Molecular Biology of the Cell
Vol. 16, 4572–4583, October 2005
TOR Complex 2 Integrates Cell Movement during
Chemotaxis and Signal Relay in Dictyostelium
Susan Lee,* Frank I. Comer,†Atsuo Sasaki,* Ian X. McLeod,‡Yung Duong,*
Koichi Okumura,§John R. Yates III,‡Carole A. Parent,†and Richard A. Firtel*
*Section of Cell and Developmental Biology, Division of Biological Sciences and Center for Molecular
Genetics, University of California, San Diego, La Jolla, CA 92093-0380;†Laboratory of Cellular and Molecular
Biology, Division of Basic Sciences, NCI/National Institutes of Health, Bethesda, MD 20892-4256;
‡Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037; and§Ludwig Institute for Cancer
Research, School of Medicine, University of California, San Diego, La Jolla, CA 92093-0660
Submitted April 25, 2005; Revised July 8, 2005; Accepted July 21, 2005
Monitoring Editor: Anne Ridley
Dictyostelium cells form a multicellular organism through the aggregation of independent cells. This process requires
both chemotaxis and signal relay in which the chemoattractant cAMP activates adenylyl cyclase through the G protein-
coupled cAMP receptor cAR1. cAMP is produced and secreted and it activates receptors on neighboring cells, thereby
relaying the chemoattractant signal to distant cells. Using coimmunoprecipitation and mass spectrometric analyses, we
have identified a TOR-containing complex in Dictyostelium that is related to the TORC2 complex of Saccharomyces
cerevisiae and regulates both chemotaxis and signal relay. We demonstrate that mutations in Dictyostelium LST8, RIP3,
and Pia, orthologues of the yeast TORC2 components LST8, AVO1, and AVO3, exhibit a common set of phenotypes
including reduced cell polarity, chemotaxis speed and directionality, phosphorylation of Akt/PKB and the related PKBR1,
and activation of adenylyl cyclase. Further, we provide evidence for a role of Ras in the regulation of TORC2. We propose
that, through the regulation of chemotaxis and signal relay, TORC2 plays an essential role in controlling aggregation by
coordinating the two essential arms of the developmental pathway that leads to multicellularity in Dictyostelium.
TOR, Target of rapamycin, is a member of the PI3K-related
family of serine/threonine protein kinases and a known
regulator of cell growth in eukaryotic cells (Lorberg and
Hall, 2004). The protein complex that controls this pathway,
termed TOR Complex 1 (TORC1), is highly conserved in
evolution and contains the conserved proteins LST8 and
KOG1/Raptor complexed with TOR (Loewith et al., 2002;
Jacinto et al., 2003; Hay and Sonenberg, 2004). In addition,
TOR controls other pathways, including regulated polarized
actin assembly in S. cerevisiae (Schmidt et al., 1996). A distinct
TOR complex, TORC2, contains TOR, LST8, and three other
proteins: AVO1, AVO2, and AVO3. TORC2 regulates this
cytoskeletal rearrangement (Loewith et al., 2002), and a re-
lated TORC2 has been identified in mammalian cells that
contains, in addition to TOR and LST8, the AVO3 ortholog
mAVO3/Rictor (Jacinto et al., 2004; Sarbassov dos et al.,
2004). AVO1 and AVO3 are orthologues of two previously
identified Dictyostelium proteins, RIP3 (Ras interacting pro-
tein 3) and Pia (Pianissimo) (Chen et al., 1997; Lee et al.,
1999). Disruption of either of these gene products in Dictyo-
stelium (pia null [pia?] and rip3 null [rip3?] cells) gives rise to
serious developmental defects, because they are unable to
activate the aggregation-stage adenylyl cyclase ACA in re-
sponse to chemoattractant stimulation and are defective in
In Dictyostelium, the activation of ACA and chemotaxis are
closely integrated responses, although they have been
thought to be independently regulated (Comer et al., 2005).
On starvation, Dictyostelium cells initiate a developmental
program that requires chemotaxis toward extracellular
cAMP emitted by neighboring cells, which leads to the
formation of multicellular aggregates (Parent and Dev-
reotes, 1996; Aubry and Firtel, 1999; Kimmel and Parent,
2003). The chemoattractant cAMP binds to the heterotri-
meric G-protein-coupled cAMP receptor cAR1, which acti-
vates multiple intracellular signaling events that control che-
motaxis, the activation of adenylyl cyclase A (ACA), and
gene expression required for aggregation (Aubry and Firtel,
1999; Kimmel and Parent, 2003). The activation of ACA
produces more cAMP, which acts both intracellularly to
activate protein kinase A (PKA) and mediated downstream
effectors and extracellularly to initiate chemotaxis and to
relay the chemoattractant signal to distal cells (signal relay).
The concerted regulation of chemotaxis and signal relay
comprises a system that efficiently culminates with the de-
velopment of a true, differentiated multicellular organism.
In this article, we demonstrate that Dictyostelium TORC2
functions as an integrator of multiple distinct signaling path-
ways to control aggregation and the formation of the mul-
ticellular organism. Cells lacking any of the TORC2 proteins
LST8, RIP3 (AVO1), and Pia (AVO3, mAVO3, Rictor) exhibit
strong chemotaxis defects including loss of speed, cell po-
larity, and directionality, and are unable to fully activate
Akt/PKB and the related kinase PKBR1, both of which are
required for cell polarity and chemotaxis and are defective
This article was published online ahead of print in MBC in Press
on August 3, 2005.
Address correspondence to: Richard A. Firtel (email@example.com).
4572© 2005 by The American Society for Cell Biology
in their capacity to activate adenylyl cyclase in response to
chemoattractant stimulation. Further, we demonstrate that
RIP3 carrying a point mutation that abrogates Ras-GTP
binding is unable to fully complement the null mutation,
suggesting that Ras plays a role in mediating TORC2 func-
MATERIALS AND METHODS
Biochemical and Chemotaxis Assays
To obtain developmentally competent cells capable of responding to cAMP as
a chemoattractant, log-phase vegetative cells were washed twice and resus-
pended at a density 5 ? 106cells/ml with 12 mM Na?/K?phosphate buffer,
pH 6.2, and pulsed with 30 nM cAMP for 5 h at 6-min intervals. Cells were
then washed and resuspended in 12 mM Na?/K?phosphate buffer.
The chemoattractant receptor-mediated activation of adenylyl cyclase was
carried out as previously described (Parent and Devreotes, 1995). Briefly,
differentiated cells were stimulated with 50 ?M cAMP and filter lysed at the
indicated time points into Tris buffer containing
unlabeled ATP to a final concentration of ?150 Ci/mol. The reaction was
allowed to proceed for 1 min, stopped with SDS/ATP, and the radiolabeled
cAMP was purified by column chromatography. Adenylyl cyclase assays
exhibit considerable variability between experiments. Although the relative
extent of activation of different cell lines or treatment conditions is highly
reproducible between experiments, the absolute activity can vary significantly
(2–3-fold at times) from day to day. Therefore we, and others in the field, have
chosen to present adenylyl cyclase activation data as representative of results
from at least 3–5 independent experiments, each performed in duplicate on a
given day. Furthermore, comparisons between different cell lines or treatment
conditions are made on samples assayed on the same day.
For direct G-protein stimulation of adenylyl cyclase, differentiated cells
were filter lysed with GTP?S (50 ?M final concentration) and, after 5 min,
adenylyl cyclase activity was assayed for 2 min. To prepare cytosolic extracts
for the in vitro complementation experiments, cells were suspended at 8 ? 107
cells/ml in simple lysis buffer (SLB, 10 mM Tris, pH 7.5, 0.2 mM EGTA, 200
mM sucrose), filter lysed, and centrifuged at 9500 ? g for 20 min. The in vitro
complementation was performed in essentially the same manner as the direct
GTP-?-S-stimulated adenylyl cyclase activation assay, except that cells were
lysed directly into cytosolic extract.
The PKB and PKBR1 kinase activities were assayed as described previously
(Meili et al., 1999, 2000). Briefly, unstimulated differentiated cells (0 time
point) and differentiated cells stimulated with cAMP for various times were
lysed with an equal volume of 2? NP-40 lysis buffer (2? phosphate-buffered
saline, 100 mM NaF, 2% NP-40, 4 mM EDTA, 2 mM pyrophosphate, 2 mM
Na3VO4, and protease inhibitors leupeptin and aprotinin) on ice for 10 min.
The lysate was precleared by centrifugation for 10 min. To immunoprecipitate
PKB or PKBR1, the anti-PKB or anti-PKBR1 antibody and 30 ?l of slurry of
protein A-Sepharose were added into the supernatant. The beads were
washed twice with lysis buffer and twice with kinase buffer (25 mM MOPS,
pH 7.4, 25 mM ?-glycerophosphate, 20 mM MgCl2, and 1 mM dithiothreitol
[DTT]). We incubated beads with 75 ?l of kinase buffer containing 5 ?M cold
ATP, 10 ?Ci of ?-32P-ATP, and 5 ?g of H2B as substrate at room temperature
for 15 min. The reaction was stopped by the addition of 25 ?l of 5? sample
buffer (250 mM Tris, 500 mM DTT, 10% SDS, 50% glycerol, and 0.5% bromo-
phenol blue) and boiled for 2 min. Samples were separated by 12% SDS-
PAGE, blotted onto a polyvinylidene difluoride (PVDF) membrane, and
exposed to film. After the autoradiography, the lower part of the membrane
containing the phosphorylated H2B was cut off and the upper portion with
the Akt/PKB or PKBR1 was subjected to Western blot analysis to quantify the
level of Akt/PKB or PKBR1 in each lane. Experiments were repeated inde-
pendently at least three times, always assaying wild-type cells as a control for
comparison in each experiment. The wild-type samples were run on the same
gel as one of the experimental strains, and all gels and membranes were
Ras activation was performed as previously described (Sasaki et al., 2004).
F-actin polymerization and myosin II assembly were assayed as described
previously (Lee et al., 2004; Park et al., 2004). Experiments were repeated
independently at least three times, always including a wild-type control for
Chemotaxis analysis was performed as described previously (Park et al.,
2004; Sasaki et al., 2004) and analyzed using DIAS software (Wessels et al.,
1998). Differentiated cells were plated in Na?/K?phosphate buffer at a
density of 6 ? 104cells/cm2onto a plate with a hole covered by a 0.17-mm
glass coverslip. An Eppendorf Patchman micromanipulator with a glass
capillary filled with 150 ?M cAMP solution was brought into the field of view
of an inverted microscope. The response of the cells was recorded by time-
lapse video. Experiments were performed at least three times on different
days, always including a wild-type control strain in the analyses.
32P-?ATP diluted with
Cell Growth in the Presence of Rapamycin
Cells growing in log phase (?3–4 ? 106cells/ml) were diluted to 4 ? 105
cells/ml in HL5 medium. Rapamycin (Sigma Chemical Co, St. Louis, MO)
was dissolved in dimethyl sulfoxide as a 1 mM stock and added to the
cultures to the indicated concentration. Cell number in the suspension cul-
tures was monitored over 4 d, with no further addition of the drug.
The chemoattractant-mediated recruitment of the CRAC PH domain
(PHCRAC-GFP) to the plasma membrane was measured biochemically as
previously described in response to 20 ?M cAMP (Parent et al., 1998), except
that protein samples were subjected to PAGE analysis using NuPage 4–12%
Bis-Tris protein electrophoresis gradient gels, according to the manufacturer-
recommended protocol (Invitrogen, Carlsbad, CA). For microscopic observa-
tion of translocation, cells expressing PHCRAC-GFP were stimulated with 20
?M cAMP and fixed (1% formaldehyde, 0.125% glutaraldehyde, 0.01% Triton
X-100 in PB) after 5 s of stimulation. Cells were viewed with an inverted Zeiss
Axiovert S100 microscope (Thornwood, NY) equipped with automated filter
wheels (Ludl Electronic Products, Hawthorne, NJ). Images were recorded
with a CoolSnap HQ CCD camera (Roper Scientific, Trenton, NJ) operated by
IPLab software (Scanalytics, Fairfax, VA). GFP-PhdA translocation was ex-
amined by real-time fluorescent video microscopy as described previously
(Funamoto et al., 2001).
Detection of the Dd-TOR Complex
Aggregation-competent cells were washed with Na/K phosphate buffer and
resuspended at a density of 1 ? 108cells/ml in Na/K phosphate buffer
containing 20 mM MOPS (pH 7.0) and 1 mM phenylmethylsulphonyl fluo-
ride. Cells were mechanically lysed by filtration (Nuclepore Track Etch mem-
brane, 3 ?m; Whatman, Clifton, NJ). Subsequently, cell lysates were incubated
in 2.5 mg/ml DSP (ditho-bis-succinimidylpropionate, Calbiochem, La Jolla,
CA) for 5 min. The cross-linking reactions were quenched with 200 mM
Tris-HCl (pH 7.4). A sample was mixed with an equal volume of 2? lysis
buffer A (1% NP-40, 300 mM NaCl, 40 mM MOPS, pH 7.0, 20% glycerol, 2 mM
Na3VO4, 2 ?g/ml leupeptin, 5 ?g/ml aprotinin) and centrifuged. Total cell
extracts and immunoprecipitates with 25 ?l resin of anti-V5 (V5–10) antibody
agarose conjugate (Sigma) were analyzed by silver staining or immunoblot-
ting with anti-T7 (Novagen, Madison, WI) and anti-V5 antibodies. For iden-
tifying the Dd-TOR complex, the anti-V5 antibody immunoprecipitated prod-
ucts were eluted with the buffer containing 4% SDS and 0.1 M glycine (pH
2.5). The protein complex was incubated with 100 mM DTT and then precip-
itated with methanol/chloroform.
The samples were washed with acetone, resuspended in Tris buffer, 8 M urea,
pH 8.6, reduced with 100 mM TCEP, and alkylated with 55 mM iodoacet-
amide. Trypsin digest was done in the presence of 1 mM CaCl2for tryptic
specificity. Peptide mixtures were loaded onto a triphasic LC/LC column
with the following steps of 500 mM ammonium acetate bumps: 25, 35, 50, 80,
and 100%. Tandem mass spectra were analyzed using DTA Select and the
Dictyostelium sequence database with the following filtering parameters for
cross correlation scores 1.8 (?1), 2.8 (?2), and 3.5 (?3) (Washburn et al., 2001).
Identities of specific bands were confirmed by sequence analysis.
The LST8 knockout construct was made by inserting the blasticidin resistance
(Sutoh, 1993) cassette into a BamHI site created at nucleotide 500 of the LST8
genomic DNA sequence. A 5?-fragment was amplified from genomic DNA by
PCR by using the primers GTTTTTGAATTCAGTTTTGATGGTCATA-
AAGGTAATG and GTTTTTGGATCCGGTCAATGATGTTATACC and sub-
sequently digested with enzymes EcoRI and BamHI. A 3? fragment was
amplified by using the primers GTTTTTGGATCCTCAAGTGATGGTGGTT-
TAG and GTTTTTCTCGAGTTTTTATCTTGGTAAATCATTTAAAGCAAC
subsequently digested with BamHI and XhoI. The vector was digested with
EcoRI and XhoI and the DNA was transformed into Dictyostelium cells. The
knockout clones were confirmed by Southern and Northern blot analysis.
Two independent clones were picked and examined. Both showed the same
chemotaxis defects described in the results.
A T7-tagged full length LST8 ORF (open reading frame) clone was ampli-
fied from genomic DNA by PCR using primers 5?- GTTTTTGAAT-
TATTATATTGGCAACAGCATC and GTTTTTCTCGAGTTTTTATCTTGG-
TAAATCATTTAAAGCAAC-3? subsequently digested with EcoRI and XhoI
and subcloned to expression vector Exp4(?).
A 3? V5-tagged, full-length Pia clone was amplified from genomic DNA by
TAGTAGTG and GTTTTTCTCGAGTTTTTAAGTTGAATCTAAACCTAA-
TG and subsequently digested by EcoRV and XhoI and subcloned into the
expression vector Exp4(?).
TORC2 Controls Dictyostelium Aggregation
Vol. 16, October 20054573
A 5? T7-tagged clone of RIP3 from ATG to internal BamHI site at nucleotide
1490 was amplified by PCR from the full-length RIP3 (Lee et al., 1999) by using
ACAAATGGGTTCAGTTTATTGTGAATTAGTTGATG and ATTTGGACA-
AAGTACCAATACTTC. The full length of 5? T7-tagged RIP3 clone was
obtained by digesting the PCR product with EcoRI and BamHI and the
full-length RIP3 with BamHI and XhoI and the fragments cloned into the
expression vector Exp4(?).
All constructs were sequenced.
Dictyostelium Expresses Orthologues of the Yeast and
Mammalian TOR Complex 2
The previously identified Dictyostelium proteins RIP3 and
Pia are orthologues of the TORC2 components AVO1 and
AVO3/mAV03/Rictor in S. cerevisiae and mammals, respec-
tively (Chen et al., 1997; Lee et al., 1999; Loewith et al., 2002).
Previous studies indicated that both Dictyostelium proteins
are required for efficient chemotaxis and the activation of
ACA to relay the cAMP signal to neighboring cells. The
WD40-repeat protein LST8 is a known component of both
yeast and mammalian TORC1 and TORC2. We identified a
single gene in the Dictyostelium database (GenBank acces-
sion no. DDB0184464) that encodes an LST8 ortholog. We
created an LST8 gene knockout by homologous recombina-
tion. The knockout strain (lst8?cells) was confirmed by
Southern and RNA blot analyses (see Materials and Methods).
Analysis of the lst8?, pia?, and rip3?cells showed that these
cells were the same size and exhibited the same growth
kinetics as the parental wild-type cells (unpublished data),
indicating that these genes do not regulate Dictyostelium
growth under the conditions tested (rich axenic medium
containing 1% yeast extract, 1% proteose peptone, and 1%
dextrose or in association with bacteria as a food source).
Because RIP3, Pia, and LST8 are homologues of proteins
complexed with TOR in yeast and mammalian cells, we
examined whether rapamycin, which inhibits TORC1 but
not TORC2 (Loewith et al., 2002; Jacinto et al., 2004), inhibits
growth. Addition of rapamycin to concentrations of 50 nM
dramatically inhibited cell growth, whereas 500 nM rapa-
mycin resulted in an almost complete cessation of growth,
consistent with the involvement of a rapamycin-sensitive
TORC1 in controlling cell growth in Dictyostelium (Figure 1).
When plated for development on nonnutrient agar in the
absence of exogenous pulses of cAMP, we found that, sim-
ilar to rip3?and pia?cells, lst8?cells do not spontaneously
aggregate, but remain as a smooth monolayer of cells (Fig-
ure 2A; Chen et al., 1997; Lee et al., 1999). When provided
with exogenous pulses of cAMP, which mimic the endoge-
nous cAMP signaling of wild-type cells, and then plated for
development, lst8?and pia?cells still do not develop. In
contrast, rip3?cells form mounds, although very ineffi-
ciently (Figure 2A; Lee et al., 1999), suggesting that the rip3?
defect can be partially overridden, a phenotype that is also
shared with the Ras GEF (guanine nucleotide exchange fac-
tor) Aimless (Insall et al., 1996; Lee et al., 1999). Defects in the
ability to aggregate can result in an inability to induce the
genes required for this process.
To assess whether LST8, Pia, or RIP3 regulates the expres-
sion of early genes required for development, we performed
an RNA blot analysis examining the expression of the cAMP
receptor cAR1 and csA (gp80), a cell adhesion molecule,
both of which are required for aggregation. We find that
cAR1 and csA transcripts are normally induced in response
to exogenous cAMP in lst8?, pia?, and rip3?cells (Figure
2B). These data suggest that the developmental defects of
lst8?cells, like rip3?and pia?cells, may arise from deficien-
cies in chemotaxis and/or chemoattractant signal relay.
Dictyostelium TORC2 Exists in a Preformed Complex
That Regulates Adenylyl Cyclase Activation
To investigate the underlying developmental defect of lst8?
cells, we measured their capacity to activate ACA in re-
sponse to chemoattractant stimulation. In this activation trap
increases in cell number of wild-type cells in the absence and
presence of different concentrations of rapamycin are shown. See
Materials and Methods for details.
Inhibition of Dictyostelium cell growth by rapamycin. The
opment of rip3?, pia?, and lst8?and wild-type cells plated on
nonnutrient agar (left side). The cells in the right-hand panels were
first pulsed for 6 h with 30 nM cAMP, to mimic the natural cAMP
oscillations that induce aggregation-stage responses, before plating.
All pictures were taken at 24 h. (B) RNA blot showing the expres-
sion of two pulse-induced genes (cAR1 and csA) at 0 time and after
5 h of cAMP pulsing.
Development of rip3?, pia?, and lst8?cells. (A) Devel-
S. Lee et al.
Molecular Biology of the Cell 4574
assay (Parent and Devreotes, 1995; Lee et al., 1999), chemoat-
tractant receptor-mediated ACA activation is measured by
stimulating cells with a saturating dose of cAMP, rapidly
lysing aliquots at specific time points, and determining the
amount of new cAMP synthesis. Wild-type cells display a
rapid rise in enzyme activity, peaking ?1 min after addition
of chemoattractant, followed by a return to basal levels
within 7–10 min. A representative experiment (see Materials
and Methods) is shown in Figure 3A. Cells lacking LST8 are
defective in this response, showing dramatically reduced
ACA activation (Figure 3A). Parallel experiments on rip3?
and pia?cells reveal a similar loss of receptor-stimulated
ACA activity, consistent with previous findings (Figure 4A;
Chen et al., 1997; Lee et al., 1999). We also found a similar
defect in ACA activation in response to GTP-?-S, which
directly and constitutively activates G proteins downstream
of chemoattractant receptors (Figure 3B). From these results,
we conclude that LST8 must act downstream of G protein
activation and that the developmental defect of lst8?cells is
at least partially due to defective chemoattractant signal
relay through ACA.
To gain more insight into the mechanisms by which com-
ponents of TORC2 regulate ACA activity, we performed a
series of in vitro reconstitution experiments. Previous work
indicated that, in addition to receptor linked heterotrimeric
G proteins, chemoattractant-mediated activation of ACA re-
quires multiple cytosolic factors, including CRAC (cytosolic
regulator of adenylyl cyclase), Pia, and RIP3 (Insall et al.,
1994; Chen et al., 1997; Lee et al., 1999). One can achieve in
vitro complementation of CRAC null (crac?) cells, thus re-
storing GTP?S-stimulated ACA activity, by combining a
crac?cell lysate with cytosol derived from wild-type cells
(Lilly and Devreotes, 1995). We have utilized this approach
to further examine the activation of adenylyl cyclase in
rip3?, pia?, and lst8?cells. We hypothesized that if these
components exist in a stable, preformed complex, one may
be able to reconstitute GTP?S-stimulated ACA activity with
wild-type cytosol, but may not be able to cross-complement
between different mutant strains. As depicted in Figure 3B,
addition of wild-type cytosol to lysates from any of the
putative TORC2 mutant or crac?cell lines results in strong
reconstitution of GTP?S-stimulated ACA activity. Likewise,
we found significant reconstitution of crac?cell lysates with
cytosolic fractions from any of the TORC2-deficient cell
lines, although the rescue is more modest with cytosol from
rip3?cells than with cytosol from pia?or lst8?cells. In
contrast, addition of cytosol from any of the TORC2 mutants
to lysates from any of the other TORC2 mutants reconsti-
tutes little or no GTP?S-stimulated ACA activity. These data
support the hypothesis that RIP3, LST8, and Pia all function
together in a preformed complex to promote ACA activa-
tion. Furthermore, our observations suggest that these pre-
formed TORC2 complexes either cannot exchange compo-
nents rapidly or are unstable in the absence of all of the
Dictyostelium TORC2 Components Form a Complex
Our data suggest that RIP3, Pia, and LST8 regulate common
biochemical pathways. To examine whether these proteins
are found in a complex, we coexpressed two different pair-
wise combinations of RIP3, LST8, and Pia harboring differ-
ent epitope tags in wild-type cells and performed coimmu-
noprecipation experiments. Figure 4, A and B, show that
T7-tagged RIP3 coimmunoprecipitates with V5-tagged Pia
(V5-Pia) and that T7-tagged LST8 (T7-LST8) immunoprecipi-
tates with V5-Pia, suggesting that they can form a TORC2-
like complex in vivo.
To further characterize the components of such com-
plexes, we used anti-V5 antibody cross-linked to Sepharose
beads to purify Pia and associated proteins from cytosolic
extracts of Dictyostelium. This purified sample was subjected
to mass spectrometric (MS) analysis. Cytosolic extracts from
untransformed cells were used as a control. The MS analysis
showed that LST8 and TOR associate with V5-Pia (Figure
3C). 14-3-3, and heat shock proteins were preferentially
found in the experimental but not the control sample. Al-
though Dictyostelium encodes a Raptor/KOG1 ortholog
paired in TORC2 mutant cell lines. (A) Adenylyl cyclase activity of
each of the indicated cell lines was measured as de novo synthesis
of32P-labeled cAMP following the addition of the chemoattractant
cAMP to a final concentration of 50 ?M. Data are representative of
results from 3 to 4 independent experiments, each performed in
duplicate. (B) Cytosolic extracts of wild-type KAx-3 and each of the
TORC2 mutant cell lines were prepared as described in the Materials
and Methods. Direct G protein-mediated activation of adenylyl cy-
clase was measured after stimulation of cell lysates with GTP?S in
the absence or presence of the indicated cytosolic extract. The data
set for each combination is listed in the format: mutant cell lysate/
cytosolic extract. The data presented are representative of three
independent experiments, each performed in duplicate.
Receptor-mediated activation of adenylyl cyclase is im-
TORC2 Controls Dictyostelium Aggregation
Vol. 16, October 20054575
(GenBank accession no. DDB0191024), it is not present in
these immunoprecipitates, suggesting that Dictyostelium
forms a separate TORC1. Interestingly, we did not observe
RIP3 in the complex, although it coimmunoprecipitates with
Pia. These differences between the coimmunopreciptation
data and the MS analysis could be due to instability of the
complex and differential loss of some components during
the purification and/or differences in the efficiency of pep-
tide ionization in the MS, leading to different efficiencies of
peptide recovery in the analysis, although this is unlikely
given the dynamic range and sensitivity of the MS. Never-
theless, these findings indicate that, similar to the yeast and
mammalian counterparts, Dictyostelium LST8, RIP3, and Pia
form one or more TORC2 complexes (Loewith et al., 2002;
Jacinto et al., 2004). We did not identify any orthologues to
yeast AVO2 and no clear ortholog is found in the Dictyoste-
lium genome database.
Dictyostelium TORC2 Regulates Cell Movement during
An aggregation-deficient developmental phenotype can
arise from defects in either ACA activation or chemotaxis,
which are functionally independent (Comer et al., 2005).
Previous studies suggested that pia?cells exhibit chemotaxis
defects (Chen et al., 1997) and demonstrated that rip3?cells
have reduced cell polarity and a lower chemotaxis index
(Lee et al., 1999). To assess the ability of the lst8?cells to
chemotax, we examined their chemotaxis toward a micropi-
pette filled with cAMP. We quantified their behavior using
DIAS computer software (Wessels et al., 1988, 1998) and
compared this strain to wild-type cells and pia?and rip3?
cells, whose chemotaxis parameters had not been previously
measured. We found that, although the lst8?cells can mi-
grate toward the micropipette, they do so with reduced
speed and directionality and with significant loss in polarity
compared with wild-type cells (Figure 5, A and B; Table 1).
rip3?cells showed similar defects, consistent with previous
findings (Lee et al., 1999). The chemotaxis defects of pia?
cells were not as strong as those for lst8?and rip3?cells. To
further characterize the chemotaxis defects of rip3?, pia?,
and lst8?cells, we analyzed the mutants using the under-
agarose assay (Laevsky and Knecht, 2001; Comer et al.,
2005), in which cells migrate under a layer of agarose in a
gradient of chemoattractant. Under these conditions, the
lst8?cells, as well as the rip3?and pia?cells, do not migrate
as far toward the cAMP source as wild-type cells and, most
notably, do not organize into streams, a process that requires
ACA activation (Kriebel et al., 2003; unpublished data).
These data are consistent with the notion that LST8, RIP3,
and Pia function together in a complex that regulates both
ACA activation/signal relay and chemotaxis.
Proper chemotaxis requires the concerted regulation of
anterior F-actin extension and posterior myosin II contrac-
tion (Ridley et al., 2003). We therefore examined whether
deficiencies in LST8, RIP3, or Pia result in defective chemoat-
tractant-mediated F-actin polymerization or myosin II as-
sembly. The lst8?cells exhibit a nominal but reproducible
reduction in the extent of chemoattractant-stimulated F-ac-
tin polymerization in both the first (?5-s) and second
(?45-s) peaks of activation, which correlate with the initial
cringe response and the subsequent pseudopod extension,
respectively, observed after uniform chemoattractant stim-
ulation (Hall et al., 1988; Figure 5C). The rip3?and pia?cells
experience a similar modest decrease in the first peak, but
the level of the second F-actin peak is indistinguishable from
that of wild-type cells. We do not think that any change in
coimmunoprecipitation of V5-tagged Pia
with T7-tagged RIP3 (A) or T7-tagged LST8
(B). Cells were cotransformed with expres-
sion vectors expressing V5-Pia and T7-RIP3
or V5-Pia and T7-LST8 or expression on T7-
RIP3 or only T7-LST8. The immunoprecipita-
tion was carried out using anti-V5 antibody
(see Materials and Methods) and the Western
blot was probed (IB) with either anti-T7 or
anti-V5 antibody as shown. TCL shows the
protein in total cell lysates. (C) Partial list of
peptides identified in the mass spectrometry
analysis of the V5-Pia-containing complex.
Proteins found in common with the control
sample are not shown.
TORC2 complex. (A and B) The
S. Lee et al.
Molecular Biology of the Cell4576
the first F-actin peak that is observed is at all responsible for
the null polarity and chemotaxis defects. All three mutant
strains exhibit moderately altered patterns of chemoattrac-
tant-mediated myosin II assembly. In wild-type cells, the
levels of myosin II associated with the cytoskeleton undergo
an initial slight decrease 5–10 s after uniform chemoattrac-
shown are taken at the time the micropipette containing the chemoattractant is inserted (0 time) and at 15 min. See Materials and Methods for details.
(B) Images of chemotaxing cells obtained using DIAS computer software (see Materials and Methods). The overlapping images are taken at 1-min
intervals. (C) Chemoattractant stimulated F-actin polymerization and myosin II assembly as described in the Materials and Methods.
Chemotaxis properties of TORC2 mutant strains. (A) Chemotaxis images of wild-type (KAx-3) and lst8?cells using DIC optics. Images
Table 1. DIAS analysis
Dir ch (deg)
9.96 ? 0.70
24.1 ? 4.06
52.4 ? 3.04
0.78 ? 0.01
5.39 ? 0.38
40.4 ? 6.62
75.8 ? 3.46
0.57 ? 0.10
7.41 ? 0.25
41.1 ? 0.56
66.3 ? 1.29
0.48 ? 0.01
5.36 ? 1.97
46.8 ? 9.84
62.2 ? 3.62
0.53 ? 0.12
10.2 ? 2.12
16.0 ? 3.10
54.3 ? 3.93
0.84 ? 0.02
9.37 ? 0.18
30.9 ? 5.64
60.5 ? 5.78
0.74 ? 0.04
6.70 ? 0.82
32.2 ? 4.09
71.0 ? 2.48
0.57 ? 0.04
DIAS analysis of chemotaxis. Numbers are mean values ? SD. Speed indicates speed of cell’s centroid movement along the total path.
Direction change is a relative measure of the number and frequency of turns the cell makes. Larger numbers indicate more turns and less
efficient chemotaxis. Directionality is a measure of the linearity of the pathway. Cells moving in a straight line to the needle have a
directionality of 1.00. Roundness is an indication of the polarity of the cells. Larger numbers indicate the cells are more round (less polarized).
TORC2 Controls Dictyostelium Aggregation
Vol. 16, October 20054577
S. Lee et al.
Molecular Biology of the Cell4578
tant stimulation and then increase about twofold, peaking at
?30 s, before decreasing to basal levels (Steimle et al., 2001;
Park et al., 2004). The rip3?cells exhibit the strongest phe-
notype, with a significantly reduced peak of myosin II as-
sembly upon chemoattractant stimulation (Figure 5C). The
essentially wild-type F-actin response and moderate defects
in myosin II assembly suggest that these cells do not have a
fundamental inability to undergo cytoskeletal changes in
response to chemoattractant. However, all three null strains
simultaneously extend random pseudopodia from multiple
points around the entire periphery of the cell, in contrast to
wild-type cells, which predominantly extend pseudopodia
in the direction of the chemoattractant gradient. The poor
directionality of chemotaxing of the rip3?, pia?, and lst8?
cells supports the hypothesis that at least part of the loss of
directionality results from more random movement inherent
in cells that form multiple lateral pseudopodia. Our data,
however, do not exclude the possibility that these cells show
a defect in gradient interpretation or spatial-temporal sig-
naling to the cytoskeleton. This possibility is strongest for
lst8?cells as some of these cells exhibit directionality defects
that are stronger than those exhibited by the other strains
and for other apolar mutants such as myoII?or pakB/
pakC?/?cells (Wessels et al., 1988; Lee et al., 2004).
Ras Is Involved in the Regulation of RIP3
RIP3 was identified in a yeast two-hybrid screen using con-
stitutively active human H-Ras as bait and was found to
interact with GTP-bound forms of Dictyostelium RasG and
human H-Ras (Lee et al., 1999). Sequence comparison iden-
tified a putative Ras-binding domain (RBD) in RIP3 (Figure
6A). The Arg residue at position 681 is conserved in many of
these domains, suggesting it might be important for medi-
ating interactions between the RBD and Ras-GTP. We exam-
ined this by mutating this Arg and the preceding Lys to Glu
(K680E, R681E) and found that the RIP3 RBD carrying these
substitutions no longer interacted with RasG-GTP in a two-
hybrid assay (Figure 6B). Expression of wild-type RIP3 com-
plemented the developmental and chemotaxis defects of
rip3?cells (Figure 6C; Table 1). The chemotaxis parameters
were slightly reduced compared with wild-type cells, but
they were similar to wild-type cells overexpressing RIP3,
suggesting any changes were due to an overexpression of
RIP3 protein (Table 1; Figure 5A). In contrast, when
RIP3K680E,R681Ewas expressed in rip3?cells, it only partially
complemented the developmental and chemotaxis defects.
rip3?cells expressing RIP3K680E,R681Eform aggregates at
higher cell densities where cell-cell contacts can assist in
aggregate formation but the organisms exhibit developmen-
tal defects and are delayed in morphogenesis. The cells
cannot aggregate at lower cell densities where chemotaxis
plays an increasingly important role. Similar effects have
been noted for several signaling mutants, including akt/pkb
null cells (Meili et al., 1999). Further, rip3?cells expressing
RIP3K680E,R681Eexhibit only a small improvement in chemo-
taxis speed, and the directionality of movement, an indicator
of chemotaxis index, is similar to that of rip3?cells. These
results suggest that a functional RBD is important for full
Dictyostelium TORC2 Acts Downstream of Ras and PI3K
Numerous studies have shown that PI3 kinases (PI3Ks) play
multiple roles in chemoattractant signaling in Dictyostelium
and mammalian leukocytes (Funamoto et al., 2001; Stephens
et al., 2002; Merlot and Firtel, 2003; Van Haastert and Dev-
reotes, 2004). Cells lacking PI3K function exhibit chemotaxis
defects similar to those of the rip3?, pia?, and lst8?cells
(Funamoto et al., 2001, 2002; Sasaki et al., 2004). In Dictyoste-
lium, PI3K1 and PI3K2 translocate from the cytosol to the
leading edge membrane during chemotaxis and removal of
the membrane-targeting domain impairs chemoattractant
signaling through PI3K (Funamoto et al., 2002). Ras-medi-
ated activation of PI3K is thought to preferentially promote
localized activation of the chemotactic machinery, thus
sharply amplifying chemoattractant signaling at the leading
edge of chemotaxing cells (Sasaki et al., 2004). To gain insight
into the mechanism by which TORC2 regulates chemotaxis
and signal relay, we studied the impact of TORC2 on PI3K
signaling pathways. We first assessed the extent of chemoat-
tractant-stimulated Ras activation in the TORC2 mutants
using a pulldown assay in which GTP-bound Ras is isolated
through its binding to the RBD of human Raf1 and then
quantified using a Western blot assay (Sasaki et al., 2004).
This assay examines the activation of RasG, RasD, and RasB
with the majority of the Ras-GTP bound to the Raf1-RBD in
aggregation-competent cells attributed to RasG (Sasaki et al.,
2004). As depicted in Figure 7A, all strains exhibited kinetics
and extent of Ras activation similar to those of wild-type
cells. We next assessed the role of the TORC2 components
RIP3, Pia, and LST8 in the cellular distribution of PI3K. We
transformed wild-type, rip3?, pia?, and lst8?cells with GFP-
N-PI3K1, which is necessary and sufficient for PI3K1 cortical
localization, and studied its distribution in chemotaxing
cells. As previously reported, we observed that the majority
of the cortically localized GFP-N-PI3K1 is found at the lead-
ing edge of wild-type chemotaxing cells. Because of im-
proved imaging technology, we now also find that a smaller
fraction of GFP-N-PI3K1 localizes at the cell posterior (Fig-
ure 8). Both localizations are sites of enriched F-actin poly-
merization, consistent with our demonstration that en-
hanced GFP-N-PI3K cortical localization is dependent on
new F-actin (Sasaki et al., 2004). As expected, wild-type cells
have a ruffled leading edge with numerous protrusions. In
contrast, rip3?, pia?, and lst8?cells have broader GFP-N-
PI3K1-containing domains with multiple lateral pseudopo-
dia and broader posteriors, illustrating their loss-of-polarity
phenotype (Figures 5, A and B, and 8; data for pia?cells are
not published). Nevertheless, we find that GFP-N-PI3K1 is
targeted to the cortex in the mutants. Furthermore, we ob-
served that the mutant cell lines have normal kinetics of
GFP-N-PI3K1 translocation to the cortex in response to uni-
form chemoattractant stimulation (unpublished data). These
data demonstrate that the TORC2 components are not re-
quired for Ras activation or for the spatial targeting of PI3K.
We next evaluated whether the components of TORC2
regulate PI3K activation, using translocation of the CRAC
and PhdA PH domain to the plasma membrane as markers
for the accumulation of PI(3,4,5)P3, the major product of
PI3K. Earlier studies demonstrated that chemoattractant-
mediated translocation of CRAC and PhdA to the plasma
membrane is a G protein-, PI3K-, and PH domain-dependent
Figure 6 (facing page).
son of RIP3 Ras-binding domain. RIP3 (AAD43567, D. discoideum);
AVO1 (NP_014563, S. cerevisiae); RMIL/v-Raf1 (EAL24023, Homo
(AAP03432, H. sapiens); RGSE-1h/RGS14 (043566, H. sapiens); AAD/
CG5248-PL (NP_732776, Drosophila melanogaster); STEF (NP_036008,
Mus musculus); STL (NP_524647, D. melanogaster). (B) Two-hybrid
interaction of wild-type RIP3 and RIP3K680E,R681Ewith constitutively
active Dictyostelium RasG (RasGQ61L). (C) Development of rip3?cells
expressing RIP3 or RIP3K680E,R681Eplated on nonnutrient agar at
high (3 ? 106/cm2) and low (0.75 ? 106/cm2) density.
Analysis of RIP3. (A) Sequence compari-
TORC2 Controls Dictyostelium Aggregation
Vol. 16, October 20054579
event (Parent et al., 1998; Funamoto et al., 2001, 2002). Figure
7, B–D, depict the plasma membrane translocation of
PHCRAC-GFP in rip3?, pia?, and lst8?cells following a uni-
form, saturating dose of chemoattractant, as judged by both
fluorescence microscopy and Western blot analysis of mem-
brane preparations (data for PhdA not published). All three
mutant cell lines retain the capacity to generate membrane
binding sites for PHCRAC-GFP and PhdA, displaying normal
kinetics and an extent of translocation similar to that of
wild-type cells. From these data, we infer that RIP3, Pia, and
LST8 are not directly required to activate PI3K.
To gain further insight into the impact of TORC2 on PI3K
signaling pathways, we examined activation of the PI3K-
regulated effector PKB in rip3?, pia?, and lst8?cells by
directly measuring the chemoattractant-mediated activation
of PKB. Surprisingly, we found that in rip3?and pia?cells
exhibit a significant impairment in PKB activation compared
with wild-type cells (Figure 7D). PKB activation was less
impaired in lst8?cells, although the activation was still
reproducibly lower than that of wild-type cells (Figure 7D).
Combined with our observation that CRAC and PhdA trans-
location/PI3K activation is not impaired in the TORC2 mu-
tants, these data are consistent with a model in which
TORC2 acts either downstream or parallel to PI3K in the
chemoattractant-mediated activation of PKB.
Dictyostelium cells express two distinct PKB-related ki-
nases, PKB (the ortholog of mammalian PKB) and PKBR1.
PKBR1 is required for morphogenesis during the multicel-
lular stages of Dictyostelium development (Meili et al., 2000).
It harbors highly conserved kinase and C-terminal hydro-
phobic domains that include conserved sites of phosphory-
lation equivalent to T308and S473in human PKB. However,
in contrast to PKB, PKBR1 lacks a true PH domain (it has an
N-terminal, PH-like domain) and localizes constitutively to
the plasma membrane via an N-terminal myristoylation
modification. PKBR1 is activated by chemoattractants in a
PI3K-independent manner, presumably because PKBR1 is
constitutively associated with the plasma membrane and
does not require de novo PtdIns(3,4,5)P3synthesis for its
membrane localization. pkbr1?cells exhibit very weak che-
motaxis phenotypes but a double PKB/PKBR1 null strain
has strong growth and chemotaxis defects, indicating that
both kinases control chemotaxis pathways. These pheno-
types are more severe than those of rip3?, pia?, and lst8?
cells and significantly more severe than those of pkb?cells.
We therefore examined the extent of chemoattractant-in-
duced PKBR1 phosphorylation in rip3?, pia?, and lst8?cells.
Remarkably, as with PKB, PKBR1 phosphorylation is im-
paired in rip3?and pia?cells and less affected in lst8?cells
(Figure 6E), further indicating that the defect in PKB phos-
phorylation is not a result of improper targeting to the
plasma membrane or defects in PI3K activation. Rather,
these results are consistent with the Dictyostelium TORC2
regulating the phosphorylation of PKB and PKBR1. Al-
though the effects on cell movement are strongest in lst8?
cells, these cells exhibit the weakest effect on PKB and
PKBR1 activation. These observations suggest that different
components of the TORC2 complex have different effects on
TOR activity in different pathway. Our data do not exclude
the possibility of multiple TORC2 complexes with different
or overlapping functions or activities.
The Composition of Dictyostelium TORC2
We demonstrate here that Dictyostelium cells express a
TORC2 that is essential for multicellular development, but
not growth. As in S. cerevisiae, Dictyostelium Pia, RIP3, and
LST8, the orthologues of yeast AVO3, AVO1, and LST8,
form a complex in vivo. Our MS analyses of Pia-containing
Ras activation in wild-type and lst8?, rip3?, and pia?cells is shown.
(B and C) Translocation of PH-CRAC-GFP to the cortex examined
biochemically (B) or by fluorescence microscopy (C). (D and E)
Chemoattactant-mediated activation (kinase assay) of Akt/PKB (D)
and PKBR1 (E) in wild-type and lst8?, rip3?, and pia?cells. Activa-
tion of PKBR1 in pi3k1/2 null cells is also shown (E). Histone 2B
(H2B) is used as a substrate. Upper lanes show the kinase assay.
Lower lanes show a Western blot of Akt/PKB or PKBR1 protein in
the immunoprecipitate. See Materials and Methods for details.
Regulation of the PI3K pathway in TORC2 mutants. (A)
S. Lee et al.
Molecular Biology of the Cell 4580
complexes identified TOR and LST8 but not Raptor, which is
present in the Dictyostelium genome and is a known compo-
nent of rapamycin-sensitive TORC1 in other systems (Lor-
berg and Hall, 2004). Although RIP3 was not observed in the
MS analysis of Pia-containing complexes, coimmunoprecipi-
tation experiments determined it is associated with Pia in
vivo. Although rapamycin inhibits growth in Dictyostelium,
presumably through TORC1, our studies indicate that
TORC2 is rapamycin-insensitive, because the signaling
pathways that are genetically controlled by TORC2 are
not inhibited by the drug. This is consistent with the
findings in yeast and mammalian cells, although we have
not formally proven the existence of an independent
TORC1 biochemically (Loewith et al., 2002; Jacinto et al.,
2004; Sarbassov dos et al., 2004). As cells lacking Pia, RIP3,
or LST8 do not have growth defects, it is clear that a
rapamycin-sensitive TOR complex that does not contain
Pia, RIP3, or LST8 controls growth in Dictyostelium (Lor-
berg and Hall, 2004). Furthermore, our attempts to create
a cell line lacking the single TOR gene found in the
Dictyostelium genome (http://dictybase.org) were unsuc-
cessful, consistent with our rapamycin results and show-
ing that TOR is required for growth.
Our attempts to purify Dictyostelium TORC2 suggest that
the complex is not very stable, and we obtained the best
coimmunoprecipitation results using chemical cross-linking.
Our biochemical findings, combined with the common phe-
notypes of rip3?, pia?, and lst8?cells, provide strong indi-
cations of a Dictyostelium TORC2 that minimally contains
TOR, LST8, RIP3, and Pia. Intriguingly, our MS analyses
revealed that 14-3-3 and several heat-shock proteins are
associated with Pia, the latter of which we assume function
as molecular chaperones, possibly to help form or stabilize
TORC2. 14-3-3 may further stabilize the complex or TOR by
bridging phosphoserine residues.
TORC2 Integrates Signaling Pathways Regulating
Previous studies and those presented here show that rip3?,
pia?, and lst8?null strains all share common phenotypes:
the inability to activate adenylyl cyclase in response to che-
moattractant signaling; and a severe loss of cell polarization,
directionality in movement, and chemotaxis speed (Chen et
al., 1997; Lee et al., 1999). Using reconstitution of these null
cell lines, we provide strong evidence that Pia, RIP3, and
LST8 function together in a preformed complex to regulate
ACA. The mechanism by which this occurs remains to be
determined. Our reconstitution experiments demonstrate
that TORC2 is formed in the absence of CRAC, a PI3K
effector required for the activation of adenylyl cyclase. It was
previously established that ACA activity can be reconsti-
tuted in lysates derived from cells lacking both Pia and
CRAC only when both proteins are added back (Chen et al.,
1997), suggesting that ACA activation requires an input
from both TORC2 and CRAC.
We also demonstrate that rip3?, pia?, and lst8?cells ex-
hibit a reduction in chemoattractant-mediated activation of
Akt/PKB and PKBR1, a PKB-related kinase that is also
activated by phosphorylation of a conserved site in the
activation loop and C-terminal hydrophobic residues corre-
sponding to T308 and S473, respectively, in human PKB
(Alessi et al., 1996, 1997; Williams et al., 2000). It was recently
shown that TORC2 directly phosphorylates PKB on S473 in
Drosophila Kc167cells as well as in a variety of human cell
lines (Sarbassov et al., 2005). Our data strongly suggest that
TORC2 acts in a similar manner in Dictyostelium, in which
the reduced chemoattractant-mediated phosphorylation of
PKB and PKBR1 could due to the specific loss of phosphor-
ylation in the C-terminal domain. Consistent with this
model, we find that although Akt/PKB activation in Dictyo-
stelium is PI3K-dependent (Meili et al., 1999), as it is in
mammalian cells, the PI3K pathway and the activation of
Ras, which lies upstream of PI3K, is not detectably affected
in lst8?, pia?, and rip3?cells. A complete loss of both Akt/
PKB and PKBR1 in Dictyostelium results in chemotaxis phe-
notypes significantly more severe than those of rip3?, pia?,
and lst8?cells (Meili et al., 1999, 2000). It is currently difficult
to determine if the observed partial loss of PKB and PKBR1
phosphorylation is responsible for the chemotaxis and po-
larity defects of the rip3?, pia?, and lst8?cells, as we expect
the TORC2 pathway to control other chemoattractant sig-
nals. Furthermore, we do not know whether TORC2 activity
is constitutive or stimulated in response to chemoattractant.
Although we expect the latter, presently we have no way of
directly assaying this.
A commonality of the yeast, mammalian cell, and Dictyo-
stelium TORC2 pathways is a loss of cell polarity (Schmidt et
al., 1996; Chen et al., 1997; Lee et al., 1999; Jacinto et al., 2004;
Sarbassov dos et al., 2004). Dictyostelium TORC2 mutants
produce multiple pseudopodia simultaneously along the
cells. Fluorescent images of GFP-N-PI3K1, a
reporter for the localization of PI3K, are
taken from real-time digital recordings of
lst8?, rip3?, and wild-type chemotaxing cells
(Funamoto et al., 2001; Sasaki et al., 2004). The
micropipette is located to the left of the cells
off the field of view.
PI3K localization in chemotaxing
TORC2 Controls Dictyostelium Aggregation
Vol. 16, October 20054581
cell’s cortex when placed in a chemoattractant gradient. We
find that lst8?, rip3?, and pia?cells have reduced chemoat-
tractant-mediated F-actin polymerization and myosin II as-
sembly, with the strongest defect in myosin II assembly
observed in rip3?cells. The reduced myosin II assembly
may be due to the reduction in Akt/PKB activity, which is
required for maximal myosin II assembly in Dictyostelium
(Chung et al., 2001). In yeast and mammalian cells, TORC2
functions, at least in part, by controlling protein kinase C
and/or Rho GEF activity, although a direct link between
these signaling pathways, TORC2, and polarity has yet to be
established (Schmidt et al., 1997; Jacinto and Hall, 2003;
Jacinto et al., 2004; Lorberg and Hall, 2004). In Dictyostelium,
loss of cell polarity during chemotaxis is at least partially
due to reduced Akt/PKB and PKBR1 activation.
Aggregation to form a multicellular organism in Dictyo-
stelium requires the integration of signal relay (the activation
and secretion of the chemoattractant cAMP) with chemo-
taxis (Aubry and Firtel, 1999; Kimmel and Parent, 2003). We
have shown that TORC2 is required for chemotaxis, cell
polarity, and activation of ACA. TORC2 therefore coordi-
nates the activation of distinct signaling pathways required
for a common biological response (see model in Figure 9).
We propose that, in vivo, TORC2 provides a rapidly mobi-
lizable module that can respond quickly to extracellular
chemoattractant signals, thus providing an efficient way to
modulate complex responses. Interestingly, ACA and PKB
activation requires input from PI3K and TORC2, which are
both regulated by Ras-GTP (Funamoto et al., 2002; Sasaki et
al., 2004). We previously discovered that the RIP3 RBD binds
RasG-GTP, which is required for proper cell polarity and
chemotaxis (Tuxworth et al., 1997; Lee et al., 1999; Funamoto
et al., 2002; Sasaki et al., 2004). We now demonstrate that
RIP3 carrying a point mutation that abrogates RasG-GTP
binding cannot fully complement the rip3?mutation, sug-
gesting that Ras-GTP/RIP3 interaction is required for full
RIP3/TORC2 function. A recent study indicates that strains
in which Ras function is severely abrogated display a loss of
directionality and ability to directionally respond to the
chemoattractant gradient during chemotaxis (Sasaki et al.,
2004). The fact that this phenotype is significantly stronger
than those of cells lacking PI3K1 and PI3K2 is consistent
with Ras playing other parts during chemotaxis. We pro-
pose that TORC2 is one of these Ras-regulated processes.
This complex level of coordinated control of chemotaxis and
signal relay is consistent with other complex feedback loops
that regulate leading edge formation in both neutrophils and
Dictyostelium cells (Weiner et al., 2002; Park et al., 2004; Sasaki
et al., 2004). Regulatory controls, such as those imposed by
TORC2, thus provide cells with additional layers of flexibil-
ity for the coordination of complex physiological responses
such as Dictyostelium aggregation.
F.I.C. is a recipient of the National Institute of General Medical Sciences
Pharmacology Research Associate Training Fellowship. This work was sup-
ported in part by US Public Health Service Grants RR11823-09 to J.R.Y. and
GM37830 to R.A.F.
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