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INTRODUCTION |
Cell division, motility, and shape determination depend on
reorganization of the actin cytoskeleton. Actin cytoskeletal dynamics is precisely regulated by a variety of actin-binding proteins involved
in polymerization/depolymerization, capping, and bundling of actin
filaments (1, 2). Extensive remodeling of the actin cytoskeleton with
exposure to extracellular signals is regulated by Rho family GTPases
(including Rho, Rac, and Cdc42), and each member participates in the
specific regulation of the actin cytoskeleton and various cell
adhesion-related events (3-6). Several direct or indirect downstream
effectors of Rho family GTPases have been identified, but signal
transduction pathways by which Rho family GTPases evoke specific actin
reorganization are not completely understood.
Small actin-binding proteins of the actin-depolymerizing factor/cofilin
family play a definitive role in depolymerization of actin filaments
(7, 8) and, hence, are potential downstream effectors of signaling
cascades that evoke actin cytoskeletal reorganization. A mechanism
involved in regulating cofilin function is phosphorylation of an
N-terminal serine residue of cofilin, through which its
F-actin-depolymerizing activity is inactivated (9, 10). Studies showed
that LIM-kinase (LIMK),1 LIM
domain-containing serine/threonine/protein-tyrosine kinase (11-17),
phosphorylates cofilin downstream of Rho family GTPases (18-20). We
report that two types of LIMKs, LIMK1 and LIMK2, are regulated in a
distinct manner downstream of Rho family GTPases. LIMK1 is specifically
activated downstream of Rac1 and Cdc42 but not RhoA, whereas LIMK2 is
specifically activated downstream of RhoA and Cdc42 but not Rac1 (20).
LIMKs may be indirectly activated by Rho family GTPases, because LIMKs
are not a direct target of these GTPases.
Several target proteins of Rho are involved in regulating the actin
cytoskeleton. Among them, ROCK, an isoform of Rho- kinase, plays a key
role in formation of actin stress fibers downstream of Rho (21-26).
Previous reports showed that the constitutive active form of ROCK
activates both LIMK1 and LIMK2 (27) and that LIMK1 is activated by
wild-type ROCK (28). However, these reports contradict a previous
notion that LIMK1 is specifically activated by Rac but not by Rho
(18-20). In the present study, we asked if ROCK could specifically
activate LIMK2 downstream of RhoA. We found that signal transduction
pathways wherein ROCK directly phosphorylates and activates LIMK2, but
not LIMK1 downstream of RhoA resulted in phosphorylation and
inactivation of the actin-depolymerizing factor cofilin.
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EXPERIMENTAL PROCEDURES |
Materials--
Fluorescein isothiocyanate-conjugated anti-mouse
IgG, anti-hemagglutinin (HA) monoclonal antibody (12CA5), and anti-Myc
monoclonal antibody (9E10) were purchased from Roche Molecular
Biochemicals. Anti-HA (Y-11), anti-Myc (A-14) polyclonal antibody and
rhodamine-conjugated phalloidin were, respectively, purchased from
Santa Cruz Biotechnology (Santa Cruz, CA) and Molecular Probes, Inc.
(Eugene, OR). Anti-LIMK2 polyclonal antibody was generated as described
elsewhere (29). LipofectAMINE and Opti-MEM were purchased from Life
Technologies, Inc. Y-27632 was generously provided by WelFide Co.
(Osaka, Japan).
Construction of Expression Plasmids and Preparation of
Recombinant Protein--
The expression plasmids for Rho family
GTPases (pEF-BOS-myc), HA-tagged LIMK1, LIMK2 and its mutants, and
glutathione S-transferase (GST)-fused cofilin were
constructed as described (20). Expression plasmids for Myc-tagged ROCK
and its mutants were kindly provided by Dr. S. Narumiya (Kyoto
University, Kyoto, Japan). The cDNA for T494V and T494E mutants of
LIMK2 were constructed to introduce substitution of Thr-494 by Val and
Glu residues, respectively, using a site-directed mutagenesis kit
(CLONTECH, Palo Alto, CA). The plasmids for T505V,
T505E, and T494V/T505V mutants of LIMK2 were generated in a similar
manner. To generate the plasmid encoding for the GST-fused protein
kinase domain of the kinase-dead LIMK2 (GST-PK) and its T494V/T505V
mutants, the cDNA fragment of the kinase-dead LIMK2 was amplified
by polymerase chain reaction using a set of the following primers:
forward, 5'-GCGGATCCCCCTGTGACCTGATCCAC-3', and the reverse,
5'-GCGGATCCCTAGGGTGGCGAGTCCC-3'. The polymerase chain reaction product
digested with BamHI was ligated to the BamHI-digested pGEX-6P-2 vector (Amersham Pharmacia
Biotech). The authenticity of these expression plasmids was confirmed
by nucleotide sequence analysis. The plasmids coding for GST-fused cofilin, GST-PK, and its mutants were transformed into
Escherichia coli BL-21. Expression and purification of
recombinant protein were done using GST purification modules (Amersham
Pharmacia Biotech), according to the manufacturer's protocol.
Transfection--
COS-7 cells were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum.
HeLa cells were maintained in minimal essential medium supplemented
with 10% fetal bovine serum and non-essential amino acids.
Subconfluent COS-7 cells were trypsinized and resuspended in
phosphate-buffered saline (PBS), and 106 cells were
transfected with 10 µg of plasmid DNA by electroporation using a Gene
Pulser (Bio-Rad) according to the manufacturer's instructions. Cells
were cultured for 36 h in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum.
HeLa cells were plated on a glass coverslip at a density of 6 × 103/cm2, cultured for 12 h, then further
cultured for 16 h in serum-free minimal essential medium. The
cells were transfected in Opti-MEM containing 1 µg of plasmid DNA
complexed with LipofectAMINE. After a 2-h incubation, the medium was
replaced with serum-free minimal essential medium, and the cells were
further cultured for 22 h, then fixed and stained.
Immunoprecipitation, Immunoblot Analysis, and Protein Kinase
Assay--
COS-7 cells were transiently transfected with expression
plasmid, as described above, then cultured for 36 h. The cells
were lysed in 1 ml of lysis buffer consisting of 50 mM
Tris-HCl (pH 7.5), 0.5 M NaCl, 25 mM
-glycerophosphate, 10 mM NaF, 1 mM
Na3VO4, 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin and
aprotinin, and incubated on ice for 30 min. After centrifugation, the
supernatant was preadsorbed with 15 µl of protein G-Sepharose
(Amersham Pharmacia Biotech) for 1 h at 4 °C and centrifuged to
remove protein G-Sepharose, and the supernatant was incubated for
3 h at 4 °C with anti-HA or anti-Myc antibody and 5 µl of
protein G-Sepharose. Protein G-Sepharose beads were washed three times
with lysis buffer and dissolved in the sample buffer for
SDS-polyacrylamide gel electrophoresis. The immunoprecipitates and cell
lysates were, separated by SDS-polyacrylamide gel electrophoresis and
electroblotted onto a polyvinylidene difluoride membrane (Bio-Rad) and
probed with anti-HA, anti-Myc, or anti-LIMK2 antibody. Proteins
reacting with these antibodies were detected using ECL enhanced
chemiluminescence (Amersham Pharmacia Biotech).
For protein kinase assay, immunoprecipitates bound to protein
G-Sepharose as described above were washed three times with kinase
buffer consisting of 50 mM Hepes-NaOH (pH 7.5), 25 mM
-glycerophosphate, 5 mM
MgCl2, 5 mM MnCl2, 10 mM NaF, 1 mM Na3VO4 and
then incubated for 20 min at 30 °C in 15 µl of kinase buffer
containing 50 µM ATP, 5 µCi of
[
-32P]ATP (6000 Ci/mmol; PerkinElmer Life
Sciences) and 6 µg of GST-cofilin or 2 µg of GST-PK as
substrate. After incubation for 20 min at 30 °C, the reaction was
terminated by heat treatment (100 °C for 3 min) in sample buffer for
SDS-polyacrylamide gel electrophoresis, subjected to SDS-polyacrylamide
gel electrophoresis, and analyzed by autoradiography.
Phosphoamino Acid Analysis--
Phosphoamino acid analysis was
done as described (30). The region of the polyacrylamide gel containing
the radioactive protein was excised and eluted, protein from the gel
was incubated with 6 N HCl at 110 °C for 1 h, and
hydrolysates were separated on a thin-layer cellulose plate using of
the Hunter thin-layer electrophoresis system (HTLE-7000; CBS
Scientific, Del Mar, CA). The 32P-labeled phosphoamino
acids were detected by autoradiography, then compared with the
ninhydrin-stained phosphoamino acid standards.
Immunofluorescence Analysis--
HeLa cells were fixed with 4%
paraformaldehyde in PBS for 20 min and treated with PBS containing
0.2% Triton X-100 for 3 min at room temperature. After washing three
times with PBS, the cells were incubated with anti-HA for 1 h and
subsequently with fluorescein isothiocyanate-conjugated anti-mouse IgG
and rhodamine-conjugated phalloidin for 1 h. The cells were then
washed three times with PBS, mounted on glass slides, and analyzed with
use of a LSM 410 confocal laser scanning microscopy (Carl Zeiss,
Oberkochen, Germany).
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RESULTS |
Specific Activation of LIMK2 by ROCK in the Downstream of
Rho--
To determine if LIMKs could be activated by ROCK, wild type
and mutant types of this protein kinase were, respectively,
co-expressed with HA-tagged LIMK1 or LIMK2 in COS-7 cells, then we
measured LIMKs activity using GST-fused cofilin as substrate (Fig.
1). ROCK-
3 contains the protein kinase
domain and half of the coiled-coil domain, which served as the
constitutively active form of ROCK (24). LIMK1 and LIMK2 activities
toward cofilin phosphorylation in cells co-expressing ROCK-
3 were,
respectively, 5.3-fold and 7.3-fold higher than those seen in control
cells expressing LIMK1 or LIMK2 alone (Fig. 1A). Likewise,
autophosphorylation of LIMK1 and LIMK2 was also slightly enhanced by
co-expression with ROCK-
3 (Fig. 1A, arrows).
Importantly, however, when LIMK1 or LIMK2 was co-expressed with
wild-type ROCK, LIMK2 activity toward cofilin phosphorylation was
enhanced 2.8-fold, whereas LIMK1 activity was not stimulated (Fig.
1B). Stimulatory effects on LIMK1 and LIMK2 activities were
not seen when LIMK1 or LIMK2 was co-expressed with the protein
kinase-deficient form of ROCK (KDIA) (Fig. 1A). Similar
results were obtained when these proteins were transiently expressed in
HeLa and NIH3T3 cells (data not shown). The results indicate that
wild-type ROCK activates LIMK2 but not LIMK1 in the cells, depending on
the protein kinase activity, whereas deletion of the C-terminal half of
ROCK results in a non-selective activation of LIMKs.

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Fig. 1.
Specific activation of LIMK2 kinase activity
by ROCK. A, activation of LIMK1 and LIMK2 kinase
activity by dominant active ROCK (ROCK- 3). COS-7 cells were
co-expressed with HA-tagged LIMK1 or LIMK2 and empty vector
(Mock), Myc-tagged ROCK- 3, or KDIA. B,
activation of LIMK2 by wild-type (w.t.) ROCK. COS-7 cells
were co-expressed with HA-tagged LIMK1 or LIMK2 and empty vector
(Mock) or Myc-tagged wild-type ROCK. After transient
expression, immunoprecipitated LIMK1 and LIMK2 were used for in
vitro kinase assay using GST-cofilin as substrate (top
panel) and for anti-HA immunoblots (middle panel). Cell
lysates (50 µg) were also used for anti-Myc immunoblots (bottom
panel). The arrow indicates autophosphorylated LIMK1
and LIMK2, respectively. Arrowheads indicate phosphorylated
cofilin. Cofilin phosphorylation was estimated using an image analyzer
(model BAS-2000; Fuji Film, Kanagawa Japan), and the amount of cofilin
phosphorylated by LIMK1 or LIMK2 in Mock cells was taken as 1.0. Each
value represents the mean ± S.E of three independent
experiments.
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To further confirm the specific activation of LIMK2 by ROCK, we
examined effects of Y-27632, a specific inhibitor of ROCK (31), on the
activation of LIMKs by Rho family GTPases (Fig. 2). We reported that LIMK2 activity was
regulated by RhoA and Cdc42 but not Rac1, whereas LIMK1 activity was
regulated by Cdc42 and Rac1 but not RhoA (20). Consistent with these
data, LIMK2 activity was enhanced 2.4-fold by the co-expression of
dominant active RhoA (RhoV14) (Fig. 2A). The activation of
LIMK2 by RhoV14 was completely blocked by treatment of the cells with
Y-27632. Suppression of kinase activity in cells expressing LIMK2 alone by Y-27632 may be due to inhibition of endogenous ROCK, since Y-27632
did not directly inhibit the protein kinase activity of LIMK2 (data not
shown). These findings indicate that the activation of LIMK2 by RhoA
required ROCK activity. In contrast, when LIMK1 was co-expressed with
the dominant active Rac1 (RacV12), LIMK1 activity toward to cofilin
phosphorylation was stimulated to a 3.3-fold higher level than was the
basal LIMK1 activity (Fig. 2B). However, the addition of
Y-27632 did not affect the LIMK1 activity enhanced by RacV12. This
means that the activation of LIMK1 by Rac1 is not mediated by ROCK.
Taken together, these observations strongly indicate that ROCK
specifically activates LIMK2, but not LIMK1 downstream of RhoA, whereas
the Rac1-dependent activation of LIMK1 is not mediated by
ROCK.

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Fig. 2.
Effect of the ROCK inhibitor Y-27632 on
RhoA-activated LIMK2 (A) and Rac1-activated LIMK1
activity (B). A, COS-7 cells were
co-expressed with HA-tagged LIMK2 and empty vector (Mock) or
Myc-tagged RhoV14, respectively. B, COS-7 cells were
co-expressed with HA-tagged LIMK1 and empty vector (Mock) or
Myc-tagged RacV12, respectively. Transiently transfected COS-7 cells
were cultured for 36 h and incubated for 30 min with or without 10 µM Y-27632. Immunoprecipitated LIMK1 and LIMK2 from cell
lysates were, respectively, used for in vitro kinase assay
using GST-cofilin as substrate (top panel) and for anti-HA
immunoblots (middle panel). Cell lysates (50 µg) were also
used for anti-Myc immunoblots (bottom panel). The
arrow indicates autophosphorylated LIMK1 and LIMK2,
respectively. Arrowheads indicate phosphorylated cofilin.
The amount of cofilin phosphorylated by LIMK1 or LIMK2 in Mock cells
was taken as 1.0. Each value represents the mean ± S.E. of three
independent experiments.
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ROCK-dependent LIMK2 Activation through Protein Kinase
Domain--
We reported that the activity of LIMK2 is regulated by
RhoA and Cdc42 through its protein kinase domain of LIMK2 (20). Here, we asked if the activation of LIMK2 activity by ROCK is also through the kinase domain (Fig. 3). Consistent
with data in Fig. 1A, the wild-type LIMK2 activity was
enhanced 10.5-fold by the co-expression with ROCK-
3 (Fig.
3A). Similar to findings with the wild-type LIMK2, the
kinase activity of PK mutant (which is deleted with N-terminal half
containing LIM domains) was also enhanced to about 10-fold by the
co-expression with ROCK-
3, indicating that LIMK2 through its protein
kinase domain is activated by ROCK.

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Fig. 3.
Activation and phosphorylation of LIMK2
protein kinase domain (PK) by ROCK. A, activation of
LIMK2 and PK toward cofilin phosphorylation by ROCK. COS-7 cells were
co-expressed with HA-tagged LIMK2 or its protein kinase domain (PK) and
empty vector (Mock) or Myc-tagged ROCK- 3. After transient
expression, immunoprecipitated LIMK2 and PK were used for in
vitro kinase assay using GST-cofilin as substrate (top
panel) and for anti-HA immunoblot (middle panel). Cell
lysates (50 µg) were also used for anti-Myc immunoblot (bottom
panel). The arrow indicates autophosphorylated
full-length LIMK2. The amount of cofilin phosphorylated by LIMK2 in
Mock cells was taken as 1.0. Each value represents the mean ± S.E. of
three independent experiments. B, phosphorylation of PK by
ROCK. COS-7 cells were transfected with expression vector for
Myc-tagged ROCK- 3 or KDIA. After transient expression,
immunoprecipitated ROCK- 3 and KDIA were used for in vitro
kinase assay using GST or GST-PK as substrate (top panel)
and for anti-Myc immunoblot (middle panel). Substrates were
stained by Coomassie Brilliant Blue (C.B.B.) (bottom
panel). The arrow indicates autophosphorylated
ROCK- 3. C, phosphoamino acid analysis of phosphorylated
PK. The 32P-labeled GST-PK band was excised from the
polyacrylamide gel prepared as in B, hydrolyzed, and
analyzed by thin-layer electrophoresis. Positions of phosphoserine
(pSer), phosphothreonine (pThr), phosphotyrosine
(pTyr), sample origin (Ori), and free phosphate (Pi) are
indicated.
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To further address the mechanism of ROCK-dependent LIMK2
activation, protein kinase domain of the kinase-dead LIMK2 (PK) (which did not undergo autophosphorylation in the in vitro
phosphorylation assay) was prepared as the GST-fused protein, and we
determined if ROCK directly phosphorylates this recombinant GST-fused
PK as a substrate. As shown in Fig. 3B, ROCK-
3 induced
phosphorylation of PK, and this phosphorylation depended on the protein
kinase activity of ROCK, since the kinase-defective mutant ROCK (KDIA) did not phosphorylate PK. These results indicate that LIMK2 is directly
phosphorylated and is activated by ROCK through the protein kinase
domain of LIMK2. To analyze the phosphorylated amino acid residues in
PK, PK was phosphorylated by ROCK-
3 in vitro and subjected to thin-layer electrophoresis. A phosphoamino acid analysis revealed that LIMK2 was phosphorylated by ROCK on threonine residues (Fig. 3C).
ROCK Activates LIMK2 through Phosphorylation on Threonine
505--
Many protein kinases are phosphorylated on a residue(s)
located in a particular segment in the kinase domain termed the
activation segment, and phosphorylation of this segment is associated
with the stimulation of kinase activity (32). In the corresponding activation segment of LIMK2, two threonine residues at positions 494 and 505 are conserved among species (Fig.
4A). Since sequences KKRT and
KRYT surrounding the Thr-494 and Thr-505, respectively, are
consistent with the consensus recognition sequence for Rho-kinase/ROCK, (R/KX0-2S/T) (single letter code in which
X denotes any residue) (33), we considered that these two
threonine residues might be potential phosphorylation sites for LIMK2
activation by ROCK. To address this issue, we prepared mutant protein
kinase domains of LIMK2 in which Thr-494 and Thr-505 were,
respectively, replaced with valine residues, and we asked if ROCK could
phosphorylate these mutant PKs (Fig. 4B). When these
proteins were incubated with ROCK-
3, both wild-type PK and
PKT494V mutants were phosphorylated by ROCK-
3, depending
on the protein kinase activity. In contrast, there was a remarkable
decrease in phosphorylation by ROCK-
3 with the PKT505V
mutant, indicating that Thr-505 is a major site phosphorylated by ROCK.
However, since marginal phosphorylation was still seen in
PKT505V, ROCK may weakly phosphorylate the kinase domain of
LIMK2 in addition to Thr-505. Consistent with this notion, a double
mutant of PK (in which both Thr-494 and Thr-505 were replaced with
valine) was not phosphorylated by ROCK (data not shown).

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Fig. 4.
Phosphorylation of the threonine 505 residue
of LIMK2 by ROCK. A, schematic diagram of LIMK2 showing
the two LIM domains (hatched), PDZ domain
(white), and protein kinase domain (PK;
black) and the partial amino acid sequence for the
activation segment in PK. Thr-494 and Thr-505 in PK are
boxed, and consensus amino acid sequences for
phosphorylation sites by Rho-kinase/ROCK are also indicated.
B, phosphorylation of PK and its mutant (T494V and T505V) by
ROCK. COS-7 cells were transfected with expression vector for
Myc-tagged ROCK- 3 or KDIA. After transient expression,
immunoprecipitated ROCK- 3 and KDIA were used for in vitro
kinase assay using GST-PK, T494V, or T505V as substrate (top
panel) and for anti-Myc immunoblot (middle panel).
Substrates were stained by Coomassie Brilliant Blue (C.B.B.)
(bottom panel). The arrow indicates
autophosphorylated ROCK- 3.
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To further examine the requirement for Thr-494 and Thr-505 as
regulatory phosphorylation sites in LIMK2 by ROCK, we constructed the
expression plasmids for mutated full-length LIMK2 in which Thr-494 was
substituted by Val (LIMK2T494V) or by Glu
(LIMK2T494E) and Thr-505 was substituted by Val
(LIMK2T505V) or by Glu (LIMK2T505E), and we
examined the protein kinase activity. As shown in Fig. 5A, wild-type LIMK2 exhibited
autophosphorylation and kinase activity toward cofilin. The
LIMK2T505V mutant resulted in a significant decrease in
LIMK2 kinase activity, whereas the kinase activity of
LIMK2T505E mutant was 3.7-fold higher than that of
wild-type LIMK2. The replacement of Thr-505 for Glu may mimic the
phosphorylated state of LIMK2, which means a constitutive activation of
LIMK2. In contrast, the protein kinase activity of
LIMK2T494V and of LIMK2T494E was not
significantly changed compared with findings with the wild-type LIMK2.
Thus, Thr-505 in the activation segment is likely to be an essential
phosphorylation site for activation of LIMK2 by ROCK, whereas Thr-494
is not.

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Fig. 5.
Effect of Thr-494 and Thr-505 mutation on
LIMK2 activity and LIMK2-induced actin cytoskeletal organization.
A, protein kinase activity of LIMK2 and its Thr-505 and
Thr-494 mutants. COS-7 cells were transfected with expression
vectors for HA-tagged LIMK2 and its Thr-494 and Thr-505 mutants.
After transient expression, immunoprecipitated LIMK2 and its mutant
were used for in vitro kinase assay using GST-cofilin as
substrate (top panel) and for anti-LIMK2 immunoblot
(middle panel). The bottom panel shows the
protein kinase activity toward cofilin phosphorylation. Protein kinase
activity of wild-type LIMK2 was taken as 1.0. Each value represents the
mean ± S.E. of three independent experiments. The arrow
indicates autophosphorylated LIMK2. B, C, and
D, effect of LIMK2 and its mutants on actin cytoskeletal
organization. HeLa cells were transfected with expression vectors for
HA-tagged wild-type LIMK2 (B), LIMK2T505V
(C), and LIMK2T505E (D). Cells were
fixed and double-stained with anti-HA antibody (inset panel)
and phalloidin. Confocal images were obtained as described under
"Experimental Procedures." Bar, 20 µm. The
arrows indicate transfected cells.
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We next analyzed the potential of LIMK2 and its mutants to induce actin
cytoskeletal changes. HA-tagged LIMK2 and its mutants were transfected
in serum-starved HeLa cells, and the actin filaments were visualized by
making use of rhodamine-conjugated phalloidin. Ectopic expression of
wild-type LIMK2 enhanced the formation of actin stress fibers compared
with findings in non-transfected cells (Fig. 5B). Expression
of the LIMK2T505E mutant induced a dramatic formation of
actin stress fibers (Fig. 5D), which thickened and increased
in number over those found in the wild-type LIMK2-expressed cells. In
contrast, the LIMK2T505V mutant did not induce changes in
the actin cytoskeleton (Fig. 5C). Taken together, these
results suggest that phosphorylation of Thr-505 is essential for
protein kinase activity toward cofilin and thus for induction of actin
cytoskeletal changes by LIMK2.
To determine if ROCK regulates LIMK2 activity through direct
phosphorylation of Thr-505 in vivo, we expressed ROCK-
3
together with LIMK2T505V or LIMK2T505E and
examined the potential of ROCK-
3 to activate each mutant LIMK2 (Fig.
6). The kinase activity of wild-type
LIMK2 was enhanced to a 9.6-fold higher level by co-expression with
ROCK-
3. Similar results were also obtained in both
LIMK2T494V and LIMK2T494E mutants co-expressing
ROCK-
3 (data not shown). In contrast, neither LIMK2T505V
nor LIMK2T505E was enhanced by co-expression with
ROCK-
3. ROCK can thus exert its effect only if there is a
phosphorylatable threonine residue at position 505 within the
activation segment of LIMK2, and ROCK apparently directly regulates
LIMK2 activity through the phosphorylation of Thr-505.

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Fig. 6.
Requirement of Thr-505 for activation of
LIMK2 by ROCK. COS-7 cells were co-expressed with HA-tagged LIMK2
or its Thr-505 mutants and empty vector or Myc-tagged ROCK- 3. After
transient expression, immunoprecipitated LIMK2 and its Thr-505 mutants
were used for in vitro kinase assay using GST-cofilin as
substrate (top panel) and for anti-HA immunoblot
(upper middle panel). Cell lysates (50 µg) were also used
for anti-Myc immunoblot (lower middle panel). The
bottom panel shows changes in protein kinase activity toward
cofilin phosphorylation. Protein kinase activity of wild-type
(w.t.) LIMK2 in control cells was taken as 1.0. Each value
represents the mean ± S.E. of three independent experiments. The
arrow indicates autophosphorylated LIMK2.
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DISCUSSION |
We reported that LIMK1 and LIMK2 function downstream of distinct
Rho family GTPases (20). In the present study, we found that the LIMK2
activity was specifically activated by ROCK downstream of RhoA and that
ROCK directly phosphorylated at Thr-505 in the activation segment of
LIMK2. Moreover, phosphorylation of this site was essential for actin
cytoskeletal change and cofilin phosphorylation by LIMK2. On the other
hand, it has been reported that LIMK1 is phosphorylated and activated
by Pak1 (34). Taken together, these observations suggest that LIMK1 and
LIMK2 are regulated by distinct upstream protein kinases downstream of
distinct Rho family GTPases.
LIM-kinase is the LIM domain-containing serine/threonine/tyrosine
kinase composed of closely related LIMK1 and LIMK2 (11-17). Recent
studies showed that LIMK1 and LIMK2 play a role of actin cytoskeletal
reorganization through cofilin phosphorylation downstream of distinct
Rho family GTPases (18-20). LIMK1 acts downstream of Rac1 but is not a
direct target of this GTPase. It has been shown that LIMK1 is directly
phosphorylated and activated by Pak1, an effector protein kinase of
Cdc42/Rac and is essential for Pak1-induced dorsal membrane ruffles
formation (34, 35). On the other hand, Maekawa et al. (27)
showed that ROCK can activate LIMK1 and LIMK2. However, they used the
constitutive active form of ROCK (C-terminally deleted ROCK including
ROCK-
3), which resulted in a non-selective activation of LIMK1 and
LIMK2. We found that the full-length ROCK specifically activates LIMK2
but not LIMK1. Thus, the C-terminal-half region containing Rho-binding
and PH domain has a definite role in the substrate-targeting mechanism for specifying actions of ROCK toward LIMK2. Ohashi et al.
(28) reported that wild-type ROCK phosphorylates and activates LIMK1. Although we have no explanation for the discrepancy in all these results, their data contradict the proposal that LIMK1 is specifically regulated by Rac1 but not RhoA and plays a role in the Rac1-induced lamellipodia formation (18-20), as do our present results.
The mechanisms that determine the specificity of Rho-kinase/ROCK action
toward its multiple kinase substrates have only been partly
characterized. In cells, this kinase, once activated, translocates from
the cytosol to the plasma membrane (23). It was also reported that both
Rho and Rho-kinase are translocated from the cytosol to the cleavage
furrow and play a critical role in inducing and maintaining the
contractile ring during cytokinesis (36, 37). The target proteins of
Rho-kinase/ROCK, including the ezrin/radixin/moesin family,
myosin light chain, myosin binding subunit, glial fibrillary acidic
protein, vimentin, and desmin, accumulate at these area, where their
phosphorylation occurs specifically (33, 37-43). However, the dominant
active form of Rho-kinase C-terminally deleted form)
non-selectively phosphorylates vimentin and desmin in the cytoplasm of
interphase cells (42, 43). These observations suggest that the
C-terminal region of Rho-kinase containing PH and Rho binding domains
may regulate its subcellular localization and, hence, provide
susceptibility to specific substrates. The PH domain has a role in the
signal-dependent membrane localization of several proteins
(44). Rho binding domains of Rho-kinase/ROCK is essential for
activation of protein kinase activity through binding to Rho and
translocation to the membrane by forming a complex with activated Rho
(22, 23). Therefore, even though potential substrates are
non-selectively phosphorylated in vitro, susceptibility of
Rho-kinase/ROCK to substrates at specific loci may determine if
Rho-kinase/ROCK could act on substrate proteins within cells. Although
the intracellular localization of endogenous LIMK2 remains to be
determined, all these observations lead to the notion that the
activated native form of ROCK may translocate to specific sites where
LIMK2 co-localizes, then preferentially phosphorylate LIMK2, the result
being LIMK2-dependent cofilin phosphorylation and
stabilization of actin filaments.
The spatial and temporal organization of actin filaments plays an
essential role in cellular locomotion, and actin cytoskeletal reorganization is distinctly regulated both spatially and temporally within migrating cells (45, 46). In these cells, membrane ruffling and
filopodial protrusion, respectively, regulated under Rac and Cdc42 are
observed in the leading edge, whereas actin-myosin contraction
regulated under Rho is observed in the ruffling area and posterior
regions of cells (3-5). Thus, actin cytoskeletal reorganization, which
occurs in a distinct spatiotemporal manner in distinct subcellular
loci, appears to be regulated through Rho family GTPases, which utilize
different sets of downstream effectors. In terms of actin filament
depolymerization, independent pathways, i.e. Rac1-Pak1-LIMK1
and RhoA-ROCK-LIMK2, may play a role in distinct regulation of
cofilin-mediated actin filament depolymerization such that it occurs in
a distinct spatiotemporal manner. Alternatively, different expression
patterns of LIMK1 and LIMK2 in tissues and cells (12-17, 47) may
specify the tissue- or cell type-specific patterns of actin filament organization.