabilizes the actin cytoskeleton and the polarized structure of both types of cells. In HL-60 cells, this treatment also abolishes the persistent accumulation of PtdIns(3,4,5)P3 at the leading edge induced by a constitutively active Rac or by a chemoattractant stimulation (Srinivasan et al., 2003Go; Wang et al., 2002Go). An F-actin-based pathway might thus control PtdIns(3,4,5)P3 diffusion away from the leading edge; alternatively, it might directly activate PI3K at the leading edge. The activation of PI3K also involves Ras and possibly other upstream pathways. Further work is needed to identify missing components and to define, at a biochemical level, the signaling pathways involved in amplification and maintenance of the PtdIns(3,4,5)P3 gradient.


    Conclusion and perspectives
 Top
 Summary
 Introduction
 Directional sensing and cell...
 Role of class I...
 PTEN downregulates the...
 PI3K effectors and their...
 A PtdIns(3,4,5)P3 positive...
 Conclusion and perspectives
 References
 
Upon chemoattractant stimulation, a steep PtdIns(3,4,5)P3 gradient is created and maintained in the leading edge of motile cells such as leukocytes and Dictyostelium. PtdIns(3,4,5)P3 subsequently recruits a subclass of PH domain proteins that are important for actin remodeling at the leading edge and myosin II assembly in the back of the cell. The PtdIns(3,4,5)P3 gradient is formed and maintained by the concomitant localization and activation of PI3K at the leading edge and the dephosphorylation by PTEN of PtdIns(3,4,5)P3 that might diffuse to the sides and the back of the cell.

The localization and activation of PI3K at the leading edge appears to be a primary event in the signal transduction pathway that mediates directional sensing and cell polarization. The very rapid localization and activation of PI3K in response to chemoattractant stimulation indicates that these two events need to be tightly coordinated. This could be achieved if chemoattractant sensing triggers the formation of a PI3K signaling complex. We speculate that this signaling complex might also contain {gamma}, the regulatory subunits of PI3K, RacGEF, and the GTPases Rac and Ras. The stability of such a complex might be maintained by an F-actin-based mechanism, direct interaction with PtdIns(3,4,5)P3, and/or interactions with PH-domain-containing proteins.

Although most studies have focused on the role of PtdIns(3,4,5)P3 at the leading edge, the uropod also appears to be highly regulated and partially controlled by the PI3K pathway in some cell types. The examples of the Dictyostelium proteins PAKa, adenylyl cyclase, and PTEN support this suggestion (Chung et al., 2001bGo; Funamoto et al., 2002Go; Iijima and Devreotes, 2002Go; Kriebel et al., 2003Go). Yin and Jamney have proposed that PtdIns(4,5)P2 is responsible for localization of PTEN to the plasma membrane at the back of the cell and regulates F-actin assembly/disassembly in part through its inhibitory action on cofilin (Yin and Janmey, 2003Go). Such models imply that PtdIns(4,5)P2 is depleted from the leading edge in response to chemoattractant stimulation. Monitoring PtdIns(4,5)P2 localization in vivo using the PH domain of PLC{delta} fused to GFP could test this hypothesis. The available data indicate that the depletion of PtdIns(4,5)P2 from the leading edge probably does not result from its phosphorylation by PI3K, but from the activation of a PtdIns(4,5)P2-specific phospholipase C by chemoattractant signaling. The combination of genetic studies and in vivo imaging in motile cells such as Dictyostelium and neutrophils should help answer the remaining questions.


    Acknowledgments
 
We thank members of the Firtel laboratory for helpful suggestions and J. Roth for critically reading this manuscript. The work was supported by grants from USPHS to RAF.


    References
 Top
 Summary
 Introduction
 Directional sensing and cell...
 Role of class I...
 PTEN downregulates the...
 PI3K effectors and their...
 A PtdIns(3,4,5)P3 positive...
 Conclusion and perspectives
 References
 

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