Developmental Cell, Vol.
2, 1–20, May, 2002, Copyright 2002 by Cell Press
Modulation of CREB Activity by the Rho GTPase
Regulates Cell and Organism Size
during Mouse Embryonic Development
Raffaella Sordella,1,4 Marie Classon,1,4
Kang-Quan Hu,1 Stephen F. Matheson,1
Madeleine R. Brouns,1 Barry Fine,1 Le Zhang,1
Hiroya Takami,2 Yoshihiko Yamada,2
and Jeffrey Settleman1,3
1
MGH Cancer Center and
Harvard Medical School
Charlestown, Massachusetts 02129
2
National Institute of Dental
and Craniofacial Research
National Institutes of Health
Bethesda, Maryland 20892
Summary
Rho GTPases regulate several aspects of tissue mor-
phogenesis during animal development. We found that
mice lacking the Rho-inhibitory protein, p190-B Rho-
GAP, are 30% reduced in size and exhibit develop-
mental defects strikingly similar to those seen in mice
lacking the CREB transcription factor. In p190-B Rho-
GAP-deficient mice, CREB phosphorylation is sub-
stantially reduced in embryonic tissues. Embryo-
derived cells contain abnormally high levels of active
Rho protein, are reduced in size, and exhibit defects in
CREB activation upon exposure to insulin or IGF-1.
The cell size defect is rescued by expression of consti-
tutively activated CREB, and in wild-type cells, expres-
sion of activated Rho or dominant-negative CREB re-
sults in reduced cell size. Together, these results
suggest that activity of the Rho GTPase modulates a
signal from insulin/IGFs to CREB that determines cell
size and animal size during embryogenesis.
Introduction
The Rho GTPases mediate signaling pathways that reg-
ulate a variety of cellular activities, including cytoskeletal
reorganization, gene expression, and cell cycle progres-
sion (Van Aelst and D’Souza-Schorey, 1997). They are
also essential regulators of tissue morphogenesis during
embryonic development (Settleman, 1999). Like all
GTPases, the Rho proteins cycle between active, GTP-
bound and inactive, GDP-bound states. This cycling is
largely regulated by two classes of proteins, the guanine
nucleotide exchange factors (GEFs) and the GTPase
activating proteins (GAPs; Symons and Settleman,
2000). The RhoGEFs function to promote the conversion
of inactive Rho to active Rho, while the RhoGAPs stimu-
late the intrinsic GTP hydrolysis activity of Rho proteins,
thereby converting them to a GDP-bound, inactive form.
Accumulating evidence suggests that the RhoGEFs are
rapidly activated upon ligand-receptor interactions at
the cell surface, and that the subsequently activated
3
Correspondence: settleman@helix.mgh.harvard.edu
4
These authors contributed equally to this work.
GTPases elicit their biological actions through direct
interactions with a variety of so-called effector targets.
The biological function of the RhoGAPs is somewhat
less clear. These negative regulators of Rho GTPases
have been postulated to play a role in precisely modulat-
ing Rho-mediated signaling in vivo, and there are several
reports indicating that these proteins, when overex-
pressed, can inhibit GTPase-mediated signaling path-
ways (Symons and Settleman, 2000). However, there
has been relatively little analysis of these proteins in a
truly in vivo context. Among the various RhoGAPs that
have been identified is a family of widely expressed
potent Rho-specific GAPs, referred to as the p190 Rho-
GAPs. These GAPs are unusual in that they contain
an amino-terminal GTPase domain in addition to their
carboxy-terminal RhoGAP catalytic domain (Burbelo et
al., 1995; Settleman et al., 1992). In mammals, there are
two p190 RhoGAPs, referred to as p190 RhoGAP and
p190-B RhoGAP. Closely related orthologs of these pro-
teins are present in Drosophila and C. elegans as well.
Recently, RNA interference studies have revealed a role
for the Drosophila p190 RhoGAP in neurite retraction in
the developing nervous system (Billuart et al., 2001),
although a loss-of-function mutant of this gene has yet
to be reported. As Rho activation has previously been
demonstrated to promote neurite retraction in cultured
neurons (Jalink et al., 1994), this result suggests that
loss of p190 RhoGAP activity in vivo leads to deregu-
lated Rho GTPase function. Disruption of the p190 Rho-
GAP gene in mice by gene targeting revealed that it is
an essential protein that mediates an adhesion signaling
pathway downstream of Src kinases that functions in
neural development (Brouns et al., 2000, 2001).
Although the biological function of p190-B RhoGAP
is not clear, it has been reported that this RhoGAP ac-
counts for the majority of Rho-inhibitory activity in cul-
tured fibroblasts, suggesting that it is a major down-
modulator of Rho GTPase activity in vivo (Vincent and
Settleman, 1999). To determine the biological require-
ment for p190-B RhoGAP, we have disrupted the gene
in mice. Surprisingly, we found that these mice exhibit
phenotypes that are remarkably similar to those seen
in mice lacking the CREB transcription factor, including
a substantial reduction in animal size (Rudolph et al.,
1998). Additional studies revealed that cell size is re-
duced in these animals and that Rho and CREB are
important novel regulators of cell size in a developing
mouse. The results also suggest a cytoskeleton-inde-
pendent role for Rho GTPase signaling in embryonic
development.
Results
Mice Lacking p190-B RhoGAP Closely Resemble
Mice Lacking CREB
To explore p190-B RhoGAP function in vivo, we dis-
rupted the mouse gene using homologous recombina-
tion in embryonic stem (ES) cells (Figure 1A). Two lines
of properly targeted ES cells were used to establish
Developmental Cell
2

Figure 1. Mice Lacking p190-B RhoGAP Resemble CREB-Deficient Mice

(A) Targeting strategy for disruption of the p190-B RhoGAP gene. Schematic representations of the 5⬘ genomic region of the p190-B RhoGAP
gene (upper line), the targeting vector (middle line), and the mutated allele (lower line) (gray bar depicts exon I). Bar under the upper line
represents the probe used for the identification of heterozygous ES lines. S, StuI; H, HindIII; B, BamHI; N, NotI; NEO, neomycine resistance
gene; TK, thymidine kinase gene.
(B) An immunoblot of embryonic tissue from wild-type (⫹/⫹), heterozygous mutant (⫹/⫺), and homozygous mutant (⫺/⫺) tissue probed with
antisera that specifically recognize either p190-B RhoGAP (top panel) or p190 RhoGAP (bottom panel).
Rho and CREB Regulate Embryonic Cell Size
3

germline transmission of the targeted allele in mice, and
the resulting homozygous mutant mice fail to express
any detectable p190-B RhoGAP (Figure 1B). Homozy-
gous mutant mice die immediately after birth, and,
grossly, they always exhibit a substantial and uniform
reduction in size (30% reduction in mass) relative to
their littermates (more than 100 examined; Figure 1C).
Histological analysis revealed several completely pene-
trant (30/30 examined) defects in the mutant mice. At
birth, they exhibit a severe reduction in thymus size
(disproportionate to the reduction in body size; Figure
1D), their lungs fail to inflate properly (not shown), and
in brain, they exhibit axon defects that include a severe
reduction in the major midline forebrain crossing tracts
(corpus callosum and anterior commissure), as well as
a thinner cortex, associated with partial loss of cortical
axon tracts (Figures 1E and 1F). The lateral ventricles
in brain are substantially enlarged in mutants (Figures
1E and 1F). Aside from a small decrease in overall size,
all other major organs appear to be unaffected in the
mutants.
The observed spectrum of defects in mutant animals
strikingly resembles defects previously reported in mice
lacking the CREB transcription factor (Rudolph et al.,
1998). CREB-deficient mice also die immediately after
birth, are 30% reduced in size, fail to inflate their lungs,
have a small thymus, and exhibit brain defects that affect
the corpus callosum, anterior commissure, and lateral
ventricles (Rudolph et al., 1998). CREB, like its closely
related family members CREM and the ATFs, undergoes
phosphorylation of serine 133 when activated (Shaywitz
and Greenberg, 1999). Significantly, we determined that
while CREB protein levels are unaffected in mutant tis-
sues, phospho-CREB and phospho-ATF-1 are virtually
absent in mutant brain, are substantially reduced in thy-
mus, and are also reduced in stomach and heart (Fig-
ure 1G).
Loss of p190-B RhoGAP Causes a Cell Size
Reduction that Is Rescued by Activated CREB
To determine the role of p190-B RhoGAP in CREB regu-
lation, we established stable fibroblast lines from wild-
type and mutant embryos. All results were confirmed
with three independently derived litter-matched wild-
type and mutant pairs of spontaneously immortalized
fibroblast (3T3) lines. Morphologically, wild-type and
mutant cells appear similar, although mutant cells, as
would be expected as a consequence of losing a Rho-
inhibitory protein, contain significantly more polymer-
ized actin (data not shown). By comparing the growth
properties of these cells, we determined that while they
exhibit equivalent doubling times, saturation density is
significantly increased in the mutants (Figure 2A). For-
ward light scatter FACS analysis, which indirectly mea-
sures changes in cell volume, revealed that this differ-
ence reflects an approximately 30% reduction in cell
size in the mutants (Figures 2B and 5D). Thus, they pack
more densely in a monolayer. This result was indepen-
dently confirmed by comparing protein:cell number and
protein:DNA ratios between wild-type and mutant cells
(Figures 2C and 2D), as well as by morphometric analysis
(not shown). Changes in cell size observed by these first
three criteria would not be influenced by changes in the
cytoskeleton, indicating that the reduced cell size in
mutants is not due to Rho-induced effects on actin as-
sembly.
The small size of mutant animals could potentially
reflect this cell size phenotype seen in 3T3 lines, or
it could be due to either reduced cell proliferation or
increased apoptosis in developing tissues. Indeed,
CREB has previously been implicated as a cell survival
factor (Walton and Dragunow, 2000). However, in mutant
embryonic tissues (E12–E18) analyzed at several levels
of sectioning, we were not able to detect any significant
increase in apoptosis by TUNEL staining (data not
shown). Moreover, no evidence of reduced cell prolifera-
tion was seen by BrdU incorporation into embryonic
tissues (Figures 2E and 2F). Then, by comparing pro-
tein:DNA ratios, we found that in most tissues there is
a significant reduction in cell size in animals lacking
p190-B RhoGAP (Figure 2G). Together with the observa-
tion that mutant 3T3 cells are reduced in size, these
results suggest that the small size of the mutant mice
reflects a cell-autonomous defect in cell size regulation.
Significantly, p190-B RhoGAP is expressed in all tissues
during embryonic development (Figure 2H).
To determine whether the cell size defect that results
from loss of p190-B RhoGAP is due to impaired CREB
activation, we generated p190-B RhoGAP-deficient 3T3
lines that stably express a constitutively activated form
of CREB (CREBFY; Du et al., 2000). These cells are
significantly larger than the parental p190-B RhoGAP-
deficient lines, indicating that CREB activation is suffi-
cient to rescue the cell size defect in mutants, and that
CREB is probably functioning downstream of p190-B
RhoGAP (Figures 3A–3C). In addition, we determined
that CREBFY rescues the cell size defect in both G1-
and G2-selected cell populations, indicating a cell cycle-
independent role for CREB in cell size regulation (Fig-
ures 3D–3G).
Rho and CREB Activity Modulate Cell Size
To address the mechanism by which loss of p190-B
RhoGAP affects CREB activation, we considered the

(C) Newborn wild-type (⫹/⫹) and homozygous mutant (⫺/⫺) animals illustrating the size defect seen in animals lacking p190-B RhoGAP.
(D) Thymuses dissected from newborn wild-type (⫹/⫹) and homozygous mutant (⫺/⫺) animals revealing the substantial reduction in the size
of mutant thymus.
(E and F) Coronal forebrain sections from wild-type (E) and mutant (F) embryos at E18.5 stained with Nissl reveals the reduction in the thickness
of the corpus callosum (black arrows) in the mutant as well as the enlargement of lateral ventricles in these animals. Note that the thickness
of the cortex is also reduced in the mutants, and this appears to reflect a reduction of the major forebrain cortical axon tracts (white arrows).
(G) Immunoblots of various tissues from wild-type (⫹/⫹) and mutant (⫺/⫺) E18.5 animals with antibodies that specifically recognize the
phosphorylated form of CREB and ATF-1 (upper panels) or total CREB protein (lower panels).
Developmental Cell
4

Figure 2. Cell Size Is Reduced in Fibroblasts and Tissues from Mice Lacking p190-B RhoGAP
(A) Proliferation curve of wild-type (⫹/⫹) and p190-B RhoGAP mutant (⫺/⫺) 3T3 cells, indicating similar proliferative rates in the two populations
and increased saturation density of the mutant cells.
(B) Cell size distribution of wild-type (⫹/⫹) and p190-B RhoGAP mutant (⫺/⫺) 3T3 cells determined by forward light scatter FACS analysis,
indicating the mean reduction in cell size in a population of G1-selected (2N content) mutant cells.
(C) Ratio of total protein to cell number in a population of confluent wild-type (⫹/⫹) and p190-B RhoGAP mutant (⫺/⫺) cells confirming the
reduced cell size of mutant cells.
(D) Ratio of total protein to total DNA content in a population of confluent wild-type (⫹/⫹) and p190-B RhoGAP mutant cells, again confirming
the reduced cell size of mutant cells. Values in (C) and (D) reflect the average of three independent experiments, and error bars represent
standard error of the mean.
(E and F) BrdU incorporation of intestinal sections of wild-type (E) and p190-B RhoGAP mutant (F) E18.5 embryos (following 4 hr intrauterine
BrdU injection), indicating approximately equivalent levels of cell proliferation. Red, BrdU positivity by anti-BrdU antibody fluorescence; blue,
DAPI staining.
(G) Ratio of total protein to total DNA content in various tissues derived from E18.5 wild-type (⫹/⫹) and p190-B RhoGAP mutant (⫺/⫺) animals,
indicating that tissues from mutant embryos contain abnormally small cells. Values reflect the average of tissue measurements taken from
seven different mice, and error bars represent the standard error of the mean.
(H) Immunoblot of p190-B RhoGAP with the indicated tissues dissected from an E18.5 wild-type embryo.
Rho and CREB Regulate Embryonic Cell Size
5

Figure 3. Activated CREB Rescues the Size Defect in Cells Lacking p190-B RhoGAP
(A) Forward light scatter analysis of G1-selected p190-B RhoGAP mutant (⫺/⫺) cells and a pool of mutant cells stably transfected with an
expression vector expressing activated CREB (CREBFY), indicating that the reduced cell size of mutant cells can be rescued by activated
CREB.
(B) Immunoblot of protein lysates prepared from p190-B RhoGAP mutant 3T3 cells (left lane) and individual clonally derived cell lines of p190-
B RhoGAP mutant cells stably expressing transfected CREBFY (FLAG epitope-tagged; anti-FLAG blot).
(C) Relative cell size of G1-selected wild-type cells (⫹/⫹), p190-B RhoGAP mutant cells (⫺/⫺), and mutant clonal cell lines stably expressing
CREBFY (FY1, FY2, and FY3), indicating the rescue of reduced cell size by CREBFY.
(D and F) FACS gating of G1-selected (D) and G2-selected (F) populations of wild-type 3T3 cells.
(E and G) Forward light scatter profile of wild-type, mutant, and CREBFY-rescued mutant 3T3 cells in both G1 (E) and G2 (G). Note that
CREBFY restores cell size in both G1 and G2 populations.

known biochemical action of p190-B RhoGAP as a dow-
nmodulator of Rho. As would be expected, asynchro-
nously growing mutant cells were found to contain ab-
normally high levels of activated, GTP-bound Rho.
Moreover, these levels are maintained in the CREB “res-
cue” lines, consistent with a role for CREB downstream
of Rho (Figure 4A). To verify that levels of biologically
active CREB are in fact reduced in cells lacking p190-
B RhoGAP, we used a CRE-luciferase reporter to deter-
mine that in mutant cells, CRE reporter activity is re-
duced by 6-fold relative to wild-type cells (Figure 4B).
Moreover, introduction of constitutively activated Rho
(RhoV14) into wild-type cells results in a similar degree of
decreased CRE reporter activity (Figure 4B). Analogous
Developmental Cell
6

Figure 4. Rho Antagonizes CREB-Mediated Transcriptional Activity

(A) Anti-Rho immunoblot of total cell lysates (lys.) and GST-Rhotekin “pull-downs” (GTP-Rho) from wild-type (⫹/⫹), p190-B RhoGAP mutant
(⫺/⫺), and CREBFY-transfected mutant cells, indicating that levels of activated Rho are substantially increased in the mutant cells and that
the proportion of Rho in the active, GTP-bound state is similarly increased in the CREBFY-transfected cells.
(B) CRE-mediated transcriptional activity (CRE-luciferase reporter plasmid) in transiently transfected wild-type (⫹/⫹) and p190-B RhoGAP
mutant (⫺/⫺) 3T3 cells, and in mutant cells cotransfected with activated Rho (RhoV14). The results indicate that the transcriptional activity
of CREB is reduced by approximately 6-fold in mutant cells and is similarly reduced in wild-type cells transfected with RhoV14.
(C) GAL4-CREB activation of a UAS-luciferase reporter confirming the requirement for ser133 phosphorylation and demonstrating the specific
inhibitory effect of Rho on wild-type GAL4-CREB.
(D) Immunoblot of cytochrome c in wild-type, mutant, and two CREBFY-rescued mutant 3T3 lines, indicating reduced expression in mutant
cells that is restored in the CREBFY lines. Normalization is with an anti-actin blot.
(E) Immunoblot of cytochrome c in wild-type and mutant brain (Br.), stomach (St.), and heart (Hrt.), indicating reduced expression in mutant
tissues (from E18.5). Normalization is with an anti-actin blot.
Rho and CREB Regulate Embryonic Cell Size
7
experiments with an E2F-luciferase reporter as a control
revealed similar levels of transcriptional activity in wild-
type and mutant cells (data not shown). To address
specifically the ability of Rho to regulate CREB transcrip-
tional activity via phosphorylation of ser133, we used a
GAL4-CREB reporter assay. In those experiments, Rho
antagonizes transcriptional activity of wild-type GAL4-
CREB, but does not affect the residual activity of GAL4-
CREB containing a ser133 substitution (Figure 4C). We
also determined that expression of one of the well-docu-
mented CREB-regulated genes, cytochrome c (Herzig
et al., 2000), is reduced in mutant 3T3s and embryonic
tissues, and is restored in mutant 3T3s that express
activated CREB (Figures 4D and 4E). Together, these
observations indicate that at least some of the conse-
quences of hyperactivation of Rho in cells lacking p190-
B RhoGAP are likely to be mediated through specific
inhibitory effects on CREB-dependent transcriptional
activity.
These results implicate both Rho and CREB in cell
size regulation. However, in the context of the p190-B
RhoGAP mutant cells, it is difficult to make this conclu-
sion definitively because it requires the assumption that
increased Rho activity is the only significant conse-
quence of losing p190-B RhoGAP. Therefore, to analyze
more directly the role of Rho and CREB in cell size
regulation, we examined transiently transfected Rat-1
fibroblasts. We observed by FACS analysis that cells
transfected with RhoV14 are significantly smaller than
nontransfected cells, while transfection with CREBFY
results in cells that are abnormally large (Figure 5A). In
addition, transfection of wild-type cells with a dominant-
negative form of CREB (Walton et al., 1992) gives rise
to cells that are reduced in size (Figure 5A). As before,
these effects appear to be independent of potential ef-
fects on cell cycle because the analysis was restricted
to cells in G1. These results, which suggest that Rho
activation inhibits cells growth and that CREB activation
promotes cell growth, are consistent with the observa-
tion that cell size and CREB phosphorylation are both
reduced in mice lacking p190-B RhoGAP.
Rho Regulates Insulin Signaling to CREB via
an Inhibitory Effect of Rho-Kinase on IRS
Previous studies in both vertebrate and invertebrate or-
ganisms revealed that cell size and animal size are regu-
lated by components of the insulin/IGF signaling path-
way (Stocker and Hafen, 2000). Significantly, we found
that wild-type fibroblasts undergo a substantial induc-
tion of CREB phosphorylation following exposure to
IGF-1, and that this response is diminished in cells lack-
ing p190-B RhoGAP (Figure 5B). Similar results were ob-
served with insulin (data not shown). We then determined
that expression of RhoV14 (Sullivan and Finkel, 2000) in
wild-type cells is sufficient to reduce levels of IGF-1-
induced phospho-CREB (Figure 5B). Significantly, levels
of IGF-1-induced phospho-ERK and phospho-MEK1 are
not reduced in mutant cells, indicating that the observed
effect is specific (data not shown). In addition, we found
that introduction of the activated Rac GTPase into wild-
type cells does not inhibit insulin-induced CREB phos-
phorylation, and in fact, causes an increase in CREB
phosphorylation (data not shown). Together, these re-
sults indicate that insulin/IGF signaling promotes CREB
activation, and that this pathway can be specifically
downmodulated by increased levels of Rho GTPase ac-
tivity.
One of the major downstream targets of insulin/IGF
receptor activation is the insulin receptor substrate IRS,
which plays a role in cell size regulation in Drosophila
(Bohni et al., 1999). Interestingly, one of the major Rho
targets, Rho-kinase (ROK; Leung et al., 1995), can re-
portedly antagonize insulin signaling through an associ-
ation with the major insulin receptor substrate IRS (Be-
gum et al., 2001; Farah et al., 1998) and an inhibitory
phosphorylation (Paz et al., 1997). Therefore, we hypoth-
esized that increased ROK activity in mutant cells could
account for the observed defects. Indeed, we found that
when mutant cells are exposed to a specific pharma-
cological inhibitor of ROK (Y-27632), levels of IGF-1-
induced CREB phosphorylation are restored to levels
seen in wild-type cells (Figure 5C). In addition, when
mutant cells are exposed to Y-27632, cell size is in-
creased nearly to wild-type size (Figure 5D). Thus, ROK
inhibition can “rescue” defects in cell size and IGF-1
responsiveness in mutant cells. In addition, exposure
of Rat-1 cells to Y-27632, in the absence of other treat-
ments, is sufficient to promote increased cell size (data
not shown). This suggests that ROK is an important
Rho target in regulating cell size, and that the observed
growth inhibition seen upon Rho activation is not simply
a consequence of cell toxicity caused by active Rho,
but rather that this reflects a physiological consequence
of modulating Rho activity.
To further establish the role of ROK and IRS in a cell
size pathway, we examined the IRS status in primary
MEFs lacking p190-B RhoGAP. After IGF-1 treatment,
levels of tyrosine phosphorylation of the IGF-1 receptor
are normal, as expected; however, in mutant cells, there
is a significant decrease in tyrosine phosphorylation of
IRS-1, suggesting that IRS-1 does not interact as effi-
ciently with the activated IGF receptor in these cells (Fig-
ure 5E). Next, we tested the hypothesis that this reduction
is associated with an increase in the “inhibitory” phosphor-
ylation of IRS-1 (ser612) in mutant cells. This was found
to be the case (Figure 5F), and additionally, we found
that treatment of mutant cells with Y-27632 “rescues” the
excessive levels of serine phosphorylation of IRS-1, con-
sistent with the postulated role for ROK in antagonizing
insulin/IGF signaling through an inhibitory IRS phosphory-
lation in mutant cells (Figure 5F).
Reduced MAP Kinase Signaling Links Rho-Mediated
Inhibition of Insulin Signaling to CREB
We next tested the possibility that known downstream
components of insulin/IGF signaling that could poten-
tially provide a link to CREB are affected in cells lacking
p190-B RhoGAP. For these studies, we compared pri-
mary MEFs and embryonic tissues from wild-type and
mutant animals. In MEFs, cell size is also substantially
reduced in the mutants (data not shown). In these pri-
mary cell cultures, we confirmed the defect in CREB
activation in response to IGF-1 in mutants, and addition-
ally, we found that phosphorylation of several down-
stream pathway components, including two kinases that
can promote CREB activation (JNK, not shown and p38;
Developmental Cell
8

Figure 5. A Rho-ROK (Rho-Kinase) Pathway Modulates Cell Size and IGF-Induced CREB Phosphorylation
(A) Relative cell size of G1-selected, GFP-positive Rat-1 fibroblasts transiently transfected with either RhoV14, CREBFY, or CREB-DN, indicating
that expression of activated Rho or dominant-negative CREB results in reduced cell size, and expression of activated CREB promotes
increased cell size.
(B) IGF-1-induced phospho-CREB (after 15 min) as revealed by immunoblotting with a phospho-CREB antibody (upper panel) of protein lysates
prepared from wild-type (⫹/⫹) and p190-B RhoGAP mutant (⫺/⫺) 3T3 cells, and wild-type cells infected with an adenovirus that expresses
RhoV14 (48 hr before IGF-1 treatment). RhoV14 expression blocks approximately 50% of the IGF-induced CREB phosphorylation seen in
wild-type cells. All non-RhoV14-expressing cells were infected with a GFP-expressing adenovirus as a negative control. Middle panel represents
an immunoblot of the same lysates probed with anti-CREB antibody as a normalization control. Lower panel represents an immunoblot of
Rho protein, indicating the size-shifted band corresponding to RhoV14 in the right-most lane.
(C) IGF-induced CREB phosphorylation (after 15 min) similar to that shown in (B), and rescue of IGF-induced CREB phosphorylation in mutant
cells by pretreatment with the Rho-kinase inhibitor Y-27632.
(D) Relative cell size (by forward light scatter) of G1-selected wild-type (⫹/⫹) and p190-B RhoGAP mutant (⫺/⫺) 3T3 cells, and mutant cells
treated for 48 hr with Y-27632, indicating the partial rescue of cell size in mutant cells by inhibition of Rho-kinase. Values in (A) and (D) reflect
the average of three independent experiments, and error bars represent standard error of the mean.
(E) Immunoprecipitation of IGF-1 receptor and IRS-1 from wild-type and mutant 3T3 cells before and after 15 min of IGF-1 treatment, followed
by immunoblot with anti-phosphotyrosine antibodies. Samples were also immunoblotted for total IGF-1 receptor and IRS-1 for normalization.
(F) Immunoprecipitation of IRS-1 from wild-type and mutant 3T3 cells before and after 15 min of IGF-1 treatment, followed by immunoblot with
phospho-IRS-1 (phospho-ser612) antibodies. Y-27632 was included where indicated. Immunoblot of total IRS-1 is included for normalization.

Shaywitz and Greenberg, 1999), as well as Akt, is sub-
stantially reduced in mutant MEFs (Figure 6A). More-
over, expression of RhoV14 in wild-type MEFs is suffi-
cient to inhibit IGF-1-induced phosphorylation of these
proteins, and treatment of mutant MEFs with Y-27632
can restore normal levels IGF-1-induced phosphoryla-
tion of these kinases (Figure 6A). Together, these results
are consistent with a model wherein increased ROK
activity in mutant cells downmodulates insulin/IGF sig-
naling to several downstream pathway components, in-
cluding MAP kinases that can promote CREB activity
(Figure 7A). Consistent with this model, we find that
levels of phosphorylated p38, JNK, and Akt are substan-
tially reduced in embryonic tissues (Figures 6B and 6C),
Rho and CREB Regulate Embryonic Cell Size
9

Figure 6. Downstream Components of the Insulin/IGF Signaling Pathway Are Affected in Mutant Primary MEFs and Tissues
(A) Immunoblots with the indicated antibodies of lysates from wild-type and mutant MEFs upon treatment with IGF-1 (15 min), Y-27632, and/
or adenovirus RhoV14 infection, as indicated.
(B) Immunoblots with the indicated antibodies of tissue lysates from wild-type and mutant embryos (E18.5).
(C) Immunoblot of phospho-Akt in the indicated wild-type and mutant tissues (E18.5).

and, as would be expected, there is a significant in-
crease in ser612 phosphorylation of IRS (data not
shown). The expected reduction in levels of active Rac
GTPase in mutant cells was also confirmed (data not
shown).
Discussion
Taken together, our observations indicate that activity
of the Rho GTPase and the CREB transcription factor
are important determinates of cell and animal size during
embryonic development, thereby defining a novel bio-
logical function for both of these widely expressed regu-
latory proteins. In fibroblasts derived from mice lacking
p190-B RhoGAP, it appears that Rho is performing a
largely cell-autonomous role that can influence respon-
siveness to insulin/IGFs through activation of the Rho
target ROK. However, the fact that mutant fibroblasts
are not completely defective in insulin/IGF-1 respon-
siveness suggests that at least some aspect of insulin-
promoted growth is Rho insensitive. Moreover, the ob-
servation that mice lacking p190-B RhoGAP appear to
be uniformly reduced in size, while phospho-CREB lev-
els are not substantially reduced in every tissue, raises
the possibility that there is additional complexity in-
volved in determining organism size.
It is possible, for example, that modulation of CREB
is a more significant determinant of cell size at earlier
stages of development in some tissues (we focused on
E18.5). In addition, there may be both cell-autonomous
and -nonautonomous mechanisms involved in vivo. In-
deed, PI-3 kinase, which appears to play a role in the
pathway proposed here, has been previously implicated
in cell size regulation, and has been reported to have
both cell-autonomous and -nonautonomous functions
in C. elegans (Hsin and Kenyon, 1999). In addition, while
IRS plays a well-documented cell-autonomous role in
regulating cell size in Drosophila (Bohni et al., 1999),
studies in mice reveal an additional cell-nonautonomous
role in controlling animal size (Withers, 2001). It is also
worth noting that both insulin and IGF-1 gene expression
have been found to be CREB responsive (Philippe and
Missotten, 1990; Thomas et al., 1996), raising the possi-
bility that impaired CREB activity can also contribute in
Developmental Cell
10
a cell-nonautonomous manner to growth regulation by
affecting levels of secreted and circulating insulin/IGF.
Such findings point to a complex regulatory system for
the control of cell, organ, and animal size that involves
both cell-autonomous and -nonautonomous mecha-
nisms that may require an overlapping set of proteins.
In fact, there may be additional compensatory mecha-
nisms that serve to uniformly adjust the size of organs
to accommodate changes that may be limited to a sub-
set of organs in the mutant animals.
Rho GTPase signaling has not previously been directly
implicated in cell size regulation. In fact, the vast majority
of biological functions associated with the Rho GTPase
in vivo appear to reflect the ability of Rho proteins to
regulate the actin cytoskeleton (Settleman, 2000). How-
ever, there is indirect evidence to suggest a potential
role for Rho in cell size regulation. The human tumor
suppressor TSC1 encodes a protein that reportedly pro-
motes Rho activation (Lamb et al., 2000) and which,
when disrupted in Drosophila, results in tissue out-
growth due to a substantial increase in cell size (Tapon
et al., 2001). Significantly, overexpression of TSC1, like
Rho activation, reportedly causes reduced cell growth
by inhibiting insulin signaling (Gao and Pan, 2001; Potter
et al., 2001). In addition, the PKA kinase, which, when
disrupted in Drosophila, results in a small fly phenotype
(Lane and Kalderon, 1993), can directly phosphorylate
Rho, resulting in Rho inactivation (Dong et al., 1998).
Both of these results are consistent with the role of Rho
in cell size regulation that has been proposed here, and
significantly, this role for Rho appears to be independent
of its ability to regulate the cytoskeleton.
Previous studies to address the in vivo function of
CREB have highlighted its role in cellular differentiation
and protection form apoptosis (survival), as well as in
learning and memory (Mayr and Montminy, 2001). Con-
sistent with this, we also see evidence of differentiation
defects in brain and thymus of mice lacking p190-B
RhoGAP (S.F.M., R.S., and J.S., unpublished observa-
tions). However, we do not see evidence of any signifi-
cant increase in the level of apoptosis in our mutant
animals, despite extensive analysis of embryos at sev-
eral developmental stages. Thus, it is possible that a
Figure 7. Model Depicting the Proposed
Role of p190-B RhoGAP in Modulating Insu-
lin/IGF-1 Signaling to CREB
CREB-related protein provides some redundant func-
tion for cell survival in this setting, or that there is a
phosphorylation-independent function for CREB in pro-
moting survival. Interestingly, it has been reported that
while mutational loss of one of the CREB homologs in
Drosophila, dCREB2, results in a lethal phenotype, the
few “escapers” that survive to adulthood are substan-
tially reduced in size (Belvin et al., 1999). This suggests
that a role for CREB in determining animal size has been
evolutionarily conserved. It is also notable that CREB is
a direct phosphorylation target of PKA (Colbran et al.,
1992), which, as described above, regulates animal size
in Drosophila. Possibly, crosstalk between Rho- and
CREB-mediated pathways occurs at multiple levels.
The vast majority of studies of cell size regulation in
both vertebrates and invertebrates have focused on the
insulin/IGF cell size pathway, and accumulating evi-
dence suggests that an important “endpoint” of this
pathway is the upregulation of components of the pro-
tein translational machinery (Stocker and Hafen, 2000).
However, there is also evidence that transcriptional reg-
ulation plays a role in regulating cell size, and it appears
that one of the relevant transcription factors is Myc,
whose expression can promote cell growth in a cell
cycle-independent manner (Iritani and Eisenman, 1999).
Our results indicate that CREB also plays a significant
role in regulating cell growth, presumably through its
ability to regulate transcriptionally the expression of one
or more target genes. Thus far, the relevant transcrip-
tional target(s) have not been identified, although, as
discussed, insulin and IGF-1 may be important CREB
targets in this context. Significantly, many of the identi-
fied CREB targets are regulators of metabolism (Mayr
and Montminy, 2001), raising the possibility that CREB-
induced cell growth is due to increased expression of
several essential metabolic enzymes. Thus, these col-
lective results suggest a model wherein cellular growth
requires increased protein synthesis together with in-
creased metabolism. Significantly, CREBFY-rescued
mutant cells are resistant to the ability of the p70 S6
kinase inhibitor rapamycin to cause cell size reduction,
suggesting that CREB may be additionally required
Rho and CREB Regulate Embryonic Cell Size
11
downstream of the translational machinery for cell size
regulation (M.C. and J.S., unpublished observations).
Rho and CREB have also both been previously impli-
cated in the differentiation of thymocytes and neurons
(Henning et al., 1997; Luo, 2000), and, as described
above, we found that the brain and thymus defects in
p190-B RhoGAP-deficient mice are associated with a
failure in cell differentiation, as revealed by immuno-
staining with specific markers of differentiation. Signifi-
cantly, in CREB-deficient mice, the small thymus is simi-
larly associated with an excess of undifferentiated cells
(Rudolph et al., 1998). Thus, our results are consistent
with a role for Rho in regulating CREB function both for
differentiation as well as for determining cell size and
animal size. It is therefore interesting to note that in
a cell culture model of neuronal differentiation (serum
withdrawal from neuro-2a neuroblastoma cells), we ob-
serve, by FACS analysis, a substantial increase in cell
size during differentiation (M.C. and J.S., unpublished
observations). Such cell growth is particularly notable
when considering that it takes place in the absence
of any serum-containing factors. In similar cell culture
models of neuronal differentiation, CREB has been
found to play an important role in promoting differentia-
tion, and it will certainly be of interest in the future to
determine whether CREB utilizes a distinct or overlap-
ping set of transcriptional targets for regulating cell size
and differentiation.
Experimental Procedures
Generation of p190-B RhoGAP Mutant Mice
To construct the p190-B targeting vector, a PGK-neo cassette was
inserted in reverse transcriptional orientation relative to the p190-
B coding sequence in a BamHI site (bp 1196) of a 4.5 kb p190-B
SV129 5⬘ genomic clone, disrupting the gene immediately upstream
of the GTPase domain (Burbelo et al., 1998). The complete insert
of the resulting plasmid was subcloned as a KpnI-NotI fragment
into a PGK-neo-free pPNT vector (Tybulewicz et al., 1991), resulting
in the addition of the PGK-tk gene at the 5⬘ end of the p190-B
genomic DNA. NotI-linearized targeting vector (25 ␮g/ 8 ⫻ 106 cells)
was electroporated (400 V, 25 ␮F) into D3 ES cells of the mouse
SV129 strain. ES cells were maintained on a feeder layer of neor
embryonic fibroblasts in DMEM supplemented with 15% fetal bovine
serum (FBS) in the presence of 500 U/ml LIF (GIBCO). Transfected
ES cells were selected for growth in G418 at 300 ␮g/ml for 10
days and gancyclovir at 2 mM the first 5 days (initiated 36 hr after
electroporation). Single colonies were expanded for DNA isolation
and Southern blot analysis was performed using standard methods,
to identify correctly targeted p190-B heterozygous ES clones (14
out of 130 screened). A 500 bp HindIII fragment (bp 1088–1586) was
used to distinguish the mutated allele from the wild-type allele based
on an altered StuI restriction pattern after homologous recombina-
tion. 5⬘ and 3⬘ external probes were used to confirm the absence
of rearrangements 5⬘ and 3⬘ of the targeted region. ES cells from
three p190-B heterozygous clones were injected into the blastocoel
cavity of 3.5-day-old C57BL/6J blastocysts, which were then trans-
planted into the uteri of pseudopregnant females to generate chime-
ric mice. Germline transmission was determined by agouti color
offspring of chimeric males mated with C57BL/6J females and con-
firmed by Southern blot analysis of StuI-digested tail DNA.
Genotyping
Genotype analysis was performed by PCR using a three-primer reac-
tion containing primer 1, complementary to p190-B sequences adja-
cent to the PGK-neo gene insertion site (bp 1185–1202; 5⬘-dTAATG
ATAGGCGGATCCC-3⬘), primer 2, complementary to p190-B coding
sequence 3⬘ of the region of gene disruption (bp 1559–1577; 5⬘-dGGT
TCTTCACTTAGAACGG-3⬘), and primer 3, complementary to se-
quences at the 5⬘ end of the PGK-neo promoter (5⬘-dCGGTGGATGT
GGAATGTG-3⬘). Primers 1 and 2 were used to identify a 392 bp
fragment of the wild-type allele, whereas primers 2 and 3 were used
to amplify a 521 bp fragment of the mutated allele. PCR products
were generated after a 5 min hot start at 94⬚C by 30 cycles at 94⬚C
(1 min), 60⬚C (1 min), and 72⬚C (1 min) using Taq polymerase in the
presence of 1.25 mM MgCl2.
Immunodetection of Proteins
To detect p190 RhoGAP proteins, lysates from embryo-derived cells
were prepared in ice-cold lysis buffer (50 mM HEPES [pH 7.4], 150
mM NaCl, 1.5 mM MgCl2, 5 mM EGTA, 10% glycerol, 1% Triton
X-100) containing protease inhibitors. Debris was removed by cen-
trifugation in a microfuge at 12,000 ⫻ g for 10 min at 4⬚C. Clarified
lysates were boiled in gel loading buffer and separated by 7.5%
SDS-PAGE. Proteins were electrotransferred to nitrocellulose and
detected with monoclonal antibodies directed against p190 Rho-
GAP or p190-B RhoGAP (hybridoma supernate diluted 1:5) followed
by incubation with horseradish peroxidase-conjugated secondary
goat anti-mouse antibody (Bio-Rad; 1:5000) and development with
enhanced chemiluminescence (DuPont NEN) and autoradiography.
For immunoprecipitation, cells that were treated or not treated with
IGF-1 for 15 min were extracted with RIPA buffer, clarified by centrif-
ugation, and subjected to immunoprecipitation with IGF-1 or IRS-1
antibodies and protein A agarose for 2 hr. After washing the beads
several times with RIPA buffer, samples were subjected to immu-
noblotting. For immunoblotting of CREB, JNK, p38, Akt, IRS-1, IGF-1
receptor, cytochrome c, actin, and the various phosphoproteins,
extracts were prepared from cells and tissues in RIPA buffer and
lysates were clarified by centrifugation. Fifteen micrograms of pro-
tein of each sample were resolved by 12.5% SDS-PAGE, and the
proteins were then electrotransferred to an Immobilon P membrane.
Immunoblotting was performed as described for p190-B RhoGAP.
All antibodies were obtained from New England Biolabs.
Histology
Newborn mice were sacrificed by CO2 asphyxiation and either dis-
sected immediately or fixed by immersion in Bouin’s fixative. For
Nissl staining, E18.5 wild-type and mutant forebrains were fixed by
immersion (4% paraformaldehyde) and then cryoprotected in PBS/
20% sucrose. Coronal cryostat sections (10–14 ␮M) were stained
with cresyl violet acetate (0.5% in H2O) for 10 min.
Cell Size and Cell Cycle Analysis
For determination of protein:cell number and protein:DNA ratios,
cells were counted and lysed in RIPA buffer, and DNA and protein
content were determined as previously described (Franch et al.,
1995). For FACS analysis (forward light scatter), 3T3 cells were la-
beled with 0.1 mg/ml BrdU (cell labeling reagent; Amersham) for
30 min, harvested, and analyzed according to the manufacturer’s
instructions (Becton Dickinson). Briefly, cells were fixed in 80%
ethanol, and the DNA was denatured by treatment with 2 N HCl/
0.5% Triton X-100 at room temperature for 30 min followed by a
neutralization step in 0.1 M Na2B4O7 ⫻ 10H2O (pH 8.5). For immuno-
fluorescence staining, anti-BrdU antibodies (Becton Dickinson) and
FITC-conjugated horse anti-mouse antibodies (Vector) were used.
The immunofluorescence step was followed by incubation in propid-
ium iodide and RNase prior to two-dimensional FACS analysis using
Cell Quest software (Becton Dickinson). To measure the relative cell
size of asynchronously growing cells corresponding to the wild-
type and mutant 3T3 lines, single cells with a DNA content of 2N
(the G1 population) were gated and forward scatter height (FSC-H)
histograms were plotted. The mean FSC-H was calculated using
Cell Quest software. The analysis was performed with similar results
on three independently derived pairs of wild-type and mutant 3T3
cell lines.
For FACS analysis of transiently transfected Rat-1 cells, cells (60
hr posttransfection) were fixed in 1% formaldehyde in PBS for 30
min, and permeabilized with 0.1% Triton X-100 in PBS followed by
propidium iodide staining in the presence of RNase A, as described
above. The forward scatter height of the GFP-positive transfected
cells with a 2N DNA content was analyzed as described above.
Developmental Cell
12
Fibroblast Cell Culture Studies
To prepare embryo-derived fibroblasts, E13.5 embryos were ob-
tained from timed matings of p190-B RhoGAP heterozygotes. DNA
was extracted from tail tissue for genotype analysis. After removal
of the head and internal organs, embryos were washed in PBS under
sterile conditions, followed by trituration and 30 min trypsinization
(using 5⫻ trypsin) at 37⬚C. Trypsin was inactivated by reconstituting
the cells in DMEM containing 15% fetal calf serum (FCS) followed
by centrifugation at 1500 rpm for 5 min at room temperature. Mouse
embryonic fibroblasts (MEFs) were then resuspended in DMEM (plus
15% fetal calf serum) and plated at high density (cells derived from
one embryo in one 10 cm dish). 3T3 cell lines were established using
standard procedures (Todaro and Green, 1963). Briefly, cells were
trypsinized, counted, and reseeded in DMEM supplemented with
10% calf serum at a density of 106 cells/10 cm dish every 3 days
with a media change the subsequent day. After 30 to 50 passages,
stable lines of immortalized cells were established. Saturation den-
sity was determined by plating 104 wild-type or mutant cells in a 60
mm dish and then counting in triplicate the number of cells per dish
every day for 6 consecutive days following replating. 3T3 and Rat-1
fibroblast lines were maintained in DMEM containing 10% calf serum
or 10% fetal bovine serum, respectively, and antibiotics.
To generate “CREB rescue” 3T3 cells, homozygous mutant 3T3
cells were cotransfected (Fugene; Boehringer Mannheim) with plas-
mids encoding constitutively active CREB (CREBFY) and the puro-
mycin resistance protein (pBabe-puro), then selected with puromy-
cin (Sigma; 2 ␮g/ml) for 2 weeks, and pooled. The mean FSC-H of
the G1 population was compared to that of the homozygous mutant
parental 3T3 cells. Cells were plated at 1 ⫻ 106 cells/10 cm dish
and the G1 and G2 populations of cells were analyzed for forward
light scatter FACS analysis as described above. In addition, three
individual clonal lines of stably transfected CREBFY-expressing
cells were established, and their relative cell sizes were also deter-
mined by forward light scatter FACS analysis.
For experiments with Rat-1 cells, 106 asynchronously growing
cells were plated per 10 cm dish in DMEM containing 10% FBS the
day before transfection. The next day, the cells were transfected
with 10 ␮g of DNA (5 ␮g vector control, CMV-RhoV14, CMV-CREBDN
[dominant-negative], or CMV-CREBFY [activated], together with 5
␮g CMV-GFP vector) and 10 ␮l of Lipofectamine 2000 transfection
reagent (Life Technologies). Twelve hours posttransfection, the me-
dium was replaced with serum-free DMEM. After an additional 12
hr, the medium was replaced with DMEM containing 2.5% FBS and
2 ␮g/ml aphidicolin (to enrich for a G1 population of cells; Urbani
et al., 1995). Thirty-six hours later, the cell size of the G1 cell popula-
tion was determined by FACS as described above.
For experiments with IGF-1 treatment, 106 asynchronously grow-
ing 3T3 cells were plated in 10 cm dishes in DMEM with 10% calf
serum. After 3 days, the cells reached confluency and the medium
was replaced with serum-free DMEM. After an additional 24 hr, cells
were incubated with medium containing DMEM alone or DMEM and
80 ng/ml of IGF-1 (Life Technologies). After 15 min, cells were lysed
in RIPA buffer and 15 ␮g of protein was analyzed by immunoblotting
for the indicated proteins as described above. Adenoviral infections
with virus expressing both RhoV14 (myc epitope-tagged) and GFP
or GFP alone (negative control) were performed 48 hr before IGF-1
treatment as previously described (Sullivan and Finkel, 2000). For
treatments with the Y-27632 compound (Welfide), the drug (10 ␮M)
was added to culture media 15 min prior to IGF-1 treatment in
phospho-CREB/p38/Akt experiments, and for cell size studies, it
was added once per day on 2 consecutive days in the presence of
2.5% FBS and 2 ␮g/ml aphidicolin prior to collecting G1 cells for
forward light scatter FACS analysis as described above.
Measuring Rho Activation
Levels of activated, GTP-bound RhoA were determined using the
Rhotekin “pull-down” assay as previously described (Ren and
Schwartz, 2000). Briefly, asynchronously growing wild-type and mu-
tant cells were collected, lysed, and following clarification, lysates
were incubated with GST-Rhotekin on beads to capture GTP-bound
Rho proteins. Bound proteins were washed several times and eluted
with sample buffer before being subjected to SDS-PAGE and immu-
noblotting with an anti-RhoA antibody (Santa Cruz). Whole-cell ly-
sates (prior to incubation with GST-Rhotekin) were analyzed in paral-
lel. The amount of whole-cell lysate loaded in each case represents
approximately 5% of the input material in binding assays.
Reporter Assays
Reporter assays were performed following transfection (Fugene;
Boehringer Mannheim) of 3T3 cells with the CRE-luciferase reporter
plasmid (Stratagene; 100 ng) or the UAS-luciferase reporter plasmid
(Stratagene; 250 ng). Cotransfections were with plasmids express-
ing either RhoV14 (100 ng), wild-type GAL4-CREB, or GAL4-CREB
containing an ala133 substitution (100 ng). GAL4-CREB fusions con-
tain the entire CREB coding sequence. Twenty hours after transfec-
tion, the cells were switched to media containing 1% serum and 40
hr later luciferase assays were performed (according to the manu-
facturer’s instructions; Promega). RSV-LacZ (200 ng) was used as
an internal control for transfection efficiency. CRE-luciferase activ-
ity, as indicated in graph form, represents activity normalized to
␤-galactosidase activity from the same extracts. Results were quan-
tified using a luminometer from EG&G Berthold for luciferase and
an ELISA plate reader for ␤-galactosidase.
Acknowledgments
We thank Drs. Daniel Haber, Iswar Hariharan, and Sofie Salama for
helpful comments on the manuscript. We appreciate the generous
assistance of Dr. Nadia Carlesso with GFP FACS analysis, Dr. Fred
Preffer with interpretation of FACS data, Dr. En Li of the MGH mouse
transgenic/knockout facility with the production of mutant mice, Dr.
Thaddeus Stappenbeck with the analysis of lung tissue, and Ms.
Tracy Huang with assistance with immunoblotting. We are grateful
to members of the Settleman laboratory for advice throughout the
course of these studies. The CREB expression plasmids were kindly
provided by Dr. Marc Montminy (Salk Institute), Dr. Linda Kobierski
(MGH), and Drs. Jon Kornhauser and Michael Greenberg (Harvard
Medical School), and the RhoV14 adenovirus was a generous gift
of Drs. Enzo Calautti and Paolo Dotto (MGH). These studies were
supported by NIH and ACS awards to J.S.
Received: February 15, 2002
Revised: March 19, 2002
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