From the Division of Molecular Regenerative Medicine, Course of Advanced Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
Received for publication, April 17, 2001, and in revised form, May 2, 2001
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ABSTRACT |
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LIM kinases (LIMK1 and LIMK2) regulate
actin cytoskeletal reorganization through cofilin phosphorylation
downstream of distinct Rho family GTPases. Pak1 and ROCK, respectively,
activate LIMK1 and LIMK2 downstream of Rac and Rho; however, an
effector protein kinase for LIMKs downstream of Cdc42 remains to
be defined. We now report evidence that LIMK1 and LIMK2 activities
toward cofilin phosphorylation are stimulated in cells by the
co-expression of myotonic dystrophy kinase-related Cdc42-binding kinase
LIM kinase (LIMK)1 is a
member of a novel class of protein serine/threonine/tyrosine kinases
with characteristic structural features composed of two LIM domains and
a PDZ domain (1-7). Previous studies showed that two closely related
LIMKs, LIMK1 and LIMK2, regulate actin cytoskeletal reorganization;
LIMK-induced actin cytoskeletal rearrangement is mediated by cofilin,
an actin depolymerizing factor of actin filaments (8-10). The actin
depolymerizing factor (ADF)/cofilin family is responsible for the
turnover of actin filaments and probably is a potential downstream
effector of signaling pathways that evoke reorganization of the actin
cytoskeleton (11-13). Activated LIMK catalyzes phosphorylation of an
N-terminal third serine residue of cofilin and inhibits its activity to
depolymerize actin filaments, thereby leading to stabilization of actin
filaments. Our studies show that the XLIMK, the Xenopus
counterpart of mammalian LIMK (14), is critically involved in the
progression of progesterone-induced Xenopus oocyte
maturation through Xenopus cofilin phosphorylation (15).
Therefore, LIMKs may be a key component of a fundamental signal
transduction system that connects the extracellular stimuli that alter
actin cytoskeletal rearrangements.
The Rho family GTPases (including Rho, Rac, and Cdc42) are key
regulators in signaling pathways that link extra- and intracellular stimuli to actin cytoskeletal reorganization (16-19). Among several downstream effectors of Rho family GTPases that are involved in regulating actin cytoskeleton, LIMK1 and LIMK2 play a role in actin
cytoskeletal reorganization downstream of distinct Rho family GTPases
(8-10). LIMK1 is regulated by Rac and Cdc42, whereas LIMK2 is
regulated by Rho and Cdc42; LIMKs have a definitive role in the Rho
family GTPases-induced actin cytoskeletal rearrangement (8-10). Recent
studies revealed that Pak1 activates LIMK1 downstream of Rac1 (20), and
ROCK activates LIMK2 downstream of RhoA (21, 22). However, an effector
protein kinase that phosphorylates/activates LIMKs downstream of Cdc42
remained to be defined. We now report that myotonic dystrophy
kinase-related Cdc42-binding kinase Materials--
Anti-hemagglutinin (HA) monoclonal antibody
(12CA5) and anti-Myc monoclonal antibody (9E10) were purchased
from Roche Molecular Biochemicals. Anti-cofilin polyclonal antibody was
purchased from Cytoskeleton Inc. (Denver, CO). Y-27632 was generously
provided by WelFide Co. (Osaka, Japan).
Construction of Expression Plasmids and Preparation of
Recombinant Protein--
The expression plasmids for HA-tagged LIMK1,
LIMK2 and its mutants, glutathione S-transferase (GST)-fused cofilin,
and GST-fused LIMK protein kinase domain (PK) were constructed as
described elsewhere (10, 22). Expression plasmids for Myc-tagged ROCK and its mutants were kindly provided by Dr. S. Narumiya (Kyoto University, Kyoto, Japan). Expression plasmid for Cdc42V12 was kindly
provided by Dr. Y. Takai (Osaka University, Osaka, Japan). To generate
the plasmid encoding for full-length MRCK Expression in Cells--
COS-7 cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum (FBS). HeLa cells were maintained in minimal
essential medium (MEM) supplemented with FBS and non-essential amino
acids. Subconfluent COS-7 cells were trypsinized and resuspended in
phosphate-buffered saline, 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% FBS.
HeLa cells were cultured for 12 h at a density of 6 × 103/cm2 and then further cultured for 16 h
in serum-free MEM. The cells were transfected in Opti-MEM containing 3 µg of plasmid DNA complexed with LipofectAMINE. After a 2-h
incubation, the medium was replaced with serum-free MEM, and the cells
were cultured for 22 h.
Immunoprecipitation, Immunoblot Analysis, and Protein Kinase
Assay--
COS-7 cells were transiently transfected with expression
plasmid as described above. 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
For protein kinase assay, immunoprecipitates bound to protein
G-Sepharose were washed three times with kinase buffer consisting of 50 mM Hepes-NaOH (pH 7.5), 25 mM
Two-dimensional Gel Electrophoresis--
The transfected HeLa
cells were dissolved in two-dimensional lysis buffer composed of
9.5 M urea, 2% Triton X-100, 2% ampholine (pH 3.5-10),
and 5% 2-mercaptoethanol and subjected to non-equilibrium pH gradient
electrophoresis (NEpHGE) (24). SDS-PAGE was carried out in a 12.5%
polyacrylamide gel for the second dimension. Proteins were
electroblotted onto polyvinylidene difluoride membranes, probed with
anti-cofilin antibody, and then detected using ECL enhanced
chemiluminescence. The spot densities of immunoblots were analyzed
using NIH Image software (Wayne Rasband Analytics, National Institutes
of Health).
We reported earlier that the activity of the LIM kinases, LIMK1
and LIMK2, is distinctly regulated by Rho family GTPases (10). ROCK, a
Rho-dependent protein kinase, specifically activates LIMK2 but not LIMK1 downstream of Rho (22). To further confirm the specific
activation pathway of LIM kinases by Rho family GTPases, we examined
the effects of Y-27632, a specific inhibitor of ROCK, on the activation
of LIM kinases by Cdc42 (Fig. 1).
Consistent with our previous data (10), LIMK1 activity toward cofilin
phosphorylation was stimulated 1.8-fold when co-expressed with the
dominant active form of Cdc42 (Cdc42V12) (Fig. 1A), whereas
the addition of Y-27632 did not affect the LIMK1 activity enhanced by
Cdc42V12. Likewise, Y27632 did not significantly inhibit basal LIMK1
activity in cells expressing LIMK1 alone. On the other hand, in cells
expressing LIMK2 alone, Y-27632 inhibited LIMK2 activity to the half
level. Since Rho-dependent LIMK2 activation was
completely blocked by Y-27632 treatment (22), basal LIMK2 activity is
regulated by endogenous Rho-ROCK pathways in cells. When LIMK2 was
co-expressed with Cdc42V12, LIMK2 activity was stimulated about
1.6-fold (Fig. 1B). Activation of LIMK2 by Cdc42V12 was
inhibited by Y-27632; however, Y-27632 did not inhibit LIMK2 activity
to the level seen in Y-27632-treated cells expressing LIMK2 alone.
Thus, even under conditions wherein ROCK is inhibited in the presence
of Y-27632, expression of Cdc42V12 significantly increased LIMK2
activity. Taken together, these observations strongly suggest that LIM
kinases are regulated by another protein kinase(s) that differs from
ROCK downstream of Cdc42.
(MRCK
), an effector protein kinase of Cdc42. In
vitro, MRCK
phosphorylated the protein kinase domain of LIM
kinases, and the site in LIMK2 phosphorylated by MRCK
proved to be
threonine 505 within the activation segment. Expression of MRCK
induced phosphorylation of actin depolymerizing factor (ADF)/cofilin in
cells, whereas MRCK
-induced ADF/cofilin phosphorylation was
inhibited by the co-expression with the protein kinase-deficient form
of LIM kinases. These results indicate that MRCK
phosphorylates and
activates LIM kinases downstream of Cdc42, which in turn regulates the
actin cytoskeletal reorganization through the phosphorylation and
inactivation of ADF/cofilin.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(MRCK
), a
Cdc42-dependent protein kinase (23), activates both LIMK1
and LIMK2 downstream of Cdc42. Our results define a signal transduction
pathway wherein MRCK
activates LIMK1 and LIMK2 downstream of Cdc42,
which in turn regulates the actin cytoskeletal reorganization through
phosphorylation and inactivation of cofilin.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(amino acids 1-1732),
partial rat MRCK
cDNA fragments were cloned by reverse
transcriptase-PCR using a set of the following primers: A
fragment, forward, 5'-GAGTCACAGCAGGTCCC-3', and reverse,
5'-GGGCAGATTGCAACTCGAGTC-3'; B fragment, forward,
5'-CATGTCAGCTAGACTCGAGTT-3', and reverse, 5'-CACACACAGGCAGGACA-3'; C
fragment, forward, 5'-CGACAGCACTCTACCCC-3', and reverse,
5'-CTGCGGCCGCTCATGGATCCCAGCTCC-3'. These PCR products were digested
with the following enzymes: A fragment, AatII and XhoI; B fragment, XhoI and SacI; C
fragment, SacI and NotI. The digested fragments
were ligated to AatII- and NotI-digested
pEF-BOS-myc-MRCK-
C. To generate plasmids encoding MRCK-
C (amino
acids 1-574), rat MRCK
cDNA fragment was cloned by reverse
transcriptase-PCR using a set of the following primers: forward,
5'-GCGGATCCATGTCTGGAGAAGTGCGTTTGA-3', and reverse,
5'-GGGGATCCTCACAGTTTCCTCTGACAGTGTGCG-3'. The PCR product digested with
BamHI was ligated to the BamHI-digested pEF-BOS-myc vector (10). The cDNA for the kinase defective mutant of MRCK-
C (kinase-dead (KD)) was constructed to introduce
substitution of the 106th Lys with Ala, using a site-directed
mutagenesis kit (CLONTECH, Palo Alto, CA). The
authenticity of these expression plasmids was confirmed by nucleotide
sequence analysis.
-glycerophosphate, 10 mM NaF, 1 mM Na3VO4,
1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl
fluoride, and 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, Little
Chalfont, UK) for 1 h at 4 °C and centrifuged to remove protein
G-Sepharose, and the supernatant was then 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-PAGE. The
immunoprecipitates and cell lysates were separated by SDS-PAGE,
electroblotted onto polyvinylidene difluoride membranes (Bio-Rad), and
probed with anti-HA and anti-Myc antibody as described elsewhere (10,
22). Proteins reacting with these antibodies were detected using ECL
enhanced chemiluminescence (Amersham Pharmacia Biotech).
-glycerophosphate, 5 mM MgCl2, 5 mM MnCl2, 10 mM NaF, and 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-PAGE, subjected to SDS-PAGE, and analyzed by autoradiography.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Effect of the ROCK inhibitor Y-27632 on
Cdc42-induced LIMK1 (A) and LIMK2 (B)
activation. A, COS-7 cells were co-expressed with
HA-tagged LIMK1 and empty vector or Myc-tagged Cdc42V12,
respectively. B, COS-7 cells were co-expressed with
HA-tagged LIMK2 and empty vector or Myc-tagged Cdc42V12, respectively.
Transiently transfected COS-7 cells were cultured for 36 h and
then incubated for 30 min with or without 10 µM
Y-27632. Immunoprecipitated LIMK1 and LIMK2 from cell lysates were
used, respectively, 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).
Arrowheads indicate autophosphorylated LIMK1 and LIMK2,
respectively. The amount of cofilin phosphorylated by LIMK1 or LIMK2 in
mock cells was taken as 1.0. Each value represents the means ± S.E. of three independent experiments.
To identify the upstream protein kinase of LIM kinases, we searched for
the effector protein kinases of Cdc42. We speculated that MRCK
might be a candidate protein kinase of LIM kinase, because MRCK plays a
key role in formation of filopodial protrusion downstream of Cdc42 and
is structurally related to Rho kinase/ROCK (23). To determine whether
MRCK would activate LIM kinases downstream of Cdc42, we constructed
expression plasmids for MRCK (Fig.
2A). MRCK-
C is deleted with
the C-terminal regulatory region containing GTPase-binding and PH
domains. MRCK-
C/KD has a substituted amino acid involved in ATP
binding in the protein kinase domain, and thus MRCK-
C/KD is likely
to act in a kinase-deficient form. As shown in Fig. 2B
(left panels), co-expression of MRCK-
C stimulated LIMK1
activity toward cofilin phosphorylation. Likewise, LIMK2 activity was
stimulated by the co-expression with MRCK-
C (Fig. 2B, right
panels). These stimulatory effects of MRCK-
C on LIMK1 and LIMK2
depended on protein kinase activity, because the kinase-dead form of
MRCK-
C (MRCK-
C/KD) did not stimulate LIM kinases activity. We
recently found that the dominant active form of ROCK (ROCK-
3) nonselectively activates both LIMK1 and LIMK2, whereas the wild-type ROCK specifically activates LIMK2 but not LIMK1; thus the C-terminal half containing the Rho-binding and PH domains of ROCK is susceptible to specific substrates (22). MRCK
has similar functional domains consisting of cysteine-rich, PH, and GTPase-binding domains within the
C-terminal regulatory region (Fig. 2A); these domains play an important role in Cdc42-induced filopodial protrusion and neurite outgrowth (23, 25). Therefore, we asked whether full-length MRCK
would specifically activate either LIMK1 or LIMK2. Consistent with
previous notions (22), LIMK2 but not LIMK1 activity was specifically
activated about 2.3-fold by the co-expression of wild-type ROCK (Fig.
2C). In contrast, LIMK1 and LIMK2 activities in cells
co-expressing wild-type MRCK
were stimulated, respectively, to a
1.9- and 2.3-fold higher level than seen in cells expressing LIMK1 or
LIMK2 alone (Fig. 2C). These results indicate that MRCK
activates both LIMK1 and LIMK2 activity as an upstream protein kinase.
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Because activation of LIMK2 by ROCK is mediated by the phosphorylation
of Thr-505 in the activation segment within the protein kinase domain
of LIMK2 (22), we determined whether activation of LIM kinases by
MRCK might also be mediated through phosphorylation of the protein
kinase domain of LIM kinases. Wild-type LIMK2 activity was stimulated
6-fold with the co-expression with MRCK-
C (Fig. 3A). Similarly, the kinase
activity of the PK mutant (LIMK2 with the deleted N-terminal half
containing LIM and PDZ domains) was also stimulated by 5-fold with the
co-expression of MRCK-
C. Thus, MRCK
-dependent LIMK2
activation is apparently mediated by the protein kinase domain of
LIMK2.
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To determine whether PKs are phosphorylated directly by MRCK, we
respectively prepared recombinant GST-fused PKs (GST-PKs) of LIMK1 and
LIMK2. The GST-PKs were subjected to an in vitro phosphorylation assay (Fig. 3B). MRCK-
C directly
phosphorylated the protein kinase domain of LIMKs, whereas MRCK-
C/KD
did not induce protein phosphorylation of GST-PKs. Because MRCK
shares a similar substrate specificity with Rho kinase/ROCK (23, 26) and ROCK-
3 phosphorylates a conserved threonine residue (Thr-508 in
LIMK1, Thr-505 in LIMK2) within the activation segment of LIM kinases
(22, 27), we hypothesized that this conserved threonine residue might
be a site phosphorylated by MRCK
. To address this possibility, we
expressed MRCK-
C together with LIMK2T505V or
LIMK2T505E and examined the potential of MRCK-
C to
activate each mutant LIMK2 (Fig. 3C). The kinase activity of
wild-type LIMK2 was enhanced to a 7.5-fold higher degree over the
control by co-expression with MRCK-
C together with phosphorylation
of LIMK2. In contrast, neither activation of LIMK2T505V nor
LIMK2T505V phosphorylation was evident by co-expression
with MRCK-
C. The kinase activity of LIMK2T505E mutant
was 2.8-fold higher than that of wild-type LIMK2, presumably mimicking
the phosphorylation state of LIMK2. However, the kinase activity of
LIMK2T505E was not significantly changed by co-expression
with MRCK-
C. These results suggest that MRCK
activates LIMK2
through Thr-505 phosphorylation in the protein kinase domain of LIMK2.
It seemed important to determine whether both LIMK1 and LIMK2 would
function as downstream effectors on MRCK-mediated signal transduction toward cofilin phosphorylation. We next used extracts from
transfected HeLa cells and subjected them to two-dimensional gel
electrophoresis. The phosphorylated and nonphosphorylated ADF/cofilin
was detected using immunoblotting techniques (Fig. 4). Phosphorylated and nonphosphorylated
ADF/cofilin was clearly distinguishable as the electrophoretic mobility
was distinct. As shown in Fig. 4A, in mock-transfected HeLa
cells, ADF/cofilin was either not phosphorylated or was only marginally
phosphorylated. In contrast, when LIMK1 was expressed in cells,
phosphorylated ADF/cofilin increased to 45% of the total ADF/cofilin.
Phosphorylation of ADF/cofilin was undetectable in cells expressing the
protein kinase-deficient form of LIMK1 (LIMK1/KD). Similar results were obtained when LIMK2 was expressed in cells. Phosphorylated ADF/cofilin increased to 44% of the total ADF/cofilin, whereas phosphorylated ADF/cofilin was not evident in cells expressing LIMK2/KD. On the other
hand, when MRCK
was expressed in cells, phosphorylated ADF/cofilin
increased to 64% of the total ADF/cofilin (Fig. 4B). Because MRCK
does not directly phosphorylates ADF/cofilin (data not
shown), this MRCK
-induced phosphorylation of ADF/cofilin may
possibly be mediated via LIM kinases. Consistent with this notion,
MRCK
-induced phosphorylation of ADF/cofilin was reduced, respectively, to 24 and 18% of the total ADF/cofilin with the co-expression with LIMK1/KD and LIMK2/KD. Furthermore, when MRCK
was
co-expressed with both LIMK1/KD and LIMK2/KD, MRCK
-induced ADF/cofilin phosphorylation was almost completely inhibited. These results strongly indicate that LIMK1 and LIMK2 function as downstream effectors of MRCK
and play a role in MRCK
-induced actin
cytoskeletal reorganization through inactivation (phosphorylation) of
ADF/cofilin.
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In the present study, we found that MRCK, a downstream effector of
Cdc42, phosphorylates and activates both LIMK1 and LIMK2 and
participates in the activation of LIM kinases downstream of Cdc42.
Taken together with previous notions that LIMK1 is activated by Pak1
downstream of Rac1 (20) and that ROCK specifically activates LIMK2
downstream of RhoA (22), these observations define signal transduction
pathways through which Rho family GTPases regulate cofilin-mediated
actin filament depolymerization.
The actin cytoskeletal network in eukaryotic cells participates in
cellular processes, including locomotion, shape changes, cytokinesis,
and maintenance of polarity (28, 29). The rapid assembly and
disassembly of actin filaments are regulated both spatially and
temporally during these processes. The spatiotemporal reorganization of
actin cytoskeleton seen with extracellular stimuli is regulated through
distinct Rho family GTPases and their specific effectors (16-19). In
terms of actin filament depolymerization, signal transduction pathways,
i.e. Cdc42-MRCK-LIMKs, Rac-Pak1-LIMK1, and Rho-ROCK-LIMK2,
may play distinct role in regulating cofilin-mediated actin filament
depolymerization so that it occurs in a definite spatiotemporal manner.
By way of support for this notion, LIMK1 plays a role in Rac-induced
lamellipodia formation, whereas LIMK2 plays a role in Cdc42-induced
filopodial formation and Rho-induced stress fiber formation (8-10).
Likewise, different expression patterns of LIMK1 and LIMK2 (1-7) as
well as those of upper protein kinases for LIMKs in tissues and cells
may further specify the tissue- or cell type-specific patterns
of actin filament organization.
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ACKNOWLEDGEMENTS |
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We thank Dr. S. Narumiya (Kyoto University, Kyoto, Japan) for providing plasmids of ROCK and WelFide Co. (Osaka, Japan) for providing Y-27632. We are also grateful to Dr. Y. Takai (Osaka University, Osaka, Japan) for providing the Cdc42V12 plasmid and to M. Ohara for helpful comments and language assistance.
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FOOTNOTES |
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* This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Technology, Sports and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
81-6-6879-3783; Fax: 81-6-6879-3789; E-mail:
nakamura@onbich.med.osaka-u.ac.jp.
Published, JBC Papers in Press, May 4, 2001, DOI 10.1074/jbc.C100196200
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ABBREVIATIONS |
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The abbreviations used are:
LIMK, LIM
kinase;
ADF, actin depolymerizing factor;
PH domain, pleckstrin
homology domain;
PK, protein kinase (mutant LIMK2 with a deleted
N-terminal half containing both LIM and PDZ domains);
GST, glutathione
S-transferase;
HA, hemagglutinin;
MRCK, myotonic
dystrophy kinase-related Cdc42-binding kinase
;
PCR, polymerase
chain reaction;
KD, kinase-dead;
FBS, fetal bovine serum;
MEM, minimum
essential medium;
PAGE, polyacrylamide gel electrophoresis.
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