MOLECULAR AND CELLULAR BIOLOGY, July 2004, p. 6205–6214
Vol. 24, No. 14
Rac1 Inhibits Apoptosis in Human Lymphoma Cells by Stimulating
Bad Phosphorylation on Ser-75
Baolin Zhang,* Yaqin Zhang, and Emily Shacter
Laboratory of Biochemistry, Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug
Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892
Received 26 November 2003/Returned for modification 8 January 2004/Accepted 23 April 2004
The small GTPase Rac1 has emerged as an important regulator of cell survival and apoptosis, but the
mechanisms involved are not completely understood. In this report, constitutively active Rac1 is shown to
stimulate the phosphorylation of the Bcl-2 family member Bad, thereby suppressing drug-induced caspase
activation and apoptosis in human lymphoma cells. Rac1 activation leads to human Bad phosphorylation
specifically at serine-75 (corresponding to murine serine-112) both in vivo and in vitro. Inhibition of consti-
tutive and activated Rac1-induced Bad phosphorylation by a cell-permeable competitive peptide inhibitor
representing this Bad phosphorylation site sensitizes lymphoma cells to drug-induced apoptosis. The data
show further that endogenous protein kinase A is a primary catalyst of cellular Bad phosphorylation in
response to Rac activation, while Akt is not involved. These findings define a mechanism by which active Rac1
promotes lymphoma cell survival and inhibits apoptosis in response to cancer chemotherapy drugs.
Rac GTPases, members of the Rho family of small GTP-
binding proteins, play an important role in transducing signals
generated on the surface of a cell to the nucleus. Upon stim-
ulation by growth factors, Rac becomes activated and induces
a variety of functional responses, including endogenous super-
oxide generation, reorganization of the actin cytoskeleton,
gene transcription, cell cycle progression, and malignant trans-
formation (6, 17, 35). It is also involved in controlling apoptosis
(3, 7); expression of active Rac1 provides a survival signal to
protect tumor cell lines or transformed fibroblasts from apo-
ptosis (13, 24, 34, 36). Our recent data demonstrate that inac-
tivation of Rac1 by caspase-mediated cleavage promotes apo-
ptosis in human lymphoma cells, suggesting that native Rac1
activity interferes with the apoptotic process and needs to be
diminished in order to maximize cell killing by chemotherapy
drugs (46). However, the precise mechanism by which the
Rac1 signaling pathway transduces a survival signal that inhib-
its apoptosis needs to be established. In most human tumors,
Rac activity is upregulated either by overexpression of the
protein, by mutations that render the protein resistant to nor-
mal regulatory systems (e.g., isoform Rac1b), or by alterations
in associated regulator proteins (28, 37, 38). In addition, Rac is
a downstream effector of the oncogenic Ras protein, which is
known to participate in carcinogenesis in many human cancers
(4, 15). Therefore, understanding the molecular basis of Rac-
modulated cell survival and apoptosis might lead to strategies
to improve anticancer therapy.
Induction of apoptosis by anticancer drugs involves disrup-
tion of mitochondria and the subsequent activation of the
caspase protease cascade (25). This process is tightly regulated
by Bcl-2 family proteins (41). Bad is a proapoptotic member of
this family that has been implicated in coordinating the survival
signals and the mitochondrial cell death machinery (5, 14, 44).
Bad is normally phosphorylated and sequestered by binding to
14-3-3 in living cells. Apoptotic signals cause Bad dephosphor-
ylation and release from 14-3-3. In the unphosphorylated state,
Bad binds to Bcl-2 and Bcl-XLand inactivates their prosurvival
function, resulting in mitochondrial dysfunction, cytochrome c
release, caspase activation, and apoptotic death. The phos-
phorylation of endogenous Bad occurs under conditions in
which growth factors are present to promote cell survival, sug-
gesting that Bad phosphorylation is essential in order for sur-
vival factors to block apoptosis (10–12). Accordingly, trans-
genic mice carrying Bad mutants that lack its phosphorylation
sites exhibit defects in growth factor-dependent survival (11).
Thus, the reversible phosphorylation of Bad represents a crit-
ical sensor for survival signaling and a determinant for the
outcome of apoptotic stimuli.
A number of different Ser/Thr protein kinases are known to
phosphorylate Bad at different Ser residues in the protein. The
most-studied phosphorylation sites occur at serine residues
112, 136, and 155 in the mouse protein (10, 11, 20, 27). These
correspond to Ser-75, -99, and -118 in the human protein (16).
Cyclic AMP (cAMP)-dependent protein kinase (PKA) report-
edly phosphorylates Bad at murine Ser-112 and -155, while
phosphatidylinositol 3 (PI 3)-kinase/Akt (PKB) is reported to
phosphorylate murine Bad at Ser-136 (9, 33). The most re-
cently identified Bad kinases are p-21-activated kinases
(PAKs). While PAK1 and PAK2 were shown to phosphorylate
murine Bad on both serines 112 and 136 (22, 39), PAK4 and
PAK5 only phosphorylate murine Bad on Ser-112 (8, 19).
While informative, one limitation of much of this work has
been that it involves transfection and overexpression of Bad
and the individual kinases, often to massive levels, which pre-
cludes the understanding of how the activity of Bad is regu-
lated in the cellular context by specific signal transduction
molecules that are activated by survival signals.
We hypothesized that the Rac pathway may suppress cell
death and promote cell survival by causing Bad phosphoryla-
* Corresponding author. Mailing address: Laboratory of Biochem-
istry, FDA/CDER, 29 Lincoln Dr., Bldg. 29A, Rm. 2B-24, Bethesda,
MD 20892-4555. Phone: (301) 827-1784. Fax: (301) 480-3256. E-mail:
tion. In this report, we show that functional Rac GTPase is
required for normal Bad phosphorylation in healthy, growing
human lymphoma cells. Increased Rac1 signaling stimulates
Bad phosphorylation at human Ser-75 in vitro and in vivo.
When this phosphorylation is selectively inhibited, increased
apoptosis occurs in response to chemotherapy drugs. Thus,
Rac1 blocks drug-induced apoptosis at least in part by main-
taining Bad in the phosphorylated state. Evidence is also pro-
vided to show that PKA phosphorylates Bad in response to
activated Rac1, whereas Akt does not.
MATERIALS AND METHODS
Plasmids and transfections. The pcDNA3.1(?)-hemagglutinin (HA)-Rac1
constructs, encoding HA-tagged human wild-type Rac1, constitutively active
mutant Rac1V12, dominant-negative mutant Rac1N17, and caspase-3-resistant
mutant Rac1D11E, were prepared as previously described (46). Transfection of
Burkitt’s lymphoma BL-41 cells was carried out by electroporation as described
previously (26). Positive clones were selected with G418 (Mediatech, Herndon,
Va.) and were tested for expression of Rac1 by anti-HA Western blotting. The
stable cell lines were maintained at 37°C and 5% CO2in RPMI 1640 medium
containing 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 50 ?M
?-mercaptoethanol, and 1.5 mg of G418/ml.
Antibodies and reagents. A mouse monoclonal anti-HA antibody was pur-
chased from Covance Research Products (Berkeley, Calif.). Mouse monoclonal
antibodies against human Bad (hBad), Bcl-2, catalytic subunit of protein kinase
A (PKAC), Akt, and phospho-Akt-Ser 472/473 were purchased from BD Bio-
sciences (Lexington, Ky.). Mouse monoclonal antibody specific for phospho-
Bad-Ser-112 (7E11) and rabbit polyclonal anti-phospho-Bad-Ser-136, and -phos-
pho-Bad-Ser-155 were obtained from Cell Signaling Technology (Beverly,
Mass.). A monoclonal antibody against poly(ADP-ribose) polymerase (PARP)
was from Oncogene Research Products (Boston, Mass.). The PKA peptide
inhibitor (TTYADFIASGRTGRRNAIHD) and purified PKACwere from Pro-
mega (Madison, Wis.). Rp-cAMPS was from Alexis Biochemicals (San Diego,
Calif.). A cell-permeable PKA inhibitor, myristoylated-GRTGRRNAI-NH2
(amide 14 to 22), and an Akt-specific inhibitor (SH-5) were obtained from
Calbiochem. VP-16 (etoposide), GDP, and GTP?S were from Sigma. Bacterially
expressed, purified recombinant (His)6-fusion proteins of full-length human
Akt1/PKB, active recombinant human PAK2, and full-length murine Bad were
from Upstate Biotechnology (Lake Placid, N.Y.). Human recombinant Rac1
proteins were expressed as (His)6-tagged fusions by the pET28a expression
system and were loaded with GDP or GTP?S as described previously (45).
A peptide matching the 9 amino acids surrounding Ser-75 of hBad (amino
acids 70 to 78) and a scrambled peptide from this sequence were custom syn-
thesized by Biopeptide Corporation (San Diego, Calif.). The peptides were made
cell permeable through coupling to the human immunodeficiency virus (HIV)
Tat protein transduction domain (21). The purity of both peptides was greater
than 95%, as determined by high-performance liquid chromatography. Peptide
sequences were confirmed by mass spectrometry.
Apoptosis assay. To induce apoptosis, cells were seeded at 5 ? 105cells/ml and
were treated with VP-16 at 200 ?g/ml. At indicated time points, cells were
collected and washed sequentially with serum-containing medium, phosphate-
buffered saline, and annexin V buffer (10 mM HEPES [pH 7.4], 140 mM NaCl,
and 2.5 mM CaCl2). These samples were then incubated at room temperature for
15 min with annexin V-fluorescein isothiocyanate (FITC) (Pharmingen) and
propidium iodide (PI) at final concentrations of 2.5 and 5.0 ?g/ml, respectively.
After being washed once with annexin V buffer, the cells were resuspended in
500 ?l of annexin V buffer and were analyzed (10,000 cells per sample) by flow
cytometry. The percentage of apoptotic cells was determined by assessing the
cells that were bound to annexin V-FITC but were negative for PI staining.
In vitro kinase assays. The Bad kinase assays were carried out in the presence
of 1 ?g of recombinant murine His-Bad protein in a kinase buffer (20 mM
Tris-HCl [pH 7.5], 300 ?M ATP, 2 mM EGTA, 50 mM MgCl2, 1 mM dithio-
threitol) containing protease inhibitors (1:50 dilution of the Protease Inhibitor
Cocktail Set III; Calbiochem) and phosphatase inhibitors (25 mM NaF, 1 mM
Na3VO4, 20 mM glycerophosphate). The reaction was initiated by the addition
of cell extracts or a purified kinase. The mixture was incubated at 30°C for 10 min
and was terminated by the addition of sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) sample buffer. Phosphorylated Bad was detected
by Western blotting using antibodies specific to murine phospho-Bad at Ser-112,
-136, or -155 and to human phospho-Bad at Ser-75, -99, and -118, respectively.
To test the effect of Rac GTPase activity on the kinase activities, recombinant
glutathione S-transferase-tagged human Rac1 that was preloaded with GDP or
GTP?S was added to the reaction mixture (45).
The kinase activity of cAMP-dependent protein kinase (PKA) was determined
by a nonradioactive PepTag assay (Cat.V5340; Promega), which utilizes a
brightly colored, fluorescent peptide substrate that is highly specific for PKA.
Phosphorylation by PKA alters the net charge of the substrate from ?1 to ?1,
thereby allowing the phosphorylated and nonphosphorylated versions of the
substrate to be separated by electrophoresis on an agarose gel. The phosphory-
lated species migrates toward the positive electrode, while the nonphosphory-
lated substrate migrates toward the negative electrode. To start the reaction,
Bl-41 whole lysates (0.2 to 2 ?g in 10 ?l) were incubated with PKA reaction
mixture (25 ?l) at room temperature for 30 min. The reactions were terminated
by placing the tubes in a 95°C heating block for 10 min. After adding 80%
glycerol (1 ?l), the samples were loaded onto an agarose gel (0.8% agarose in 50
mM Tris-HCl, pH 8.0) and were separated in the same buffer at 100 V for 15 min.
The bands were visualized under UV light. For quantitation of the PepTag
results, the phosphorylated bands were cut out and heated at 95°C until the gel
slice melted. The hot agarose solution (125 ?l) was transferred to a tube con-
taining 75 ?l of Gel Solubilization Solution (Promega) and 50 ?l of glacial acetic
acid, and the absorbance was measured at 570 nm on a plate reader (Dynatech).
Akt kinase activity was measured by Stressgen’s nonradioactive PKB (Akt)
kinase assay kit (Victoria, British Columbia, Canada), which is based on a
solid-phase enzyme-linked immunosorbent assay that utilizes an Akt-specific
peptide as a substrate and a polyclonal antibody that recognizes the phosphor-
ylated form of the substrate. Briefly, purified Akt or whole-cell extract was
incubated at 30°C for 40 min in the precoated Akt substrate plate. The reaction
was terminated by the addition of a phosphospecific substrate antibody, followed
by incubation with an anti-rabbit immunoglobulin G horseradish peroxidase-
conjugated antibody. Antibody binding was quantified colorimetrically (405 nm)
by using 2,2?-Azino-bis 3-methylbenzthiazoline-6-sulfonic acid (TMB) and a 96-
well microplate reader. The assay was validated by using purified Akt and
adjusting the amount of cell extract to fall in the linear range of the standard
Western blot analysis. Equal amounts of cell lysates (20 ?g per lane) were
resolved by electrophoresis using a 4 to 12% NuPAGE Bis-Tris gel (Invitrogen)
and were transferred to nitrocellulose membranes (Millipore) for immunoblot-
ting. When necessary, the membrane was stripped by Restore Western Blot
Stripping buffer (Pierce) and reprobed with appropriate antibodies. Immuno-
complexes were visualized by enhanced chemiluminescence or SuperSignal re-
Rac1-dependent signaling pathways stimulate phosphoryla-
tion of Bad at Ser-75. The main goal of these studies was to
determine whether Rac1 inhibits cell death, at least in part, by
modulating the phosphorylation and activity of Bad. Our work-
ing hypothesis was that Rac1 is required for cell survival and
that elimination of Rac1 activity through caspase cleavage is
required for maximal apoptosis to occur. Because Bad is anti-
apoptotic in its phosphorylated state, we focused on charac-
terizing the phosphorylation state of Bad in healthy, growing
cells. Because the cellular studies employ human cells while
the cell-free studies use purified recombinant murine Bad, the
Ser residue numbers differ between experiments; i.e., Ser-112,
-136, and -155 for murine Bad and Ser-75, -99, and -118 for
hBad. However, the sequences in both species are exactly ho-
mologous and cross-react with the respective phosphorylation
We first determined whether cells containing higher or lower
levels of Rac activity phosphorylate the different phosphoryla-
tion sites in Bad to different degrees. Stable transfectants of
several Rac1 mutants were generated in human Burkitt’s lym-
phoma BL-41 cells. The genes transfected and expressed were
the dominant-negative mutant Rac1N17, the constitutively ac-
tive mutant Rac1V12, and the caspase-3-resistant mutant
6206ZHANG ET AL.MOL. CELL. BIOL.
Rac1D11 (46). Empty vector was used for control transfec-
tions. Western blot analysis of the HA-tagged mutant proteins
suggested that roughly equal amounts of Rac1N17, Rac1V12,
and Rac1D11 protein were expressed in the respective cell
lines (Fig. 1A). To examine the phosphorylation of Bad, ex-
tracts from the transfected cells were subjected to immunoblot
analysis using antibodies specific for the known phosphoryla-
tion sites in Bad. In parental BL-41 cells growing under normal
conditions, Bad phosphorylation was only detected on Ser-75.
Significantly more Ser-75-specific phosphorylation was ob-
served in cells expressing constitutively active Rac1V12 and
caspase-resistant Rac1D11, while the opposite was true in the
cells expressing the dominant-negative Rac1N17 mutant. In
contrast, neither Ser-99 nor Ser-118 phosphorylation was de-
tected in control BL-41 cells or in cells expressing the Rac1
mutants. As will be shown below, the difference in phosphor-
ylation of the different sites on Bad did not result from a
reduced sensitivity of the antibodies for p-99 and p-118 (see
Fig. 4), and all of the cell lines expressed comparable levels of
Bad protein (Fig. 1B). These results suggested that Rac1 ac-
tivity selectively upregulates hBad Ser-75 phosphorylation in
vivo. Because Ser-75 was the only residue found to be phos-
phorylated constitutively in healthy, growing cells, it is the
focus of the remainder of the studies on the regulation of Bad
phosphorylation by Rac GTPases.
We next examined the ability of Rac1 activity to modulate
phosphorylation of recombinant murine Bad protein by cell
extracts isolated from the lymphoma cells. Purified murine Bad
becomes highly phosphorylated in a dose-dependent manner
when incubated with the endogenous cellular kinases (Fig.
1C). Saturation of Ser-112 phosphorylation was achieved with
as little as 200 ng of whole lysate protein, while no Ser-136 or
Ser-155 phosphorylation was detected under these conditions.
Phosphorylation of Bad at Ser-136 and Ser-155 required
roughly 10-fold more lysate than did Ser-112 phosphorylation.
These results indicate that human lymphoma cells exhibit an
intrinsic capability to phosphorylate Bad and that the endog-
enous kinase activity is relatively specific for Ser-112.
The effect of Rac GTPase activity on endogenous Bad phos-
phorylation was examined by using purified Rac1 protein that
had either been activated by preloading with GTP?S or inhib-
ited by preloading with GDP. Addition of Rac1-GTP?S to the
FIG. 1. Phosphorylation of hBad at Ser-75 is modulated by Rac1. (A) Rac1 protein expression in human BL-41 Burkitt’s lymphoma cells stably
expressing vector alone, HA-tagged Rac1N17 (dominant-negative mutant), HA-Rac1V12 (constitutively active mutant), or HA-Rac1D11 (caspase-
3-resistant mutant). Cell extracts were separated on Bis-Tris SDS-PAGE (4 to 12% acrylamide), transferred to a nitrocellulose membrane, and
immunoblotted with antibodies against HA. (B) Levels of phosphorylated hBad in transfected cells lines. The immunoblots were probed with
antibodies that specifically recognize residues phosphorylated at Ser-75 (p-75-hBad), Ser-99 (p-99-hBad), and Ser-118 (p-118-hBad). Equal loading
was confirmed by reprobing the membrane with antibodies to Bad or ?-actin. (C) In vitro phosphorylation of Bad by cell extracts. Purified
recombinant His-tagged murine Bad (mBad; 1 ?g) was incubated in standard kinase buffer with various amounts (20 to 2,000 ng) of extracts from
healthy BL-41 cells as the source of kinase(s). The reactions were stopped by addition of SDS sample buffer and were analyzed for Bad
phosphorylation by Western blot immunoassay using site-specific anti-phospho-Bad antibodies (see Materials and Methods). (D) Effect of Rac1
GTPase activity on phosphorylation of Bad. Recombinant glutathione S-transferase-tagged Rac1 was preloaded with GDP or GTP?S and then
added (final concentration, 2.0 ?M) to a reaction mixture containing 1 ?g of His-tagged murine Bad and lysis buffer (upper panel) or 50 ng of
BL-41 extract (lower panel). The phosphorylation reactions were carried out under the conditions described for panel B. The results are
representative of three independent experiments.
VOL. 24, 2004 Rac1-REGULATED Bad PHOSPHORYLATION SUPPRESSES APOPTOSIS6207
cell lysates enhanced phosphorylation of murine Bad at Ser-
112 by approximately fivefold, whereas Rac1-GDP had no de-
tectable effect on Bad phosphorylation (Fig. 1D). In contrast,
neither Rac1 form stimulated Bad phosphorylation on Ser-75
in the absence of endogenous kinases (Fig. 1D, upper panel).
Together these results demonstrate that the active form of
Rac1 can stimulate site-specific kinases to phosphorylate Bad
both in vivo and in vitro.
Active Rac1 inhibits drug-induced caspase activation and
apoptosis. In previous studies (46) we showed that Rac1 needs
to be cleaved and inactivated by caspases in order for maximal
apoptosis to occur in response to chemotherapy drugs. The
finding that active Rac1 triggers Bad Ser-75 phosphorylation
suggested that a Rac1-dependent signaling pathway involving
Bad phosphorylation might be responsible for suppressing
drug-induced apoptosis. To investigate this possibility, we took
advantage of a transfected BL-41 cell line that expresses a
Rac1 mutant (Rac1D11E) that is resistant to caspase-3 cleav-
age and apoptosis (46). As shown in Fig. 2A, cells transfected
with Rac1D11E and treated with the chemotherapy drug
VP-16 (etoposide) show 30 and 55% less apoptosis at 3 and
6 h, respectively, than control cells, as determined by annexin
V staining. Quantitative data from several separate experi-
ments are shown in Fig. 2B and demonstrate that both
Rac1V12 and Rac1D11E prevented the cells from undergoing
apoptosis induced by VP-16. Note that Rac1V12 is also rela-
tively resistant to caspase-3 cleavage and hence remains active
in cells treated with VP-16 (46). Similar prosurvival effects of
activated Rac1 were observed previously in JLP-119 lymphoma
cells (46). Activation of caspase-3 followed a commensurate
pattern, as demonstrated by the cleavage of procaspase-3 (Fig.
2C) and PARP (Fig. 2D), which is a known caspase-3 sub-
strate. By 6 h, a significant amount caspase-3 activation had
occurred in both control cells and cells expressing Rac1N17,
and PARP cleavage was almost complete. In contrast, in cells
expressing Rac1V12 and Rac1D11E, caspase-3 activation and
PARP cleavage were significantly delayed.
The status of Bad phosphorylation during apoptosis was
FIG. 2. Rac1 activity modulates apoptosis induced by VP-16. (A) Apoptosis was assessed in control BL-41 cells and in BL-41 cells transfected
with the caspase-3-resistant Rac1 mutant Rac1D11E by fluorescence-activated cell sorting analysis using FITC-annexin V and PI (see Materials
and Methods and reference 46 for details). Cells were treated with 200 ?g of VP-16/ml for 3 or 6 h and then were harvested. The percentage of
each cell population is shown. Cells in the lower right fluorescence-activated cell sorting quadrant represent apoptotic cells. (B) Quantification of
VP-16-induced apoptosis (as described for panel A) in BL-41 cells expressing endogenous Rac1 only, dominant-negative Rac1N17, constitutively
active Rac1V12, or the caspase-3-resistant mutant Rac1D11E, as indicated. The results represent the means ? standard deviations from three
independent experiments. (C and D) Measurement of procaspase-3 and PARP cleavage in control and Rac1-transfected cell lines expressing the
various mutant proteins after treatment with VP-16 as indicated by Western blot immunoassay using anti-caspase-3 (C) and anti-PARP
(D) antibodies. Caspase activity is indicated by the appearance of the p17/p12 caspase-3 cleavage fragments (D) and the 85-kDa PARP cleavage
product. *, nonspecific band. (E) The level of endogenous Bad phosphorylation in apoptotic cells was assessed by subjecting whole-cell lysates to
Western blot immunoassay using antibodies specific for p-Ser-75. All data are representative of at least three independent experiments.
6208 ZHANG ET AL.MOL. CELL. BIOL.
assessed by immunoblot analysis using phosphorylation site-
specific antibodies (Fig. 2E). In control cells, Bad phosphory-
lation on Ser-75 decreased over time after VP-16 treatment. In
cells expressing Rac1V12, however, there was a significant
delay in the decrease in Bad Ser-75 phosphorylation. A stron-
ger delay was observed in the cells expressing Rac1D11E,
which is the most caspase-resistant mutant. These data show
that increased Rac1 function correlates with increased Bad
phosphorylation and leads to protection from apoptotic cell
Inhibition of Bad phosphorylation on Ser-75 sensitizes lym-
phoma cells to drug-induced apoptosis. If Rac1 enhances lym-
phoma cell survival by stimulating Bad phosphorylation, then
selective inhibition of Bad phosphorylation should enhance the
apoptotic response to drug treatment. To establish this linkage,
we designed a cell-permeable peptide comprised of the Ser-75
phosphorylation site (amino acids 70 to 78) of hBad coupled to
the protein transduction domain (RKKRRQRRR) (PTD) of
the HIV-1 transactivator protein TAT (Fig. 3A). This PTD has
been used previously to enhance delivery of exogenous pro-
teins or peptides into living cells (21). A second peptide con-
taining the same PTD but a scrambled Bad phosphorylation
sequence was prepared and used for control experiments. Ini-
tially, the effects of the peptides on Bad phosphorylation were
tested in vitro by using purified PKA and recombinant purified
murine Bad as a substrate. As shown in Fig. 3B, addition of the
hBad-Ser75 peptide to this assay resulted in a dose-dependent
inhibition of Bad phosphorylation at Ser-112. At 100 ?M pep-
tide, Bad phosphorylation was inhibited by ?90%, with a con-
comitant appearance of the phosphorylated peptide product.
In contrast, the control peptide with the scrambled sequence
had relatively little effect on PKA-induced Bad phosphoryla-
tion. We next determined whether the hBad-Ser75 peptide
would inhibit Bad phosphorylation and promote apoptosis in
vivo. When control cells or cells expressing Rac1D11 were
treated with 500 ?M hBad-Ser75 peptide, a significant reduc-
tion in Bad phosphorylation on serine-75 was observed (Fig.
3C). Furthermore, treatment with the hBad-Ser75 peptide in-
creased VP-16-induced apoptosis in control and Rac1D11 cells
by two- and fourfold, respectively (Fig. 3D), thereby restoring
the level of apoptosis to that observed in control VP-16-treated
cells. In sharp contrast, Rac1N17-expressing cells, which are
relatively weak in their ability to phosphorylate Bad (Fig. 1B)
and are already hypersensitive to VP-16-induced apoptosis, are
not sensitive to the hBad-Ser75 peptide. Taken together these
results indicate that Rac1-regulated Bad phosphorylation on
Ser-75 is an important mediator of the survival signaling path-
way in lymphoma cells. Cells expressing Rac1D11 are, none-
theless, still relatively resistant to apoptosis even when Bad
phosphorylation is diminished (Fig. 3D), suggesting that phos-
phorylation of Bad alone may not be sufficient for lymphoma
cell survival. It may be that additional survival signaling mol-
ecules are required to fully confer Rac1-mediated cell survival.
Site-specific phosphorylation of Bad by different protein
kinases. Having demonstrated a role for Rac1 in regulating
Bad phosphorylation, we then attempted to identify the effec-
tor proteins through which Rac1 exerts its activity. Several
different protein kinases are known to have the capacity to
phosphorylate Bad, with various levels of specificity for murine
Ser residues 112, 136, and 155 (8, 9, 19, 20, 22). These include
Akt, cAMP-dependent protein kinase (PKA), and members of
the PAKs. We compared the phosphorylation site specificity of
these kinases by using antibodies specific for phosphorylated
murine Ser-112, Ser-136, and Ser-155. As shown in Fig. 4,
purified, constitutively active Akt1/PKB? phosphorylated re-
combinant murine Bad in vitro on both Ser-112 and Ser-136
with a strong preference for the latter residue, while no Ser-
155 phosphorylation was observed. This result is consistent
with previous reports showing that Ser-136 is the major site of
phosphorylation of Bad by Akt (10, 12). In contrast, purified,
constitutively active PAK2, which was previously shown to
FIG. 3. A competitive inhibitor of hBad Ser-75 phosphorylation
sensitizes lymphoma cells to VP-16-induced apoptosis. (A) Sequence
of cell-permeable hBad-Ser-75 peptide and the control (scrambled)
peptide. The hBad-Ser-75 peptide contains the serine-75 phosphory-
lation site (amino acids 70 to 78) of hBad. The PTD (underlined)
derived from the HIV-1 Tat protein promotes cell entry. (B) Effects of
hBad Ser-75 peptide on Bad phosphorylation in vitro. Recombinant
murine Bad (mBad; 0.5 ?g) was incubated in standard PKA assay
buffer with active PKAC(20 ng) and increasing amounts (1 to 100 ?M)
of peptides in a final volume of 70 ?l for 10 min at 30°C. The reactions
were stopped by addition of SDS sample buffer and were analyzed for
Bad phosphorylation by Western blotting with anti-pSer-112-mBad
antibodies. (C) Effects of peptides on Bad phosphorylation in vivo.
Cells were treated with 500 ?M hBad-Ser75 or scrambled peptide for
2 h and were analyzed for the status of Bad phosphorylation on Ser-75.
(D) Effect of hBad Ser-75 peptide on the sensitivity of lymphoma cells
to VP-16-induced apoptosis. Cells were pretreated with 500 ?M pep-
tide for 1 h and then were incubated with VP-16 (200 ?g/ml) for an
additional 2.5 h. Apoptosis was assessed by annexin V-fluorescence-
activated cell sorting analysis. The results represent the means ?
standard deviations from three independent experiments. MW, mo-
VOL. 24, 2004Rac1-REGULATED Bad PHOSPHORYLATION SUPPRESSES APOPTOSIS 6209
phosphorylate Bad at Ser-112 and Ser-136 (22), preferentially
induced phosphorylation of Bad on Ser-112 and Ser-155 with-
out phosphorylation of Ser-136. PKAC, which was reported
previously to phosphorylate Bad on either Ser-112 (20) or
Ser-155 (27), phosphorylated all three serine residues to sim-
ilar degrees. Thus, Akt, PKA, and PAK2 exhibit differential
specificities for Bad residues Ser-112, Ser-136, and Ser-155.
Inhibition of PKA activity blocks Rac1-stimulated phos-
phorylation of Bad. To examine whether PKA might be re-
sponsible for the constitutive phosphorylation of Bad at Ser-
112 in growing, healthy lymphoma cells, two different PKA
inhibitors were employed: a PKA-specific inhibitory peptide
and the cAMP antagonist Rp-cAMPS. As shown in Fig. 5A,
when extracts from healthy BL-41 cells were assayed for in
vitro kinase activity, phosphorylation of Ser-112 was seen in
control extracts but not in extracts containing either of the
PKA inhibitors. To confirm the presence of PKA activity in the
BL-41 cell extracts, we employed a fluorescent peptide sub-
strate assay that is specific for PKA activity (PepTag A1). The
utility of the assay was validated by using PKAC. As illustrated
in Fig. 5B, nonphosphorylated substrate migrates toward the
negative electrode while the phosphorylated peptide migrates
toward the positive electrode. Addition of increasing amounts
of PKA catalytic subunit resulted in a systematic shift in mo-
bility, reflecting an increase in the phosphorylated substrate.
Whole-cell lysates from normal lymphoma cells showed a basal
PKA activity that was stimulated by addition of exogenous
cAMP. This activity was blocked completely by both PKA-
specific inhibitory peptide and Rp-cAMPS (Fig. 5C).
This PKA-specific assay was then employed to determine the
ability of Rac1 to modulate PKA activity in BL-41 cells. To this
end, we measured constitutive PKA activity levels in whole-cell
extracts from cells transfected either with constitutively active
or with dominant-negative Rac1 (Rac1V12 or Rac1D11 and
Rac1N17, respectively). As shown in Fig. 6A, PKA activity was
increased by approximately twofold in cells expressing
Rac1V12 or Rac1D11 and decreased by ?60% in Rac1N17-
expressing cells compared to corresponding levels in control
cells. These data suggested that Rac1 GTPase can activate
PKA (either directly or indirectly). In support of this conclu-
sion, addition of active Rac1-GTP?S protein to BL-41 cell
extracts stimulated basal PKA activity by approximately two-
fold. In contrast, addition of inactive Rac1-GDP had no effect
on basal PKA activity (Fig. 6B).
To determine whether PKA activity is required for Rac1-
mediated Bad phosphorylation in vivo, Rac1D11-expressing
cells were treated with a cell-permeable peptide inhibitor of
PKA. As shown in Fig. 6C, the PKA inhibitor decreased the
phosphorylation of endogenous Bad in a dose-dependent man-
ner. At 50 ?M, approximately 70% of Bad phosphorylation
was inhibited in Rac1D11 cells (based on densitometry analysis
of the bands). These results implicate PKA as a principal Bad
kinase that acts in response to Rac1 activation in lymphoma
cells. Accordingly, pretreatment with 50 ?M PKA inhibitor
resulted in significantly more apoptosis in control and
FIG. 4. In vitro phosphorylation of Bad by purified protein kinases.
Purified recombinant His-Bad protein (1 ?g) was incubated in kinase
reaction buffer (10 min at 30°C) containing the following different
purified, recombinant protein kinases (1 ?g): AKT1/PKB?, PAK2, and
the PKAC(see Materials and Methods). Phosphorylation of murine
Bad (mBad) on different serine residues was determined by immuno-
blot analysis with site-specific anti-phospho-Bad antibodies.
FIG. 5. Inhibition of cellular PKA activity blocks Bad phosphory-
lation. (A) BL-41 cell extracts (200 ng of total protein) were assayed
for Bad kinase activity in the absence or presence of a PKA peptide
inhibitor (20 ?M) or Rp-cAMPS (100 ?M). (B) PepTag assay for
determining PKA activity. PepTag A1 peptide (2 ?g), a PKA-specific
substrate, was incubated in standard PKA assay buffer with various
amounts (1 to 24 ng) of active PKACin a final volume of 25 ?l for 30
min at room temperature. The reactions were stopped and then sub-
jected to agarose gel electrophoresis. The phosphorylated peptide sub-
strate migrates toward the positive electrode (?), while the nonphos-
phorylated peptide migrates toward the negative electrode (?).
(C) Detection of PKA-specific kinase activity in cell lysates. Extracts (2
?g) from healthy BL-41 cells were subjected to the PepTag PKA assay
as described for panel B in the absence (lane 3) or presence (lanes 4 to
6) of cAMP. The PKA peptide inhibitor (20 ?M) or Rp-cAMPS (100
?M) was added where indicated (lanes 5 and 6). Lanes 1 and 2 show
the negative (buffer only) and positive controls (with 10 ng purified
PKAC). The data represent one of two independent experiments.
6210 ZHANG ET AL.MOL. CELL. BIOL.
Rac1D11 cells (Fig. 6D), respectively, reinforcing the conclu-
sion that Rac1-stimulated Bad phosphorylation plays a role in
mediating lymphoma cell survival.
Akt does not appear to be involved in Bad phosphorylation
following Rac1 activation. Akt is a Ser/Thr kinase that re-
sponds to PI 3-kinase activation and is thought to suppress
apoptosis following a variety of stimuli, in part through phos-
phorylation of Bad (9, 10, 12, 23). Rac1 was shown previously
to mediate PI 3-kinase activation of Akt and to promote cell
survival in COS-7 cells (31) or human natural killer cells (23).
In hematopoietic cells, it is the Rac2 isoform that appears to be
responsible for Akt activation and survival (43). To determine
whether Akt might be responsible for the Bad phosphorylation
observed in our experimental system, we first assessed the
levels of active (phosphorylated) Akt in BL-41 lymphoma cells
containing differing levels of Rac1 activity. No alteration in
endogenous Akt activity was observed in cells expressing
Rac1V12, Rac1D11, or Rac1N17, as determined by Western
blots using anti-phospho-Akt (Ser-473) antibodies that specif-
ically detect the active form of the kinase (Fig. 7A). Similarly,
an enzymatic assay for Akt-specific activity showed no differ-
ence between cells expressing the different forms of Rac1,
while an Akt-specific inhibitor (SH-5) almost completely in-
hibited in vitro Akt activation (Fig. 7B). This result suggested
that Rac-1-induced phosphorylation of hBad at Ser-75 is Akt
independent in human lymphoma cells. In support of this con-
clusion, immunodepletion of Akt from lysates of Rac1V12
cells had no effect on Bad kinase activity (Fig. 7C). Moreover,
addition of a selective cell-permeable Akt inhibitor did not
affect endogenous Bad phosphorylation on Ser-75, even
though Akt autophosphorylation and activation were almost
completely blocked (Fig. 7D). Accordingly, treatment with the
Akt inhibitor did not enhance the sensitivity of the cells to
VP-16 induced apoptosis (Fig. 7E). Collectively, these results
indicate that Akt is not the principal kinase responsible for the
phosphorylation of Bad in response to Rac1 activation.
In this report, we provide evidence that the Rac1 small
GTPase is a key upstream regulator of Bad phosphorylation,
thereby providing a molecular mechanism for how Rac1 inhib-
its apoptosis in response to cancer chemotherapy drugs. We
also show that the only Bad phosphorylation site relevant for
survival of these human lymphoma cells occurs on Ser-75 in the
hBad protein. Finally, we show that Rac1-induced Bad Ser-75
phosphorylation is catalyzed by PKA but not Akt. These con-
clusions are supported by the following data: (i) of the three
major Ser residues known to be phosphorylated in hBad, only
Ser-75 is found to be phosphorylated in healthy, growing Bur-
kitt’s lymphoma cells; (ii) constitutively active Rac1 mutants
(Rac1V12 or Rac1D11E) stimulate the phosphorylation of
Bad specifically on Ser-75 and inhibit apoptosis in response to
VP-16; (iii) a Rac1 dominant-negative mutant (Rac1N17) fails
to stimulate phosphorylation of Bad, and cells expressing this
mutant are more susceptible to apoptosis than cells expressing
wild-type or constitutively active Rac1; (iv) inhibition of Bad
phosphorylation by a cell-permeable competitive peptide in-
hibitor representing the Bad Ser-75 phosphorylation site sen-
sitizes lymphoma cells to drug-induced apoptosis; (v) the se-
lective inhibition of hBad Ser-75 phosphorylation by a selective
PKA inhibitor diminishes the ability of Rac1 to prevent apo-
ptosis in response to chemotherapy drugs, while the selective
inhibition of Akt has no such effect; (vi) when kinase activity in
cell extracts is studied, the selective inhibition of PKA dimin-
ishes the phosphorylation of Bad—inhibition or selective im-
munodepletion of Akt in cell extracts has no such effect; (vii)
active (GTP-bound) Rac1 stimulates PKA to phosphorylate
Bad while inactive (GDP-bound) Rac1 lacks this activity.
These findings will be discussed in greater detail below.
FIG. 6. Rac1 stimulates PKA activity. (A) Equal amounts of ex-
tracts (2 ?g) from healthy control BL-41 cells or from cell lines stably
transfected with Rac1N17, Rac1V12, or Rac1D11 were subjected to
the PepTag assay as described in the legend to Fig. 5. (B) Kinase
activity was quantified by spectrophotometric analysis (absorbance at
570 nm) of the phosphorylated bands and is expressed relative to
control (Vector alone) activity. (B) Purified Rac1 protein (2 ?g) was
preloaded with either GDP or GTP?S to inhibit or activate Rac1
activity, respectively. The Rac1 protein was then added to healthy
BL-41 extracts and tested for an effect on PKA activity, which is
expressed relative to the activity of active PKAC(10 ng). (C) Stable
Rac1D11-expressing cells were treated for 60 min with an increasing
concentration of a specific myristoylated PKA inhibitor. Whole-cell
extracts were subjected to Western blotting to assess hBad Ser-75
phosphorylation. (D) Stable cell lines were pretreated with 10 ?M
PKA inhibitor prior to adding VP-16 (200 ?g/ml). Apoptosis was
allowed to proceed for 3 h. The percentage of apoptotic cells was
assessed by fluorescence-activated cell sorting as described in the leg-
end to Fig. 2. The data are representative of three independent assays.
VOL. 24, 2004Rac1-REGULATED Bad PHOSPHORYLATION SUPPRESSES APOPTOSIS 6211
Numerous previous studies have demonstrated that the re-
versible phosphorylation of Bad plays a pivotal role in control-
ling apoptosis (5, 11, 14). This is thought to be mediated
through the reversible binding of Bad to antiapoptotic Bcl-2
family proteins. In particular, it has been shown that the phos-
phorylation of murine Bad on Ser-112 or Ser-136 prevents its
association with Bcl-2 or Bcl-XL, leaving these proteins free to
exert their antiapoptotic function (42, 44). Consistent with
these findings, Bcl-2 was enriched in the mitochondria of cells
expressing Rac1V12, in which a higher level of phosphorylated
Bad existed (data not shown). While Bad is phosphorylated in
a variety of cell contexts, the upstream regulatory mechanisms
that control Bad phosphorylation are not fully understood. A
number of cell survival pathways that lead to Bad phosphory-
lation have been described. The best characterized of these is
the PI 3-kinase/Akt pathway that has been shown to induce the
phosphorylation of Bad on Ser-136 in response to stimulation
of survival factors such as platelet-derived growth factor and
interleukin-3 (9, 12, 33). In addition to the PI 3-kinase path-
way, recent evidence has implicated the Ras/Raf cascade in the
control of Bad phosphorylation (18). The results presented
here demonstrate that Rac1 activation leads to the phosphor-
ylation of Bad at Ser-75 in human lymphoma cells. However,
while there is a connection between Rac signaling and the PI
3-kinase/Akt pathway (29), Rac appears to be activated in our
system in a PI 3-kinase-independent manner, as has been ob-
FIG. 7. Akt activity is not responsive to Rac1 activity. (A) The activation state of Akt in stable lymphoma cells expressing Rac1 mutants was
assessed by measuring the level of Akt phosphorylation by using an antibody against phospho-Akt (Ser-473). Equal loading was assessed by
reprobing the membrane with an anti-Akt antibody. (B) Akt-specific kinase activities were measured by a colorimetric Akt kinase assay as
described in Materials and Methods. The kinase reactions were carried out with equal amounts of cell extracts from the indicated cell lines. The
positive control contains purified recombinant Akt. Inhibition of Akt activity in cell extracts was accomplished with the Akt-specific inhibitor SH-5.
(C) Phosphorylation of Bad by whole (left lane) or Akt-depleted (right lane) cell extracts was assessed as described in the legend to Fig. 1.
(D) Stable Rac1D11-expressing cell lines were treated with increasing amounts of Akt inhibitor (SH-5) for 1 h. Whole-cell lysates were analyzed
by Western blotting with the indicated phosphospecific antibodies to detect Akt phosphorylation and activation and Bad phosphorylation.
(E) Effect of the Akt inhibitor on the sensitivity of the lymphoma cells to VP-16-induced apoptosis. Cells were pretreated with 10 ?M Akt inhibitor
(SH-5) for 1 h and then were incubated with VP-16 (200 g/ml) for an additional 3 h. Apoptosis was assessed by the annexin V-fluorescence-
activated cell sorting assay. The results represent the means ? standard deviations from three independent experiments.
6212 ZHANG ET AL.MOL. CELL. BIOL.
served with other cell types (1, 32). The relative contributions
and interactions that the different survival pathways make in
controlling Bad activity in different cell settings remains to be
more fully elucidated.
Rac1-stimulated phosphorylation of Bad contributes to its
antiapoptotic function; there is a significant inhibition of apo-
ptosis in BL-41 cells upon the expression of activated Rac1
mutants, and the increased cell survival correlates with the
ability of these mutants to induce the phosphorylation of Bad
on Ser-75. Inhibition of Bad phosphorylation in response to
activated Rac1 counters the antiapoptotic activity of the pro-
tein. In parallel, the caspase-3 activation triggered by VP-16 is
prevented or delayed in these cells, as determined by pro-
caspase-3 and PARP cleavage. Our previous data showed that
Rac1 is a caspase substrate that becomes cleaved during drug-
induced apoptosis (46). Hence, Rac1 prevents its own degra-
dation and inactivation by stimulating Bad phosphorylation
and inhibiting caspase activation. Interestingly, a caspase-re-
sistant mutant, Rac1D11E, appears to be constitutively active
in terms of stimulating Bad phosphorylation. Expression of
Rac1D11E leads to an even stronger protective effect than that
of the constitutively active Rac1V12, which is less resistant to
caspase-mediated cleavage (46). Because many of the growth
factors that activate Rac proteins could simultaneously induce
the phosphorylation of Bad, the Rac1-stimulated phosphory-
lation of Bad at Ser-75 may represent a general mechanism by
which growth factor receptors deliver a survival signal that
leads to the inhibition of apoptosis. However, these results do
not rule out the possibility that Rac1 promotes cell survival by
other mechanisms in addition to that mediated by the phos-
phorylation of Bad. Indeed, prevention of Rac1-stimulated
Bad phosphorylation only partially protected cells from drug-
induced apoptosis. In this respect, Rac1 and Rac2 have been
shown by others to promote cell survival through the activation
of NF-?B (24) or generation of reactive oxygen species via
NADPH oxidase (2, 13), which suggests that Rac GTPases
regulate cell survival by at least one other mechanism.
Through an effort to identify the kinases that are capable of
phosphorylating Bad at Ser-75, we show by a number of crite-
ria that PKA is likely a principal kinase responsible for the
site-specific phosphorylation of Bad in response to Rac1 acti-
vation in human lymphoma cells. Among an expanding family
of Bad kinases, PKA has been shown to phosphorylate murine
Bad at Ser-112 (8, 19, 20, 22, 39). However, the published
evidence on the site specificity of PKA is inconsistent. One
study showed that PKA primarily phosphorylates murine Bad
at Ser-112 (20), and a separate report suggested that Ser-155
was phosphorylated preferentially by PKA in vitro and was the
only residue in Bad that became phosphorylated when HEK-
293 cells were exposed to cAMP-elevating agents (27). In the
present study, we show that the three residues are almost
equally susceptible to phosphorylation by PKACbut that only
the human Ser-75 residue is phosphorylated in vivo. Specific
inhibition of the kinase activity of PKA in the cell lysates
blocked Bad phosphorylation. Intriguingly, Rac1 appears able
to modulate the activity of PKA, although the underlying
mechanism is unknown. This finding is important because it
suggests a novel mechanism for PKA activation by factors
other than cAMP.
Members of the PAK family are immediate Rac GTPase
effector proteins, and they have also been shown to limit apo-
ptosis through phosphorylation of Bad on Ser-112 and Ser-136
(8, 19, 22, 39). However, no one has yet shown which PAK
isozymes phosphorylate critical Bad serine residues in re-
sponse to Rac1 activation. In preliminary studies, we also
found that immunodepletion of PAK2 or PAK4, but not
PAK1, diminishes Bad phosphorylation in vitro (data not
shown). Moreover, constitutively active PAK2 catalyzed phos-
phorylation of recombinant murine Bad at Ser-112 and Ser-155
in vitro while Ser-136 was untouched (see Fig. 4). These pre-
liminary results confirm those of the previous reports showing
that some, but not all, of the PAK isozymes are Bad kinases.
Further experiments will be required to determine whether the
PAKs phosphorylate Bad in response to Rac1 activation in a
manner that inhibits apoptosis.
The finding that activated forms of Rac1 induce phosphor-
ylation and inactivation of Bad may be of clinical importance,
as Rac1 and its family members are frequently activated in
human cancers (28, 37, 38). Their effect on the function of Bad
may contribute to the oncogenic properties of upregulated
Rac. Therefore, it will be of interest to determine whether Bad
phosphorylation at Ser-75 is elevated in human cancers show-
ing functional activation of the Rac signaling pathway. Recent
studies suggested that the relative level of expression and phos-
phorylation of Bad might play an important role in the out-
come of cancer chemotherapy (30, 40). Our work adds to this
hypothesis by suggesting that interference with the function of
Rac proteins and their signaling components might provide a
useful anticancer strategy.
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