Download Signaling pathways at the leading edge of chemotaxing cells

Journal of Muscle Research and Cell Motility 23: 773–779, 2002.
2003 Kluwer Academic Publishers. Printed in the Netherlands.
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Signaling pathways at the leading edge of chemotaxing cells
CHANG Y. CHUNG1 and RICHARD A. FIRTEL2,*
1
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232-6600, USA; 2Section 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
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
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 forma-
*To whom correspondence should be addressed: Tel.:+1-858-5342788; Fax:+1-858-822-5900; E-mail: rafi[email protected]
tion 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
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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 and Gbc 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)P3/PI(3,4)P2 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 LY294002 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)P3 and PI(3,4)P2, 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).
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Localized activation
Phosphatidylinositol 3-kinase (PI3K) plays a central role
in local activation
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
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)P3],
is produced by two classes of PI3Ks, class IA (PI3Ka,
PI3Kb, and PI3Kd) and class IB (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
IA 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 LY294002 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)P3 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)P3 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
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)P3.
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, LY294002. In addition to C-terminal lipid
kinase and lipid-kinase-accessory domains, PI3Ks contain a Ras binding domain, a C2 domain, and a long Nterminal 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 RasGTP 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
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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)P3. 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.
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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)P3/PI(3,4)P2 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)P3 and PI(3,4)P2 (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)P3. 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)P2 binding domain of PTEN resulted in a PTENGFP 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)P2 by dephosphorylation of PI(3,4,5)P3 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 myrPI3K 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
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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 LY294002 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. Although we know the receptor and
coupled heterotrimeric G protein lie at the top of the
signaling cascade, the establishment of the initial asymmetry in the signaling pathway that leads to PI3K
localization is unknown. In addition, the issue of spatial
vs. temporal inputs in recognizing a directional signal
are still unresolved. Because of the facility of the system
and the ability to rapidly apply genetic approaches, the
Dictyostelium experimental system is poised to provide
additional insights into these important questions in cell
biology.
References
Aubry L and Firtel RA (1999) Integration of signaling networks that
regulate Dictyostelium differentiation. Ann Rev Cell Devel Biol 15:
469–517.
Chen MY, Insall RH and Devreotes PN (1996) Signaling through
chemoattractant receptors in Dictyostelium. Trends Genet 12: 52–
57.
Chung CY and Firtel RA (1999) PAKa, a putative PAK family
member, is required for cytokinesis and the regulation of the
cytoskeleton in Dictyostelium discoideum cells during chemotaxis.
J Cell Biol 147: 559–575.
Chung CY, Funamoto S and Firtel RA (2001a) Signaling pathways
controlling cell polarity and chemotaxis. TIBS 26: 557–566.
Chung CY, Potikyan G and Firtel RA (2001b) Control of cell polarity
and chemotaxis by Akt/PKB and PI3 kinase through the regulation of PAKa. Mol Cell 7: 937–947.
Clow PA and McNally JG (1999) In vivo observations of myosin II
dynamics support a role in rear retraction. Mol Biol Cell 10: 1309–
1323.
Devreotes PN and Zigmond SH (1988) Chemotaxis in eukaryotic cells:
a focus on leukocytes and Dictyostelium. Ann Rev Cell Biol 4: 649–
686.
Downey GP (1994) Mechanisms of leukocyte motility and chemotaxis.
Curr Opin Immunol 6: 113–124.
Egelhoff T, Lee RJ and Spudich JA (1993) Dictyostelium myosin heavy
chain phosphorylation sites regulate myosin filament assembly and
localization in vivo. Cell 75: 363–371.
Firtel RA and Chung CY (2000) The molecular genetics of chemotaxis:
sensing and responding to chemoattractant gradients. BioEssays
22: 603–615.
Fox JA, Ung K, Tanlimco SG and Jirik FR (2002) Disruption of
a single Pten allele augments the chemotactic response of B
lymphocytes to stromal cell-derived factor-1. J Immunol 169: 49–
54.
779
Funamoto S, Meili R, Lee S, Parry L and Firtel RA (2002) Spatial and
temporal regulation of 3-phosphoinositides by PI3-kinase and
PTEN mediates chemotaxis. Cell 109: 611–623.
Funamoto S, Milan K, Meili R and Firtel RA (2001) Role of
phosphatidylinositol 3¢ kinase and a downstream pleckstrin
homology domain-containing protein in controlling chemotaxis
in Dictyostelium. J Cell Biol 153: 795–809.
Hannigan M, Zhan L, Li Z, Ai Y, Wu D and Huang CK (2002)
Neutrophils lacking phosphoinositide 3-kinase gamma show loss
of directionality during N-formyl-Met-Leu-Phe-induced chemotaxis. Proc Natl Acad Sci USA 99: 3603–3608.
Haugh JM, Codazzi F, Teruel M and Meyer T (2000) Spatial sensing
in fibroblasts mediated by 3¢ phosphoinositides. J Cell Biol 151:
1269–1280.
Heldin CH and Westermark B (1999) Mechanism of action and in vivo
role of platelet-derived growth factor. Physiol Rev 79: 1283–1316.
Hirsch E, Katanaev VL, Garlanda C, Azzolino O, Pirola L, Silengo L,
Sozzani S, Mantovani A, Altruda F and Wymann MP (2000)
Central role for G protein-coupled phosphoinositide 3-kinase
gamma in inflammation (see comments). Science 287: 1049–1053.
Iijima M and Devreotes P (2002) Tumor suppressor PTEN mediates
sensing of chemoattractant gradients. Cell 109: 599–610.
Iijima M, Huang YE and Devreotes P (2002) Temporal and spatial
regulation of chemotaxis. Dev Cell 3: 469–478.
Insall R, Kuspa A, Lilly PJ, Shaulsky G, Levin LR, Loomis WF and
Devreotes P (1994) CRAC, a cytosolic protein containing a
pleckstrin homology domain, is required for receptor and G
protein-mediated activation of adenylyl cyclase in Dictyostelium.
J Cell Biol 126: 1537–1545.
Jin T, Zhang N, Long Y, Parent CA and Devreotes PN (2000)
Localization of the G protein betagamma complex in living cells
during chemotaxis (see comments). Science 287: 1034–1036.
Katanaev VL (2001) Signal transduction in neutrophil chemotaxis.
Biochemistry 66: 351–368.
Li Z, Jiang H, Xie W, Zhang Z, Smrcka AV and Wu D (2000) Roles
of PLC-beta2 and -beta3 and PI3Kgamma in chemoattractantmediated signal transduction. Science 287: 1046–1049.
Lilly PJ and Devreotes PN (1995) Chemoattractant and GTP gamma Smediated stimulation of adenylyl cyclase in Dictyostelium requires
translocation of CRAC to membranes. J Cell Biol 129: 1659–1665.
Maehama T and Dixon J (1998) The tumor suppressor, PTEN/
MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem 273: 13,375–13,378.
Maehama T, Taylor GS and. Dixon JE (2001) PTEN and myotubularin: novel phosphoinositide phosphatases. Ann Rev Biochem
70: 247–279.
Meili R, Ellsworth C and Firtel RA (2000) A novel Akt/PKB-related
kinase is essential for morphogenesis in Dictyostelium. Curr Biol
10: 708–717.
Meili R, Ellsworth C, Lee S, Reddy TBK, Ma H and Firtel RA (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.
Neptune ER and Bourne HR (1997) Receptors induce chemotaxis by
releasing the betagamma subunit of Gi, not by activating Gq or
Gs. Proc Natl Acad Sci USA 94: 14,489–14,494.
Parent CA and Devreotes PN (1996) Molecular genetics of signal
transduction in Dictyostelium. Ann Rev Biochem 65: 411–440.
Parent CA and Devreotes PN (1999) A cell’s sense of direction. Science
284: 765–770.
Parent CA, Blacklock BJ, Froehlich WM, Murphy DB and Devreotes
PN (1998) G protein signaling events are activated at the leading
edge of chemotactic cells. Cell 95: 81–91.
Rajagopalan S, Nicholas E, Vivancos V, Berger J and Dickson BJ
(2000) Crossing the midline: roles and regulation of Robo
receptors. Neuron 28: 767–777.
Rickert P, Weiner OD, Wang F, Bourne HR and Servant G (2000)
Leukocytes navigate by compass: roles of PI3Kgamma and its lipid
products. Trends Cell Biol 10: 466–473.
Sasaki T, Irie-Sasaki J, Jones RG, Oliveira-dos-Santos AJ, Stanford
WL, Bolon B, Wakeham A, Itie A, Bouchard D, Kozieradzki I,
Joza N, Mak TW, Ohashi PS, Suzuki S and Penninger JM (2000)
Function of PI3Kgamma in thymocyte development, T cell
activation, and neutrophil migration (see comments). Science
287: 1040–1046.
Servant G, Weiner OD, Herzmark P, Balla T, Sedat JW and Bourne
HR (2000) Polarization of chemoattractant receptor signaling
during neutrophil chemotaxis. Science 287: 1037–1040.
Servant G, Weiner OD, Neptune ER, Sedat JW and Bourne HR (1999)
Dynamics of a chemoattractant receptor in living neutrophils
during chemotaxis. Mol Biol Cell 10: 1163–1178.
Steimle PA, Licate L, Cote GP and Egelhoff TT (2002) Lamellipodial
localization of Dictyostelium myosin heavy chain kinase A is
mediated via F-actin binding by the coiled-coil domain. FEBS Lett
516: 58–62.
Stephens L, Ellson C and Hawkins P (2002) Roles of PI3Ks in
leukocyte chemotaxis and phagocytosis. Curr Opin Cell Biol 14:
203–213.
van Leeuwen FN, van Delft S, Kain HE, van der Kammen RA and
Collard JG (1999) Rac regulates phosphorylation of the myosin-II
heavy chain, actinomyosin disassembly and cell spreading. Nature
Cell Biol 1: 242–248.
Vanhaesebroeck B, Jones GE, Allen WE, Zicha D, Hooshmand-Rad
R, Sawyer C, Wells C, Waterfield MD and Ridley AJ (1999)
Distinct PI(3)Ks mediate mitogenic signaling and cell migration in
macrophages. Nature Cell Biol 1: 69–71.
Wang F, Herzmark P, Weiner OD, Srinivasan S, Servant G and
Bourne HR (2002) Lipid products of PI(3)Ks maintain persistent
cell polarity and directed motility in neutrophils. Nat Cell Biol 4:
513–518.
Weiner OD, Neilsen PO, Prestwich GD, Kirschner MW, Cantley LC
and Bourne HR (2002) A PtdInsP(3)- and Rho GTPase-mediated
positive feedback loop regulates neutrophil polarity. Nat Cell Biol
4: 509–513.
Wu W, Wong K, Chen J, Jiang Z, Dupuis S, Wu JY and Rao Y (1999)
Directional guidance of neuronal migration in the olfactory system
by the protein Slit. Nature 400: 331–336.
Xiao Z, Zhang N, Murphy DB and Devreotes PN (1997) Dynamic
distribution of chemoattractant receptors in living cells during
chemotaxis and persistent stimulation. J Cell Biol 139: 365–374.
Zhou K, Pandol S, Bokoch G and Traynor-Kaplan AE (1998)
Disruption of Dictyostelium PI3K genes reduces [32P]phosphatidylinositol 3,4 bisphosphate and [32P]phosphatidylinositol trisphosphate levels, alters F-actin distribution and impairs
pinocytosis. J Cell Sci 111: 283–294.
Zhou KM, Takegawa K, Emr SD and Firtel RA (1995) A phosphatidylinositol (PI) kinase gene family in Dictyostelium discoideum:
biological roles of putative mammalian p110 and yeast Vps34p PI
3-kinase homologs during growth and development. Mol Cell Biol
15: 5645–5656.