MOLECULAR AND CELLULAR BIOLOGY, June 2006, p. 4612–4627
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 26, No. 12
The Rho GTPase Effector ROCK Regulates Cyclin A, Cyclin D1,
and p27Kip1Levels by Distinct Mechanisms
Daniel R. Croft and Michael F. Olson*
The Beatson Institute for Cancer Research, Glasgow G61 1BD, United Kingdom
Received 24 October 2005/Returned for modification 28 November 2005/Accepted 6 April 2006
The members of the Rho GTPase family are well known for their regulation of actin cytoskeletal structures.
In addition, they influence progression through the cell cycle. The RhoA and RhoC proteins regulate numerous
effector proteins, with a central and vital signaling role mediated by the ROCK I and ROCK II serine/threonine
kinases. The requirement for ROCK function in the proliferation of numerous cell types has been revealed by
studies utilizing ROCK-selective inhibitors such as Y-27632. However, the mechanisms by which ROCK
signaling promotes cell cycle progression have not been thoroughly characterized. Using a conditionally
activated ROCK-estrogen receptor fusion protein, we found that ROCK activation is sufficient to stimulate
G1/S cell cycle progression in NIH 3T3 mouse fibroblasts. Further analysis revealed that ROCK acts via
independent pathways to alter the levels of cell cycle regulatory proteins: cyclin D1 and p21Cip1elevation via
Ras and the mitogen-activated protein kinase pathway, increased cyclin A via LIM kinase 2, and reduction of
p27Kip1protein levels. Therefore, the influence of ROCK on cell cycle regulatory proteins occurs by multiple
Rho family GTPases contribute to the regulation of many
different biological processes, including cell cycle progression,
with RhoA, Rac1, and Cdc42 being the best-studied members
(12). When bound to GTP, these proteins recruit effector pro-
teins that relay signals downstream and mediate biological
responses. RhoA and RhoC activate numerous effector pro-
teins, with the serine/threonine kinases ROCK I (also called
ROK?) and ROCK II (ROK? or Rho kinase) (55) being the
most extensively characterized. When activated by association
with Rho-GTP, ROCK phosphorylates a number of substrates,
such as LIM kinase 1 (LIMK1) and LIMK2 and regulatory
myosin light chains (MLC2). Through these phosphorylation
events, ROCK promotes the stabilization of filamentous (F)
actin and increased myosin ATPase activity, leading to the
formation of contractile actin-myosin bundles (often called
stress fibers) and integrin-containing focal adhesions (41, 75).
Although Rho GTPases have been implicated in human
cancer (21, 60), Rho mutations are not a mode of activation.
However, RhoA and RhoC protein levels are significantly el-
evated in a variety of tumors (21, 32, 38, 60). In addition to
elevated RhoA and RhoC, increased levels of ROCK I and/or
ROCK II have been found in esophageal squamous cell car-
cinoma (87) and in testicular germ cell (32), pancreatic (36),
and bladder (33) tumors. Although it has been proposed that
increased expression of the Rho and ROCK proteins contrib-
utes to the metastatic behavior of some cancers (e.g., see ref-
erence 18), elevated signaling through the ROCK pathway may
also promote tumor cell proliferation. Transformation of NIH
3T3 cells by oncogenic Ras was found to be blocked by the
ROCK-selective inhibitor Y-27632, while activated ROCK co-
operated with the Ras effector Raf-1 to promote transforma-
tion (59). In addition, proliferation of C6 glioma cells (10),
HSQ-89 oral squamous carcinoma cells (48), IMGE-5 gastric
epithelial cells (25), umbilical vein endothelial cells (66), vas-
cular smooth muscle cells (34, 44, 63, 65), prostatic smooth
muscle cells (53), atrial myofibroblast cells (52), cardiac myo-
cytes (86), glial cells (68), spleen-derived primary and Jurkat T
cells (45, 74), CD34?hematopoietic stem cells (77), corneal
stromal cells (24), chondrocytes (80), and hepatic stellate cells
(30) was inhibited by the ROCK inhibitor Y-27632. Although
it has been reported that ROCK activity is required for the
formation of actin stress fibers that contribute to the sustained
activation of Ras and the ERK mitogen-activated protein ki-
nase (MAPK) following ligand stimulation (57, 71, 83), the
possibility remains that elevated ROCK signaling might pro-
mote cell cycle progression via additional mechanisms.
The eukaryotic cell cycle is composed of the first gap phase
(G1), the DNA-synthetic phase (S), the second gap phase (G2),
and mitosis (M). Progression through G1to S phase is con-
trolled by the cyclin-dependent kinases (CDKs) in association
with cyclin regulatory subunits (67). Type D cyclins (D1, D2,
and D3) form complexes with CDK4 or CDK6, while cyclin E
and cyclin A work in combination with CDK2. The cyclin
D-CDK and cyclin E-CDK2 complexes are generally thought
to act to promote passage through G1, while cyclin A-CDK2
contributes to passage through the G1/S transition and pro-
gression through S phase. The activities of cyclin-CDK com-
plexes are modulated by two types of CDK inhibitors (CDKIs)
that have differing mechanisms of action. The INK4 CDKI
proteins (p15INK4B, p16INK4A, p18INK4C, and p19INK4D) di-
rectly interact with CDK and inhibit its activity. In contrast, the
Cip/Kip family CDKIs (p21Waf1/Cip1[p21], p27Kip1[p27], and
p57Kip2) bind to cyclin-CDK complexes. At high levels, p21 and
p27 inhibit cyclin E-CDK2 activity, leading to cell cycle arrest.
At lower stoichiometry, however, p21 and p27 promote the
assembly, stability, and nuclear retention of cyclin D-CDK4
and cyclin D-CDK6 complexes, which are inefficiently inhib-
* Corresponding author. Mailing address: The Beatson Institute for
Cancer Research, Garscube Estate, Switchback Road, Glasgow G61
1BD, United Kingdom. Phone: 44 (0)141 330 3654. Fax: 44 (0)141 942
6521. E-mail: email@example.com.
ited by associated Cip/Kip proteins. Cyclin D-CDK4 and cyclin
D-CDK6 complexes also relieve cyclin E-CDK2 complexes
from Cip/Kip-mediated inhibition by acting as a sink for p21
and p27. Therefore, the relative levels of cyclin, CDK, and
CDKI proteins are critical factors that determine whether a
cell will progress through G1toward S phase.
Initial indications that Rho contributed to cell cycle regula-
tion were the observations that Rho inactivation with the Clos-
tridium botulinum C3 exoenzyme blocked serum-stimulated
DNA synthesis and that microinjection of active RhoA was
sufficient to induce G1/S-phase progression in Swiss 3T3 fibro-
blasts (49, 85). In addition, expression of Rho from Aplysia
californica was found to oncogenically transform NIH 3T3 cells
(51). However, the means by which elevated ROCK signaling
might promote cell cycle progression, and possibly transforma-
tion, have not been thoroughly characterized. In this report, we
show that stimulation of a conditionally activated ROCK-es-
trogen receptor fusion protein (ROCK-ER) is sufficient to
stimulate G1/S cell cycle progression in NIH 3T3 cells. Further
analysis revealed that ROCK acts via independent pathways to
elevate cyclin D1 and p21 by Ras and MAPK activation, to
elevate cyclin A expression via LIMK and to reduce p27 pro-
tein levels. Therefore, the influence of ROCK on cell cycle
regulatory proteins occurs by multiple mechanisms.
MATERIALS AND METHODS
Antibodies. Antibodies to MEK1/2 (9122), phospho-MEK1/2 (Ser217/221)
(9121), phospho-MLC (Ser19) (3671), phospho-MLC (Thr18/Ser19) (3674),
LIMK1 (3842), and phospho-LIMK1 (Thr508)/LIMK2 (Thr505) (3841) were
from Cell Signaling Technologies (Beverly, MA). Antibodies against myosin light
chain (M4401) and phospho-ERK (M8159) were from Sigma (St. Louis, MO).
Antibodies against ER? (sc-543), cdk2 (sc-163), cdk4 (sc-260), cdk6 (sc-177),
cyclin A (sc-596), cyclin A1 (sc-15383), cyclin D1 (sc-450), cyclin D3 (sc-6283),
cyclin E (sc-481), lamin A/C (sc-6215), LIMK2 (sc-5577), p15 (sc-613), p18
(sc-865), p21 (sc-397G), and p107 (sc-318) were from Santa Cruz Biotechnology,
Inc. (Santa Cruz, CA). Antibodies against cdk2 (06-505), cyclin E (07-687), focal
adhesion kinase (FAK) (06-543), and Ras (05-516) were from Upstate (Lake
Placid, NY). Antibodies against paxillin (610051) and p27 (610241) were from
BD Biosciences (San Diego, CA). Additional antibodies used were against
ROCK II (ROK?, ab24843; Abcam, Cambridge, United Kingdom), cyclin D2
(RDI-CYCLD2abm-43; Research Diagnostics, Flanders, NJ), and phospho-FAK
(Tyr397) (44-624; Biosource, Camarillo, CA). Anti-ERK2 antibody (Ab122) was
provided by C. J. Marshall (Institute of Cancer Research, London, United King-
dom). Goat anti-mouse, goat anti-rabbit, and rabbit anti-goat horseradish peroxi-
dase-conjugated antibodies were from Pierce (Rockford, IL).
Generation of KD-ER and ROCK-ER cell lines. Retroviral constructs for
conditionally regulated ROCK II-ER (ROCK-ER) and the kinase-dead version
(KD-ER) were constructed as described previously (17, 18). pBABE puro
ROCK-ER and KD-ER plasmids were transfected into BOSC 23 ecotropic
retroviral packaging cells with Effectene (QIAGEN) according to the manufac-
turer’s instructions. After 36 h, supernatant was collected and centrifuged at
1,600 rpm for 15 min, and aliquots were stored at ?80°C. Exponentially growing
NIH 3T3 cells were infected with undiluted retroviral supernatant mixed with 4
?g/ml Polybrene (Sigma) and selected with 2.5 ?g/ml puromycin (Sigma) to
establish transduced pools.
Cell culture and treatments. Parental NIH 3T3 cell lines or those expressing
either kinase-dead KD-ER and ROCK-ER were maintained in Dulbecco mod-
ified Eagle medium (DMEM) supplemented with 10% donor calf serum (DCS;
Gibco, Paisley, United Kingdom) at 37°C and 10% CO2. For most experiments,
0.5 ? 106cells were plated onto 100-mm cell culture dishes containing DMEM
plus 10% DCS. After 24 h, cells were cultured in serum-free DMEM (serum
starved) and treated with or without 4-hydroxytamoxifen (4-HT; 1 ?M; Sigma)
for 16 h in the presence or absence of pharmacological inhibitors. Inhibitors used
were U0126 (Promega) dissolved in dimethyl sulfoxide and used at 10 ?M and
Y-27632 (Calbiochem) dissolved in water and used at 10 ?M. Biocoat fibronec-
tin-coated and poly-D-lysine (PDL)-coated plates were purchased from Becton
For suspension studies, nearly confluent NIH 3T3 ROCK-ER fibroblasts
grown in DMEM plus 10% DCS were trypsinized and then placed into suspen-
sion in serum-free medium. Cells (1.5 ? 106) in serum-free medium were plated
onto 100-mm cell culture dishes coated with poly-hydroxyethyl-methacrylate
(poly-HEMA; Sigma) and treated with or without 4-HT, in the presence or
absence of either Y-27632 (10 ?M) or UO126 (10 ?M), for 15 h. For studies with
actin-disrupting compounds, nearly confluent NIH 3T3 ROCK-ER fibroblasts
grown in DMEM plus 10% DCS were trypsinized and 0.5 ? 106cells were plated
onto 100-mm cell culture dishes. After 24 h, cells were serum starved and treated
with or without 4-HT (1 ?M), in the presence or absence of cytochalasin D
(CCD; 1 ?M; Sigma), latrunculin B (LTB; 0.5 ?M; Calbiochem), swinholide A
(SWA; 0.05 ?M; Sigma), or jasplakinolide (Jasp; 0.5 ?M; Molecular Probes),
for 16 h.
Cell extraction and immunoblotting. Following treatment as described above,
cells were washed with phosphate-buffered saline (PBS) and then lysed in buffer
containing 10 mM Tris (pH 7.5), 5 mM EDTA, 1% (vol/vol) NP-40, 0.5%
(wt/vol) sodium deoxycholate, 40 mM sodium pyrophosphate, 1 mM Na3VO4, 50
mM NaF, 1 mM phenylmethylsulfonyl fluoride, 0.025% (vol/vol) sodium dodecyl
sulfate (SDS), 150 mM NaCl, and protease inhibitors. Lysates were clarified by
centrifugation at 13,000 ? g for 15 min. Sixty micrograms of each whole-cell
lysate was electrophoresed on SDS-polyacrylamide gel before electrotransfer to
nitrocellulose membrane. Blots were probed with antibodies (see above) and
appropriate horseradish peroxidase-conjugated secondary antibodies (Pierce),
followed by visualization by ECL (Amersham Pharmacia) or SuperSignal West
Femto (Pierce) according to the manufacturer’s instructions and exposure to
Kodak BioMax autoradiographic film. Alternatively, for determining the levels of
MLC and phospho-MLC, cells were lysed directly in 3? Laemmli sample buffer.
Samples were sonicated and boiled for 5 min, and the supernatant was clarified
by centrifugation at 16,000 ? g for 5 min. An appropriate volume of each sample
was electrophoresed and immunoblotted as described above.
Measurement of Ras activation. Following treatment as described above, cells
were washed with PBS and then lysed by scraping in MLB buffer (25 mM HEPES
[pH 7.5], 150 mM NaCl, 1% [vol/vol] IGEPAL [CA-630], 1 mM EDTA [pH 8.0],
10 mM MgCl2, 10% [vol/vol] glycerol, 1 mM Na3VO4, 25 mM NaF, 10 ?g/ml
aprotinin, 10 ?g/ml leupeptin). Cleared lysates were incubated for 45 min at 4°C
with glutathione-agarose beads coupled to glutathione S-transferase–Raf-1 RBD
(Upstate). The beads were washed three times with MLB buffer, and bound
proteins were solubilized by boiling with 60 ?l of 3? Laemmli buffer and
separated by SDS-polyacrylamide gel electrophoresis. Ras-GTP and total Ras
were detected by Western blotting with an antibody against Ras.
Immunofluorescence. Cells were fixed for 15 min in 4% (wt/vol) paraformal-
dehyde (PFA)–PBS and then permeabilized for 15 min in 0.5% Triton X-100–
PBS. After fixation and permeabilization, cells were washed three times in PBS
and then blocked with 2% (wt/vol) BSA–PBS for 1 h. Cells were incubated with
primary antibodies (1:1,000 dilution) for 60 min, followed by three washes with
2% BSA–PBS and a 60-min incubation with the corresponding fluorochrome-
conjugated secondary antibody (Jackson Immunoresearch Laboratories, Inc.,
West Grove, PA) at a 1:200 dilution. Filamentous actin structures were stained
with a 1:250 dilution of Texas Red-conjugated phalloidin (T7471; Molecular
Probes, Eugene, OR). For visualization of focal adhesions, cells were fixed and
permeabilized in one step with 4% PFA–0.2% Triton X-100–PBS. Primary an-
tibodies were as follows: rabbit anti-MLC (Ser19) (3671; Cell Signaling Tech-
nologies), mouse anti-cyclin D1 (sc-450; Santa Cruz), goat anti-lamin B1 (sc-
6217; Santa Cruz), and mouse anti-paxillin (610051; BD Biosciences). Coverslips
were mounted in Mowiol and visualized with a Bio-Rad MRC1024 confocal
BrdU analysis by fluorescence-activated cell sorter (FACS). For cell cycle
analysis, serum-starved NIH 3T3 ROCK-ER cells were left untreated or treated
with 4-HT (1 ?M), either in the presence or in the absence of Y-27632 (10 ?M)
or U0126 (10 ?M), for 16 h. Cells were pulsed with bromodeoxyuridine (BrdU;
10 ?M) for 2 h prior to harvesting with trypsin. Cells were fixed with ice-cold 70%
ethanol for 20 min at room temperature and then treated with 3 N HCl con-
taining 0.5% Triton X-100 for 20 min. Residual acid was neutralized by incu-
bating the cell suspension with 0.1 M sodium borate (pH 8.5) for 2 min at room
temperature. Cells were then incubated with anti-BrdU monoclonal antibody
(555627; BD Biosciences Pharmingen) diluted 1:200 for 20 min, followed by
fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G
(554001; BD Biosciences Pharmingen) for 20 min. The cell suspension was
incubated with propidium iodide (5 ?g/ml) for 30 min and then analyzed with a
FACScalibur flow cytometer and CellQuest software (Becton Dickinson).
siRNA transfection. Gene silencing was achieved by transient transfection of
small interfering RNA (siRNA) duplexes (Dharmacon, Inc., Lafayette, CO).
siRNA duplexes against mouse cyclin A (CCNA2; siGenome duplexes 2
VOL. 26, 2006ROCK REGULATION OF THE CELL CYCLE4613
[D-040393-02] and 3 [D-040393-03]), cyclin D1 (CCND1; siGenome duplexes 3
[D-042441-03] and 4 [D-042441-04]), p27 (CDKN1B; siGenome duplexes 3
[D-040178-02] and 4 [D-040178-04]), lamin A/C (D-001050-01; Dharmacon),
LIMK 1 (siGenome duplexes 2 [D-043923-02] and 3 [D-043923-03]), LIMK 2
(siGenome duplexes 1 [D-043932-01] and 2 [D-043932-02]), and nontargeting
control 1 (D-001210-01) were used. In brief, NIH 3T3 ROCK-ER cells were
plated at 1.2 ? 105cells per well of a six-well plate in antibiotic-free medium.
Cells were transfected the following day with 4 ?l of Lipofectamine 2000 (In-
vitrogen, Paisley, United Kingdom) and 50 nM siRNA duplexes per well accord-
ing to the manufacturer’s instructions. After 6 h, 20% DCS was added to give a
final serum concentration of 10%. siRNA complexes were removed 16 h later,
and fresh 10% DCS was added. Twenty-four hours later, cells were trypsinized
and plated at 0.5 ? 106cells per 10-cm plate in 10% DCS. The following day,
cells were serum starved and then treated with or without 4-HT (1 ?M) for 16 h.
Cells were harvested as described above.
In vitro cyclin E-CDK2 kinase assays. Cells were washed twice in ice-cold PBS
and lysed in ELB? buffer (250 mM NaCl, 50 mM HEPES [pH 7.0], 5 mM
EDTA, 10 mM ?-glycerol phosphate, 10 mM NaF, 1 mM sodium vanadate, 0.5
mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.2% Triton X-100, 10
?g/ml aprotinin, 10 ?g/ml leupeptin). Equal amounts of cell lysate (400 ?g in 0.5
ml of ELB? buffer) were incubated with 3 ?g of anti-cyclin E (07-687; BD
Biosciences) bound to 40 ?l of protein A-agarose beads (Upstate) for 90 min at
4°C. Immunocomplexes were washed twice in ELB?, once in 50 mM HEPES
(pH 7.4), and once in kinase buffer (50 mM HEPES [pH 7.4], 10 mM MgCl2, 10
mM ?-glycerol phosphate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride, 10 ?g/ml aprotinin, 10 ?g/ml leupeptin). The washed immunoprecipi-
tates were resuspended in 40 ?l of kinase buffer containing 2 ?g of histone H1
(Upstate), 50 ?M ATP, and 5 ?Ci of [?-32P]ATP and incubated at 30°C for 15
min. Kinase reactions were stopped by addition of 10 ?l of 6? Laemmli buffer.
After SDS-polyacrylamide gel electrophoresis and transfer,32P-labeled histone
H1 was visualized by exposure to Biomax film (Kodak). Membranes were sub-
sequently blotted with antibodies against CDK2 (06-505; Upstate) and p21 (sc-
397G; Santa Cruz).
ROCK activation promotes G1/S cell cycle progression. To
determine whether the selective activation of ROCK was suf-
ficient to induce cell cycle progression, we utilized condition-
ally activated ROCK-ER, which was generated by fusing
ROCK II amino acids 5 to 553, comprising the catalytic kinase
domain and a portion of the coiled-coil domain, to enhanced
green fluorescent protein and the hormone-binding domain of
the estrogen receptor (Fig. 1A) (17). We previously used
ROCK-ER to examine the role of ROCK in keratinocyte dif-
ferentiation (46), cell invasiveness (18, 70), and apoptotic
nuclear breakdown (16). NIH 3T3 mouse fibroblasts, which
have a biallelic deletion of the INK4a locus resulting in loss of
p16 expression, were transduced with retrovirus expressing
ROCK-ER (Fig. 1B). When cell lysates were blotted with
ROCK II antibody, there was no detectable ROCK-ER pro-
tein in untransduced NIH 3T3 cells and approximately equal
levels of ROCK II and ROCK-ER in untreated ROCK-ER-
expressing cells. Consistent with previous results (17), treat-
ment with the estrogen analogue 4-HT resulted in a roughly
twofold increase in ROCK-ER expression, which was not af-
fected by the ROCK inhibitor Y-27632. Western blotting with
anti-estrogen receptor antibody (ER?) confirmed the identity
of the ROCK-ER fusion protein. To determine the functional
effects of ROCK activation, cells that were untransduced or
expressing either ROCK-ER or a kinase-dead version
(K121G) called KD-ER were treated with 4-HT and the effects
on the phosphorylation of ROCK substrates were determined
(Fig. 1C). Treatment with 1 ?M 4-HT for 16 h had no effect in
parental NIH 3T3 or KD-ER-expressing cells, but ROCK-ER-
expressing cells had increased levels of phosphorylated LIMK1
(Thr508) and/or LIMK2 (Thr505) and phosphorylated regula-
tory myosin light chain (MLC2, Ser19). Coadministration of
the ROCK-selective inhibitor Y-27632 (75) blocked the 4-HT-
induced increase in phospho-LIMK and phospho-MLC, which
indicated that these events were due to ROCK-ER activation
by 4-HT and is in agreement with the lack of effect in 4-HT-
treated parental NIH 3T3 and KD-ER-expressing cells. West-
ern blotting with ER? antibody confirmed the expression of
KD-ER and ROCK-ER. When the effects on the actin-myosin
cytoskeleton were examined, it was clear that ROCK-ER ac-
tivation by 4-HT resulted in dramatic increases in F-actin,
which colocalized with phospho-MLC in prominent contractile
stress fibers (Fig. 1D). Treatment of parental NIH 3T3 cells or
KD-ER-expressing cells with 4-HT had no effect, while the
induction of actin stress fibers in ROCK-ER-expressing cells
could be blocked by Y-27632 coadministration (17), again con-
firming that these events were dependent upon ROCK-ER
In order to determine whether ROCK activity was sufficient
to induce cell cycle progression, ROCK-ER-expressing NIH
3T3 cells were serum starved and then either left untreated or
treated with 1 ?M 4-HT alone or in combination with 10 ?M
Y-27632 for 16 h prior to a 2-h pulse with BrdU, followed by
fixation, staining with propidium iodide, and FACS analysis
(Fig. 2A, percentage of S-phase cells for each condition indi-
cated). Mean data from three experiments revealed that only
FIG. 1. ROCK-ER activation induces phosphorylation of ROCK substrates and stress fiber formation. (A) Schematic representation of ROCK
II showing functional domains (PH ? pleckstrin homology domain, CRD ? cysteine-rich domain). Conditionally regulated ROCK-ER was
generated by fusing amino acids 5 to 553 of ROCK II between the enhanced green fluorescent protein (EGFP) and the estrogen receptor hormone
binding domain (hbER). KD-ER was created by changing lysine 121 to glycine [ROCK (K121G)]. (B) NIH 3T3 cells were transduced with
retrovirus encoding the conditionally active ROCK-ER fusion proteins. Serum-starved parental NIH 3T3 or ROCK-ER-expressing cells were left
untreated or treated with 1 ?M 4-HT for 16 h as indicated, either in the presence or in the absence of ROCK inhibitor Y-27632 (10 ?M). ROCK
II and ROCK-ER protein levels were determined by Western blotting with an antibody against a common epitope. Fusion protein expression was
confirmed by blotting with ER? antibody. Results of a representative experiment of three determinations with similar results are shown. (C) NIH
3T3 cells were transduced with retrovirus encoding the conditionally active ROCK-ER or kinase-dead KD-ER fusion protein. Serum-starved
parental NIH 3T3, KD-ER-expressing, or ROCK-ER-expressing cells were either left untreated or treated with 1 ?M 4-HT for 16 h as indicated,
either in the presence or in the absence of ROCK inhibitor Y-27632 (10 ?M). Cells were lysed, and protein phosphorylation status was determined
with antibodies against phospho-MLC2 (Ser19) and phospho-LIMK1/LIMK2 (antibody recognizing a common epitope at Thr508 and Thr505,
respectively). Fusion protein expression was confirmed by blotting with ER? antibody. Results of a representative experiment of three determi-
nations with similar results are shown. (D) Serum-starved, ROCK-ER-expressing NIH 3T3 cells were either left untreated or treated with 1 ?M
4-HT for 16 h and then fixed and stained for F-actin (green) and phospho-Ser19 MLC (red). Results of a representative experiment of two
determinations with similar results are shown.
VOL. 26, 2006ROCK REGULATION OF THE CELL CYCLE 4615
FIG. 2. ROCK-ER activation promotes S-phase entry and increased cell number. (A) Serum-starved, ROCK-ER-expressing NIH 3T3 cells
were either left untreated (Control) or treated with 1 ?M 4-HT for 18 h either in the presence or in the absence of ROCK inhibitor Y-27632 (10
?M). Cells were pulsed with 10 ?M BrdU for 2 h prior to harvest and then collected by trypsinization. Cells were fixed, stained with anti-BrdU
4616CROFT AND OLSONMOL. CELL. BIOL.
5% ? 3% of the serum-starved control cells were BrdU posi-
tive, indicating passage through the DNA-synthetic S phase,
whereas a statistically significant 33% ? 3% of the 4-HT-
treated cells were in S phase, comparable to the significant
32% ? 5% S-phase cells observed following treatment with
medium containing 10% serum (Fig. 2B). Passage through to
the G2/M phases was also higher in 4-HT-treated cells (14% ?
3% for 4-HT-treated cells versus 10% ? 2% for serum-starved
cells). Consistent with the previous results for 4-HT-induced
phosphorylation of ROCK substrates (Fig. 1C), 4-HT-stimu-
lated cell cycle progression was blocked by Y-27632 (Fig. 2B).
In addition, coadministration of 4-HT with the MEK inhibitor
U0126 (10 ?M) reduced the percentage of BrdU-positive cells
to the levels observed in cells treated with U0126 alone, indi-
cating that the ROCK-stimulated cell cycle progression was
dependent on MAPK signaling.
We also wished to determine whether ROCK-ER activation
would promote cell cycle progression through to the comple-
tion of cytokinesis. ROCK-ER-expressing cells were serum
starved for 24 h and then either counted in triplicate or allowed
to proceed for another 16 h without or with the addition of
4-HT. Although the untreated group did have a significant
25% increase in cell number, activation of ROCK-ER led to a
60% increase in cell number during the same period, which
was significantly greater than either of the untreated groups
(Fig. 2C). Therefore, in addition to the increased proportion of
cells with G2/M DNA content in Fig. 2B, the increase in cell
number indicates that ROCK activation also promotes com-
pletion of cell division.
ROCK influence on cell cycle regulatory proteins. Given
that ROCK activation stimulated cell cycle progression, we
next examined how ROCK influenced the levels of key cell
cycle regulatory proteins. Consistent with previous results (Fig.
1C), 4-HT treatment of KD-ER-expressing cells did not alter
LIMK phosphorylation, whereas ROCK-ER-expressing cells
responded with increased LIMK phosphorylation that could be
blocked by coadministration of Y-27632 (Fig. 3A). Similarly,
4-HT did not alter the expression of cell cycle regulatory pro-
teins in KD-ER-expressing cells, but cyclin A2 (hereafter
called cyclin A), cyclin D1, and p21 levels increased while p27
levels decreased in 4-HT-treated ROCK-ER-expressing cells.
In addition, coadministration of Y-27632 blocked the 4-HT-
induced effects while Y-27632 treatment alone reduced basal
levels of phospho-LIMK, cyclin A, cyclin D1, and p21. No
change in the expression of cyclin E (Fig. 3A), p18INK4C,
CDK2, CDK4, or CDK6 (data not shown) was observed under
any condition, while p15INK4Band cyclins A1, D2, and D3 were
undetectable in all treatment groups (data not shown). Given
the importance of cyclin D1 nuclear localization for the stim-
ulation of cell cycle progression (20), we examined cyclin D1
localization following 4-HT treatment. Similar to total cyclin
D1 levels observed by Western blotting (Fig. 3A), immunoflu-
orescence analysis revealed that untreated serum-starved cells
had little cyclin D1 (Fig. 3B). Treatment with 4-HT signifi-
cantly increased cyclin D1 expression, which accumulated
within cell nuclei. Coadministration of 4-HT with Y-27632
inhibited both cyclin D1 expression and nuclear accumulation.
We also compared the time course of ROCK-ER activation
with the induction of cyclin D1 and found that although strong
LIMK phosphorylation was observed at all time points follow-
ing 4-HT treatment, expression of cyclin D1 lagged and was
not evident until 16 h of treatment (Fig. 3C).
ROCK induction of cyclin D1 expression via Ras/MAPK.
Arguably, the primary function of ROCK is to regulate cellular
contractility by influencing actin-myosin cytoskeletal struc-
tures. Therefore, we tested whether disrupting F-actin would
affect ROCK-ER regulation of cyclin A and D1 expression.
Cells were treated with CCD, which caps actin filaments and
stimulates ATP hydrolysis on G-actin (62); SWA, which se-
questers G-actin as dimers (8); LTB, which sequesters G-actin
monomers (69); or Jasp, which disrupts F-actin and induces
polymerization of G-actin into amorphous masses (9). Each of
these treatments did not affect cyclin E expression or LIMK
phosphorylation in response to 4-HT activation of ROCK-ER;
however, cyclin D1 expression was eliminated by the actin-
disrupting compounds (Fig. 4A). The effects on cyclin A ex-
pression were mixed; in each case, basal expression was ele-
vated, which could be further increased by ROCK-ER
activation in CCD- or LTB-treated cells. In SWA- and Jasp-
treated cells, cyclin A levels could not be increased further by
ROCK-ER activation, suggesting that maximal expression had
been achieved. Since it has been shown that the Ras/MAPK
pathway regulates cyclin D1 expression (12), we examined Ras
activation in ROCK-ER-expressing cells by using a pull-down
assay with the Ras-binding domain of Raf-1. ROCK-ER acti-
vation with 4-HT led to a significant (P ? 0.05) twofold in-
crease in Ras-GTP levels, relative to untreated cells, which
could be blocked by coadministration with Y-27632 (Fig. 4B).
Treatment of cells with CCD to disrupt actin structures in-
creased basal Ras activation and led to only an ?25% increase
antibody and propidium iodide, and then analyzed by flow cytometry. Top panels show BrdU staining versus propidium iodide staining to quantify
S-phase cells, and bottom panels show cell numbers versus propidium iodide staining to quantify DNA content. (B) Serum-starved, ROCK-ER-
expressing NIH 3T3 cells were either left untreated (Control) or treated with 1 ?M 4-HT for 18 h, either in the presence or in the absence of
Y-27632 (10 ?M) or U0126 (10 ?M). Cells were pulsed with 10 ?M BrdU for 2 h prior to harvest and then collected by trypsinization. Cells were
fixed and stained with anti-BrdU antibody and propidium iodide, flow cytometry was performed, and the cell cycle distribution was analyzed. Mean
values ? the standard error of the mean from three repetitions are shown, except for U0126 treatment groups, where mean values from duplicate
determinations are reported. Statistical significance was determined by Student’s t test, and an asterisk indicates significant difference (P ? 0.05)
from serum-starved condition. (C) Three independent pools of ROCK-ER-expressing NIH 3T3 cells were serum starved for 24 h and then either
left untreated or treated with 1 ?M 4-HT for 16 h. The numbers of cells after initial serum starvation and after the additional 16 h without or with
4-HT were determined. Mean values ? the standard error of the mean, normalized to serum-starved cells, are indicated. Statistical significance
was determined by Student’s t test, and an asterisk indicates significant difference (P ? 0.05) from the initial serum-starved condition while a double
asterisk indicates significant difference (P ? 0.05) from both serum-starved conditions. Results of a representative experiment of two determi-
nations with similar results are shown.
VOL. 26, 2006 ROCK REGULATION OF THE CELL CYCLE4617
induced by ROCK-ER that was not statistically significant,
suggesting that an intact actin cytoskeleton was required for
robust Ras activation by ROCK, consistent with previous re-
In order to determine whether cytoskeletal integrity was
necessary for ROCK-ER induction of cell cycle progression,
ROCK-ER-expressing NIH 3T3 cells were serum starved and
then either left untreated or treated with 1 ?M 4-HT alone or
in combination with CCD or Jasp for 16 h prior to a 2-h pulse
with BrdU, followed by fixation, staining with propidium io-
dide, and FACS analysis. Mean data from three experiments
revealed that only 2% ? 1% of the serum-starved controls cells
were BrdU positive, indicating passage through the DNA-syn-
thetic S phase, whereas 35% ? 4% of the 4-HT-treated cells
were in S phase (Fig. 4C). Treatment with CCD or Jasp alone
did not affect the proportion of S-phase cells but did result in
accumulation of the G2/M population, likely because of inhi-
bition of cytokinesis. Administration of CCD or Jasp resulted
in significant reductions in the percentage of S-phase cells
induced by 4-HT to 9% ? 3% and 6% ? 5%, respectively.
These results indicate that the actin cytoskeleton is the prin-
cipal mediator of ROCK influence on the cell cycle, consistent
with the key contribution of the cytoskeleton to cyclin D1
induction by ROCK (Fig. 4A).
Given the increased Ras-GTP level following ROCK acti-
vation, the sensitivity of both cyclin D1 elevation (Fig. 4A) and
Ras activation (Fig. 4B) to CCD treatment, and the inhibition
of ROCK-ER-stimulated cell cycle progression by the MEK
inhibitor U0126 (Fig. 2B), one possibility was that the regula-
tion of some cell cycle proteins by ROCK-ER might be via the
MAPK pathway. We treated ROCK-ER-expressing cells with
4-HT alone in combination with Y-27632 or with U0126 and
then Western blotted cell extracts (Fig. 4D). Although the
4-HT induction of LIMK phosphorylation was sensitive to
Y-27632, U0126 had no effect. However, inhibition of MEK
resulted in inhibition of ERK phosphorylation and loss of
cyclin D1 and p21 induction. In contrast, the induction of
cyclin A and the decrease in p27 levels were unaffected by
U0126. These data are consistent with the hypothesis that
ROCK, acting via actin cytoskeletal structures, activates Ras
and the MAPK pathway, leading to the elevation of cyclin D1
and p21 expression. Cyclin A and p27 are apparently regulated
through MAPK-independent mechanisms, which also are in-
dependent of cell cycle progression since U0126 blocked entry
into S phase (Fig. 2B) without affecting the regulation of cyclin
A or p27 (Fig. 4D).
ROCK regulation of cyclin expression is independent of
focal adhesions. In addition to regulating actin stress fibers,
RhoA signals through ROCK to promote the formation of
integrin-containing focal adhesions, leading to increased sub-
strate adherence (41, 54, 75). Therefore, we wished to deter-
mine whether the effects of ROCK-ER activation were medi-
ated through focal adhesion signaling. ROCK-ER-expressing
FIG. 3. Expression of cell cycle regulators following ROCK-ER
activation. (A) Serum-starved, KD-ER- and ROCK-ER-expressing
NIH 3T3 cells were either left untreated or treated with 1 ?M 4-HT for
16 h, either in the presence or in the absence of 10 ?M Y-27632. Cell
lysates were Western blotted for phospho-LIMK1/2, cyclin A, cyclin
D1, cyclin E, p21, and p27. Equal protein loading was confirmed by
blotting with ERK2 antibody. Results of a representative experiment
of three determinations with similar results are shown. (B) Serum-
starved, ROCK-ER-expressing NIH 3T3 cells were either left un-
treated or treated with 1 ?M 4-HT, either in the presence or in the
absence of 10 ?M Y-27632, for 16 h. Coverslips were fixed, permeabil-
ized, and then stained with anti-cyclin D1 and anti-lamin B1 antibod-
ies. Results of a representative experiment of two determinations with
similar results are shown. (C) Serum-starved, ROCK-ER-expressing
NIH 3T3 cells were treated with 1 ?M 4-HT for the times indicated.
Lysates were analyzed by immunoblotting with antibodies against cy-
clin D1, phospho-LIMK1/2, and ERK2. Results of a representative
experiment of three determinations with similar results are shown.
4618 CROFT AND OLSONMOL. CELL. BIOL.
cells were plated on untreated plastic tissue culture dishes as
before, on fibronectin-coated dishes to promote the formation
of integrin-containing focal adhesions, or on PDL-coated
dishes to allow adhesion without integrin clustering. Activation
of ROCK-ER with 4-HT consistently led to LIMK phosphory-
lation, increased cyclin D1 expression, and increased ERK and
MEK phosphorylation in cells plated on each substrate (Fig.
5A). In addition, cyclin D1 induction and ERK phosphory-
lation were blocked by U0126 under all three conditions, in-
dicating that these events were mediated by the MAPK path-
FIG. 4. ROCK-ER induction of cyclin D1 by actin-mediated Ras activation. (A) Serum-starved, ROCK-ER-expressing NIH 3T3 cells were left
untreated or treated with 1 ?M 4-HT for 16 h, either in the presence or in the absence of 1 ?M CCD, 0.05 ?M SWA, 0.5 ?M LTB, or 0.5 ?M
Jasp. Whole-cell lysates were analyzed by Western blotting for cyclin A, cyclin D1, cyclin E, and phospho-LIMK1/2. Blotting with ERK2 antibody
was used to confirm equal protein loading. Results of a representative experiment of three determinations with similar results are shown.
(B) Serum-starved, ROCK-ER-expressing NIH 3T3 cells were either left untreated or treated with 1 ?M 4-HT or 5% serum as indicated, in the
presence or absence of 10 ?M Y-27632 or 1 ?M CCD, for 16 h. Active Ras-GTP was purified with glutathione S-transferase–Raf-1 RBD agarose
beads. GTP-loaded Ras and total Ras were detected by Western blotting. Quantitation is averaged results from three separate experiments;
average values ? the standard error of the mean are shown. An asterisk indicates a significant difference, as determined by Student’s t test, at P ?
0.05. (C) Serum-starved, ROCK-ER-expressing NIH 3T3 cells were either left untreated (Control) or treated with 1 ?M 4-HT for 18 h either in
the presence or in the absence of 1 ?M CCD or 0.5 ?M Jasp. Cells were pulsed with 10 ?M BrdU for 2 h prior to harvest and then collected by
trypsinization. Cells were fixed and stained with anti-BrdU antibody and propidium iodide, flow cytometry was performed, and the cell cycle
distribution was analyzed. Mean values ? the standard error of the mean from three repetitions are shown, except for the serum treatment group,
where mean values from duplicate determinations are reported. Statistical significance was determined by Student’s t test, and an asterisk indicates
significant difference (P ? 0.05) from the 4-HT-treated condition. (D) Serum-starved, ROCK-ER-expressing NIH 3T3 cells were either left
untreated or treated with 1 ?M 4-HT for 16 h, either in the presence or in the absence of 10 ?M Y-27632 or 10 ?M U0126. Cell lysates were
immunoblotted for phospho-LIMK1/2, cyclin A, cyclin D1, cyclin E, p27, p21, and phospho-ERK. Equal protein loading was confirmed by blotting
with ERK2 antibody. Results of a representative experiment of three determinations with similar results are shown.
VOL. 26, 2006 ROCK REGULATION OF THE CELL CYCLE4619
FIG. 5. Adhesion and integrin signaling are not required for ROCK-induced cyclin D1 expression. (A) ROCK-ER-expressing NIH 3T3 cells
were plated on uncoated tissue culture dishes or dishes coated with either fibronectin or PDL. Subconfluent cells were serum starved and then left
untreated or treated with 1 ?M 4-HT for 16 h, either in the presence or in the absence of 10 ?M Y-27632 or 10 ?M U0126. Cell lysates were
Western blotted for phospho-LIMK1/2, cyclin D1, phospho-ERK, ERK2, phospho-MEK1/2 (Ser217/221), MEK1/2, phospho-FAK (Tyr397), and
FAK. Results of a representative experiment of three determinations with similar results are shown. (B) ROCK-ER-expressing NIH 3T3 cells were
plated on uncoated glass coverslips or PDL-coated coverslips. Subconfluent cells were serum starved overnight and then left in serum-free medium
for an additional 16 h. After fixation and permeabilization, cells were stained with anti-paxillin antibody to visualize focal adhesions and with
4620 CROFT AND OLSONMOL. CELL. BIOL.
way. Treatment with U0126, which works by inhibiting MEK
catalytic activity (19), blocked ERK phosphorylation but in-
creased basal MEK phosphorylation, which could be further
elevated by ROCK-ER activation. These findings are consis-
tent with previous studies that showed that treatment with
MEK inhibitors led to increased MEK phosphorylation (5, 19,
31, 76). One possible explanation is that although these inhib-
itors block MEK activity, they can actually stimulate Raf ac-
tivity (2, 82), likely by reducing the effects of ERK-induced
negative feedback mechanisms. In contrast, there was no in-
crease in phosphorylation of FAK on the Src-kinase binding
site Tyr397. In parallel, cells plated on uncoated coverslips or
PDL-coated coverslips were serum starved as in Fig. 5A and
then stained for paxillin localization to visualize focal adhe-
sions and with phalloidin for F-actin structures. As shown in
Fig. 5B, cells plated on uncoated coverslips had numerous
intense paxillin-containing focal adhesions and prominent
stress fibers. In contrast, cells on PDL had dramatically fewer
focal adhesions and rare small stress fibers, the predominant
cytoskeletal structure being cortical actin. These data suggest
that ROCK-ER stimulation of MAPK and cyclin D1 expres-
sion is independent of integrin signaling.
We extended these findings by plating ROCK-ER-express-
ing cells on either untreated plastic tissue culture dishes or
dishes that had been coated with poly-HEMA, which does not
permit adhesion. Adherent or suspended cells treated with
4-HT to activate ROCK-ER had increased LIMK phosphory-
lation and cyclin A and D1 expression, although cyclin D1
induction was slightly attenuated in suspension cells (Fig. 5C).
Interestingly, phospho-ERK levels were elevated in untreated
cells in suspension, consistent with previous results reported
for NIH 3T3 cells (84), primary mouse embryo fibroblasts (89),
ARLJ301-3 rat liver cells (28), MDCK cells (11, 15), and
growth-arrested MCF-10A mammary epithelial cells (14).
However, cyclin D1 was not induced unless ROCK-ER was
activated with 4-HT, indicating that ROCK-ER regulation of
cyclin D1 has both MAPK-dependent and -independent com-
ponents and that the influence of ROCK on the expression of
cell cycle regulatory proteins does not require adhesion.
ROCK regulates cell cycle proteins via independent path-
ways. In order to determine whether the changes in cyclin A,
cyclin D1, and p27 are independent events, we used siRNA to
knock down the expression of each protein and examined the
effects of ROCK-ER activation on the remaining proteins. We
were particularly interested in the relationship between cyclin
A induction and p27 down-regulation, given that cyclin A-
CDK2 has been reported to phosphorylate p27 on T187, lead-
ing to its ubiquitylation and degradation (47), and that cyclin A
also has been proposed to regulate p27 via a noncatalytic
mechanism (88). We were only able to partially knock down
cyclin A with two independent siRNA duplexes but observed
no significant effects on LIMK phosphorylation or on cyclin D1
and p27 levels following 4-HT activation of ROCK-ER (Fig. 6A).
Transfection with control siRNA or lamin A/C duplexes also
had no effect on cyclin D1, cyclin A, or p27 regulation in
response to 4-HT activation of ROCK-ER (Fig. 6A to C).
Similarly, the efficient knockdown achieved with either cyclin
D1 siRNA duplex had no effect on LIMK phosphorylation or
cyclin A induction following ROCK-ER activation (Fig. 6B),
consistent with the lack of effect on cyclin A induction by
reduced cyclin D1 expression following treatment with U0126
(Fig. 4D). Finally, knockdown of p27 did not change ROCK-
ER-induced LIMK phosphorylation or cyclin A and cyclin D1
elevation (Fig. 6C). These data indicate that cyclin A, cyclin
D1, and p27 are each independently regulated by ROCK.
Transcription of the cyclin A2 gene has been shown to be
regulated largely by “pocket proteins” such as Rb or p107,
which are phosphorylated by cyclin D1- and cyclin E-associated
CDKs (6). However, the actin cytoskeleton has also been
shown to be an important regulator of cyclin A expression,
independent of pocket proteins (3, 7). Given that cyclin A
induction was still observed following MEK inhibitor treat-
ment (Fig. 4D) and after cyclin D1 knockdown (Fig. 6B), we
examined the phosphorylation of p107 in response to ROCK
activation. As shown in Fig. 7A, treatment with 4-HT increased
phospho-LIMK and led to a reduced-mobility form of p107,
indicative of increased phosphorylation. Efficient knockdown
of cyclin D1 still allowed 4-HT induction of LIMK phosphory-
lation but inhibited p107 phosphorylation. Consistent with
these findings, treatment with the MEK inhibitor U0126
blocked 4-HT-induced elevation of cyclin D1 levels and p107
phosphorylation (Fig. 7B). We next determined whether
knockdown of cyclin D1 led to changes in cyclin E-CDK2
activity. As also shown in Fig. 6B and 7A, knockdown of cyclin
D1 did not affect 4-HT-induced LIMK phosphorylation; how-
ever, cyclin E-associated CDK2 activity was effectively inhib-
ited in both siRNA treatment groups (Fig. 7C). Given that one
of the primary functions of cyclin D1-CDK4 and cyclin D1-
CDK6 complexes is to titrate p21 and p27 CDKIs away from
cyclin E-CDK2 (39), inhibition of cyclin E-associated CDK2
activity would be an expected outcome. Consistent with a role
in CDKI titration, increased p21 was associated with cyclin E
complexes following cyclin D1 siRNA and 4-HT treatment. It
is possible, however, that additional factors contribute to the
decreased CDK2 activity observed following cyclin D1 knock-
down. These results are consistent with those presented in Fig.
4D and 6B, revealing that ROCK induction of cyclin A is not
only independent of cyclin D1 but also essentially independent
of pocket protein phosphorylation.
LIMK mediates ROCK induction of cyclin A. Although
ROCK phosphorylates a number of proteins (55), the only
ROCK substrates previously implicated in cell cycle regulation
were the LIMKs (58). Therefore, we used siRNA to examine
the contribution of LIMK1 and LIMK2 to ROCK-ER-medi-
phalloidin to visualize F-actin structures. The scale bar represents 35 ?m. Results of a representative experiment of four determinations with
similar results are shown. (C) Serum-starved, ROCK-ER-expressing NIH 3T3 cells were trypsinized, resuspended in serum-free DMEM, and
replated on either uncoated tissue culture dishes (adherent [Adh]) or poly-HEMA-treated dishes (suspension [Susp]). Cells were left untreated
or treated with 1 ?M 4-HT for 16 h. Cell lysates were Western blotted for phospho-LIMK1/2, cyclin A, cyclin D1, cyclin E, phospho-ERK, and
ERK2. Results of a representative experiment of three determinations with similar results are shown.
VOL. 26, 2006 ROCK REGULATION OF THE CELL CYCLE4621
ated changes in the expression of cell cycle regulatory proteins.
The profound knockdown of LIMK1 only slightly affected the
level of phospho-LIMK observed following ROCK-ER activa-
tion with 4-HT, suggesting that LIMK2 is the major protein
detected by the phospho-LIMK antibody (Fig. 8A). Consistent
with this interpretation, knockdown of LIMK2 significantly
reduced the phospho-LIMK observed after 4-HT treatment,
and LIMK1 expression was significantly lower than LIMK2
expression in Western blot assays with all of the antibodies we
have tested (data not shown). LIMK1 knockdown did not
affect the induction of cyclin A and cyclin D1 or the decrease
in p27 following ROCK-ER activation. In contrast, knockdown
of LIMK2 reduced the cyclin A induction by ROCK-ER acti-
vation. Cyclin D1 levels were elevated when LIMK2 was
knocked down and could not be raised further by ROCK-ER
activation, consistent with the reported function of LIMK as a
repressor of cyclin D1 transcriptional induction by MAPK-
independent signaling pathways (58). Similar to LIMK1,
knockdown of LIMK2 did not affect ROCK-ER-induced re-
duction in p27 protein levels. The combined knockdown of
LIMK1 and LIMK2 had the same results as knockdown of
LIMK2 alone, in that ROCK-ER induction of cyclin A expres-
sion was reduced, basal cyclin D1 expression was elevated, and
p27 regulation was unaffected.
Using a conditionally activated form of ROCK, in combina-
tion with the potent ROCK inhibitor Y-27632, we have deter-
mined that ROCK activation leads to increased levels of cyclin
A and cyclin D1 and decreased levels of p27 in NIH 3T3
fibroblasts (Fig. 8B). Each of these pathways operated inde-
pendently, since siRNA-mediated knockdown of each protein
did not alter the expression of the others (Fig. 6). Also, MEK
inhibition blocked ROCK-ER induction of cyclin D1 (Fig. 4D)
and cell cycle progression (Fig. 2B) without affecting the cyclin
A or p27 response (Fig. 4D), indicating that these proteins are
not regulated by the MAPK pathway, cyclin D1 expression,
or progression into S phase. The induction of cyclin A by
ROCK-ER was reduced by siRNA-mediated knockdown of
LIMK2, which also resulted in elevated basal cyclin D1 expres-
sion (Fig. 8A). However, given that cyclin A knockdown did
not elevate basal cyclin D1 (Fig. 6A), these results suggest that
the cyclin D1 regulation was independent of cyclin A and are
consistent with the proposed role of LIMK in repressing the
induction of cyclin D1 transcription by Rac and/or Cdc42 (58).
It has previously been shown that active Rho influences cell
cycle progression via the regulation of a number of cell cycle
regulatory proteins. One mechanism is through the regulation
of p27. Inhibition of Rho function has been reported to elevate
FIG. 6. Independent effects of ROCK-ER on cyclin A, cyclin D1,
and p27 levels. ROCK-ER-expressing NIH 3T3 cells were transfected
with control nontargeting (Con) siRNA or siRNA duplexes targeting
Lamin A/C, Cyclin A (A), Cyclin D1 (B), or p27 (C). At 24 h post-
transfection, cells were trypsinized and replated on plastic tissue cul-
ture dishes. Subconfluent cells were serum starved and then either left
untreated or treated with 1 ?M 4-HT for 16 h. Whole-cell lysates were
immunoblotted for phospho-LIMK, cyclin A, cyclin D1, p27, and lamin
A/C. Equal protein loading was confirmed by blotting with ERK2
antibody. Results of representative experiments of three or four de-
terminations with similar results are shown.
4622CROFT AND OLSONMOL. CELL. BIOL.
p27 protein levels (26, 27, 40, 56, 81), whereas expression of
active Rho decreased p27 (27, 40, 56). Although inhibition of
ROCK activity with Y-27632 has been associated with im-
paired p27 down-regulation (22, 29, 30, 35, 64), it has not been
clear whether the effect of ROCK inhibition on p27 levels is
direct or a secondary consequence of decreased proliferation.
In this study, we show that the selective activation of ROCK is
sufficient to lower p27 levels, independently of MAPK activa-
tion or the induction of cyclin A or cyclin D1. In addition to the
possibility that ROCK activity promotes p27 protein degrada-
tion, ROCK might repress p27 translation since Rho inhibition
was shown to increase p27 mRNA translation through a Rho-
responsive element in the 3?-untranslated region (78). More
research is required to elucidate the mechanism of ROCK-
induced p27 down-regulation.
Another mechanism that contributes to cell cycle regulation
is Rho-mediated suppression of p21 transcription (1, 4, 23, 42,
50). However, ROCK function was found not to be required
for p21 suppression by Rho in normal and Ras-transformed
fibroblasts or in colon carcinoma cell lines (57, 59, 61). Our
results indicate that ROCK activation increased p21 levels
(Fig. 3A), likely as a result of Ras/MAPK activation (43) and
possibly from increased cyclin D1 leading to p21 protein sta-
bilization (13). However, the induction of p21 was observed
under conditions that were permissive for G1/S progression
(Fig. 2), indicating that the extent of p21 induction was com-
patible with proliferation.
In addition to the regulation of CDKIs, Rho has been re-
ported to influence cyclin levels. Rho and ROCK are necessary
for Ras-GTP loading and sustained ERK activation, leading to
increased cyclin D1 transcription following growth factor stim-
ulation, through their contribution to the maintenance of actin
stress fibers (57, 71, 83). Our data indicate that the sustained
activation of ROCK, which leads to the formation of strong
actin stress fibers (Fig. 1D), is sufficient to induce cell cycle
progression (Fig. 2), Ras-GTP loading (Fig. 4B), ERK activa-
tion (Fig. 4D), and increased MAPK-dependent cyclin D1
expression (Fig. 4D). A model has been proposed that suggests
that ROCK activation of LIMK results in the transcriptional
FIG. 7. Cyclin D1 knockdown inhibits ROCK-ER-induced p107
phosphorylation and cyclin E-associated CDK2 activity. (A) ROCK-
ER-expressing NIH 3T3 cells were transfected with control nontarget-
ing (Con) siRNA or siRNA duplexes targeting Cyclin D1. At 24 h
posttransfection, cells were trypsinized and replated on plastic tissue
culture dishes. Subconfluent cells were serum starved and then either
left untreated or treated with 1 ?M 4-HT for 16 h. Whole-cell lysates
were immunoblotted for phospho-LIMK, p107, and cyclin D1. Equal
protein loading was confirmed by blotting with an antibody against
ERK2. Results of a representative experiment of two determinations
with similar results are shown. (B) Serum-starved, ROCK-ER-express-
ing NIH 3T3 cells were either left untreated or treated with 1 ?M
4-HT for 16 h, either in the presence or in the absence of 10 ?M
U0126. Cell lysates were immunoblotted for phospho-LIMK, p107,
cyclin D1, and phospho-ERK. Equal protein loading was verified by
blotting with an antibody against ERK2. Results of a representative
experiment of two determinations with similar results are shown. (C)
ROCK-ER-expressing NIH 3T3 cells were transfected with control
nontargeting (Con) siRNA or siRNA duplexes targeting Cyclin D1. At
24 h posttransfection, cells were trypsinized and replated on plastic
tissue culture dishes. Subconfluent cells were serum starved and then
either left untreated or treated with 1 ?M 4-HT for 16 h. Whole-cell
lysates (WCL, lower half) were immunoblotted for phospho-LIMK,
cyclin D1, cyclin E, and CDK2. Equal protein loading was confirmed
by blotting with ERK2 antibody. Cyclin E was immunoprecipitated
(IP), and associated CDK activity was assayed in vitro with histone H1
as the substrate. Cyclin E-associated CDK2 and p21 were revealed by
Western blotting (WB) the immunoprecipitated complexes that had
been assayed for kinase activity. Results of a representative experiment
of two determinations with similar results are shown.
VOL. 26, 2006 ROCK REGULATION OF THE CELL CYCLE4623
repression of Rac/Cdc42-induced cyclin D1 transcription and
that inhibition of ROCK relieves the LIMK-mediated repres-
sion (58). We did observe that siRNA-mediated knockdown of
LIMK2 resulted in increased basal cyclin D1 expression (Fig.
8A), suggesting that LIMK-mediated transcriptional repres-
sion may be independent of its ROCK-regulated catalytic
activity, possibly acting through direct actions on nuclear
NIH 3T3 cells transformed with a constitutively active ver-
sion of RhoA were reported to have elevated levels of cyclin A
by microarray analysis (73). In addition, inhibition of Rho or
ROCK function in atrial myofibroblasts inhibited proliferation
and blocked cyclin A expression (52). However, the possibility
exists that cyclin A expression was influenced indirectly
through effects on cell cycle regulation and not necessarily a
direct or specific RhoA- or ROCK-induced phenomenon in
these studies. We found that ROCK-ER activation led to in-
creased cyclin A levels, which were independent of MAPK
activity, p107 phosphorylation, or cyclin D1 and p27 changes.
We found that disruption of the actin cytoskeleton resulted in
elevated basal cyclin A expression, which could be further
increased by ROCK activation in CCD- and LTB-treated cells
(Fig. 4A). We believe that in SWA- and Jasp-treated cells, the
elevated basal cyclin A expression was maximal such that
ROCK activation could not elevate expression further. These
data suggest that the regulation of cyclin A by ROCK is inde-
pendent of actin regulation, in direct contrast to the regulation
of cyclin D1, which appears to require functional actin cy-
toskeletal structures. Knockdown of LIMK2 resulted in de-
creased cyclin A induction by ROCK-ER (Fig. 8A). Combin-
ing these results suggests that cyclin A induction by ROCK is
mediated by an actin-independent LIMK-mediated pathway,
similar to the LIMK-mediated, actin-independent repression
of Rac/Cdc42-stimulated cyclin D1 transcription (58). It re-
mains to be determined whether nuclear translocation of
LIMK2 is required for cyclin A regulation, as has been shown
for LIMK-mediated repression of cyclin D1 transcription (58).
Treatment of endothelial cells with tumor necrosis factor
alpha was recently reported to result in cyclin A transcriptional
repression, mediated by ROCK phosphorylation of ezrin and
consequent nuclear translocation of ezrin, where it repressed
transcription by binding to cell cycle homology region repres-
sor elements within the cyclin A promoter (37). In contrast, we
found that ROCK activation actually increased cyclin A ex-
pression (Fig. 4A) and have previously reported no change in
ezrin phosphorylation under conditions where tumor necrosis
factor alpha treatment of NIH 3T3 cells led to increased
ROCK-mediated phosphorylation of LIMK and MLC2 (16).
The differences between these findings may be the conse-
quence of cell-specific factors.
It has been reported that Rho activity is necessary for cyclin
E expression in rat astrocytes (72). Our findings indicate that
ROCK activity is neither necessary nor sufficient for cyclin E
expression (Fig. 3A), suggesting that there may also be cell
type-specific factors that contribute to cyclin E regulation.
We show in this study that ROCK activation is sufficient to
induce G1/S-phase cell cycle progression in p16?/?NIH 3T3
mouse fibroblasts, which is associated with increased levels of
cyclin D1 and cyclin A and lowered p27 levels. Previous re-
search has implicated Rho and ROCK signaling in the regu-
lation of these proteins; however, the experimental design has
not always allowed effects on cell cycle regulatory proteins to
be dissociated from global effects that result from cell cycle arrest.
Also, given that many of these previous studies have relied upon
inhibition of ROCK, it has not always been possible to conclude
that ROCK is both necessary and sufficient for the observed
FIG. 8. LIMK2 knockdown abrogates ROCK-ER-induced cyclin A
upregulation. (A) ROCK-ER-expressing NIH 3T3 cells were trans-
fected with control nontargeting (Con) siRNA or siRNA(s) against
LIMK1, LIMK2, LIMK1 and -2 in combination, or Lamin A/C. At 24 h
posttransfection, cells were trypsinized and replated on plastic tissue
culture dishes. Subconfluent cells were serum starved and then either
left untreated or treated with 1 ?M 4-HT for 16 h. Whole-cell lysates
were immunoblotted for phospho-LIMK, LIMK 1, LIMK 2, cyclin A,
cyclin D1, p27, and lamin A/C. Blotting for ERK2 confirmed equal
protein loading. Results of a representative experiment of three de-
terminations with similar results are shown. (B) Model of ROCK
regulation of cell cycle proteins. ROCK-ER stimulation leads to acti-
vation of Ras and MAPK, which, along with an additional independent
pathway, leads to elevation of cyclin D1 expression. Cyclin A is inde-
pendently elevated in response to ROCK activation via LIMK2. Re-
duced p27 protein levels were observed following ROCK activation,
independent of the effect on cyclin D1, cyclin A, or cell cycle
4624 CROFT AND OLSONMOL. CELL. BIOL.
effects on cell cycle regulators. Through the selective activation
of conditionally regulated ROCK-ER, we found that (i)
ROCK stimulation of actin stress fibers is sufficient to activate
the Ras/MAPK pathway and elevate cyclin D1 expression; (ii)
ROCK regulation of cyclin D1, that of cyclin A, and that of p27
are independent events; and (iii) cyclin A is regulated by
ROCK through a previously uncharacterized LIMK-mediated
mechanism. These results broaden our knowledge of Rho and
ROCK regulation of the cell cycle, which may help in the
design of anticancer therapies that target this signaling path-
way (79). Two questions that must be examined in more detail
to determine whether these results are broadly applicable are
(i) whether human cells behave in the same manner as mouse
cells and (ii) whether the absence of p16 in NIH 3T3 cells
contributes to the ability of ROCK to promote cell cycle pro-
We thank Sharon Benzeno, Alan Diehl, and Andrew Gladden for
useful discussions and reagents. We also thank Chris Marshall (ICR,
London, United Kingdom) for anti-ERK2 antibody and Martin
McMahon (San Francisco, CA) for the Raf-ER plasmid.
This research was supported by Cancer Research UK and a grant to
M.F.O. from the NIH (CA030721).
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