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)
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Summary |
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Key words: Chemotaxis, Dictyostelium, PI 3-kinase, Fibroblasts, Neutrophils, Actin
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Introduction |
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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,
1999). 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, 2002
;
Firtel and Chung, 2000
;
Parent and Devreotes,
1999
).
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.
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Directional sensing and cell polarization require PtdIns(3,4,5)P3 accumulation in the leading edge |
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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.,
1998; Meili et al.,
1999
). 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., 2001
), 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., 2001
) (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.,
2000
; Servant et al.,
2000
). 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.
|
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.,
2002). 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., 1997;
Niggli, 2000
;
Weiner et al., 2002
).
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.,
2002). 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.,
2002
) [for additional information on PI3K classes and structure
see the recent review by Foster et al.
(Foster et al., 2003
)].
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 G2 or the
Gß subunits (S. Funamoto and R. A. Firtel, unpublished)
(Lilly and Devreotes, 1995
)].
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
subunits from
G
2ß
). Recent studies
(Brock et al., 2003
) have
demonstrated that the p101 regulatory subunit of mammalian PI3K
interacts with Gß
in vivo and that this interaction is important
for PI3K activation in response to a chemoattractant. Therefore, in the case
of mammalian PI3K
, once G protein is activated, Gß
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
is directly activated both
in vitro and in vivo by Ras (Pacold et
al., 2000
; Suire et al.,
2002
). Furthermore, in Dictyostelium, a functional RBD is
necessary for PI3K activation following chemoattractant stimulation
(Funamoto et al., 2002
).
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Role of class I PI3Ks in directional sensing and cell polarization |
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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., 2002).
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., 2001a; Funamoto et al.,
2001
; Wang et al.,
2002
). 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., 1998
). Furthermore, in cells chemotaxing towards growth
factors, the actin-severing protein cofilin plays a key role in cell polarity
(Bailly and Jones, 2003
;
Dawe et al., 2003
). 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., 2000
;
Condeelis et al., 2001
;
Ichetovkin et al., 2002
;
Zebda et al., 2000
). 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.
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PTEN downregulates the PtdIns(3,4,5)P3 pathway at the back of the cell |
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Genetic studies in Dictyostelium have provided more evidence for a
direct role of PTEN in regulating chemotaxis
(Iijima and Devreotes, 2002;
Funamoto et al., 2002
). 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.
|
Iijima and Devreotes (Iijima and
Devreotes, 2002) 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,
2002
). 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.,
2002
). 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, 2002
)
(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,
2002
; Funamoto et al.,
2002
) (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.
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PI3K effectors and their roles in chemotaxis |
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A second PI3K effector in Dictyostelium is PhdA
(Funamoto et al., 2001).
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., 2001;
Pollard and Borisy, 2003
).
Members of the WASp family of proteins are activated by Rho family GTPases Rac
and Cdc42 (Etienne-Manneville and Hall,
2002
; Machesky and Insall,
1999
; Pollard and Borisy,
2003
). 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., 2002
;
Kraynov et al., 2000
).
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,
2002
). 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.,
1998
; Shinohara et al.,
2002
; Welch et al.,
2002
; Yoshii et al.,
1999
). 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.,
2002
). 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.,
2002
; Cote and Vuori,
2002
; Fukui et al.,
2001
; Meller et al.,
2002
). DOCK180 is a specific GEF for Rac
(Brugnera et al., 2002
;
Cote and Vuori, 2002
), whereas
ZIZIMIN1 is a GEF for Cdc42 (Cote and
Vuori, 2002
; Meller et al.,
2002
). Interestingly, DOCK180 has a basic domain that binds to
PtdIns(3,4,5)P3
(Kobayashi et al., 2001
) and
ZIZIMIN1 has an N-terminal PH domain
(Meller et al., 2002
). 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., 2001
). 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.
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A PtdIns(3,4,5)P3 positive feedback loop that involves Rho family GTPases |
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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.,
2000; Weiner et al.,
2002
). 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.,
2003
).
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.
Therefore, P-Rex1 appears to be particularly well-suited to play a part in
this feedback loop (Weiner,
2002
; Welch et al.,
2002
). 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.,
2003
). 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.,
2003; Wang et al.,
2002
). 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.
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Conclusion and perspectives |
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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 Gß, 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., 2001b;
Funamoto et al., 2002
;
Iijima and Devreotes, 2002
;
Kriebel et al., 2003
). 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,
2003
). 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
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.
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Acknowledgments |
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