From the Departments of Chemistry and
§ Medicine, University of California San Diego, School of
Medicine, La Jolla, California 92093
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ABSTRACT |
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LIM kinase phosphorylates and inactivates the
actin binding/depolymerizing factor cofilin and induces actin
cytoskeletal changes. Several unique structural features within LIM
kinase were investigated for their roles in regulation of LIM kinase
activity. Disruption of the second LIM domain or the PDZ domain or
deletion of the entire amino terminus increased activity in
vivo measured as increasing aggregation of the actin
cytoskeleton. A kinase-deleted alternate splice product was identified
and characterized. This alternate splice product and a kinase inactive
mutant inhibited LIM kinase in vivo, indicating that the
amino terminus suppresses activity of the kinase domain. Mutation of
threonine 508 in the activation loop to valine abolished activity
whereas replacement with 2 glutamic acid residues resulted in a
fully active enzyme. Dephosphorylation of LIM kinase inhibited cofilin
phosphorylation. Mutation of the basic insert in the activation loop
inhibited activity in vivo, but not in vitro.
These results indicate phosphorylation is an essential regulatory
feature of LIM kinase and indicate that threonine 508 and the adjacent
basic insert sequences of the activation loop are required for this
process. A combination of structural features are thus involved in
receiving upstream signals that regulate LIM kinase-induced actin
cytoskeletal reorganization.
Many protein kinases contain modular domains that regulate
catalytic activity, direct localization to specific compartments of the
cell, and dictate interactions with other components of signal
transduction complexes (1). There are 2 identified LIM kinase family
members that each contain 2 amino-terminal LIM domains, a central PDZ
domain, and a carboxyl-terminal kinase domain with predominant
serine/threonine kinase activity (2-4). During mouse development LIM
kinase is expressed early in neuroectoderm, cardiac mesoderm, and gut
endoderm and later predominantly in brain (5-7). In developing human
tissues LIM kinase is also found predominantly in brain where
hemizygous deletion of the 7q 11.23 region containing LIM kinase is
implicated in the visuospatial constructive cognition defect in
Williams syndrome (8).
Major unanswered questions have been how LIM kinase functions and how
it is regulated. Cofilin has been identified as a functionally important substrate for LIM kinase and evidence has been provided that
LIM kinase regulates actin dynamics by phosphorylation and inactivation
of cofilin (9, 10). Actin-depolymerizing factor/cofilamentous protein
(cofilin) which binds to both F-actin and actin monomers (11-13) is
essential for depolymerization of actin filaments (14). It binds more
tightly to ADP-actin than to ATP-actin and enhances the off rate of
actin monomers at the pointed end of fibers (15). At pH 8.0 and above,
cofilin depolymerizes actin stoichiometrically (16, 17). This is
essential to actin dynamics necessary for cell motility, cytokinesis,
and other cell processes (18-20). Cofilin exists in both a phospho and
dephospho form, with phosphorylation inhibiting the actin filament
severing activity (21, 22). Ser3 is the principal
inhibitory phosphorylation site in cofilin and previous studies
indicated it was a poor substrate for known kinases including protein
kinase C, cyclic AMP-dependent protein kinase, myosin light
chain kinase, and CaM kinase II (21). By using dominant negative forms
of LIM kinase and constitutively active forms of Rac, Arber et
al. (9) and Yang et al. (10) deduced that LIM kinase is
the downstream effector of Rac-dependent actin cytoskeleton
changes. Because activated Rac did not interact directly with LIM
kinase, Rac regulation of LIM kinase activity must involve intermediate
biochemical steps.
The kinase domain of LIM kinase contains significant sequence
variations compared with other serine/threonine kinases in the ATP-binding site (subdomain VIB) (23), the substrate-binding region of
subdomain VIII and in the presence of an 11-amino acid basic insert in
the activation loop between subdomains VII and VIII (2-4). Although
the sequence of the LIM domains of LIM kinase more closely resemble
those of nuclear LIM homeodomain and LIM-only proteins than those of
cytoplasmic proteins (24), the LIM domains of LIM kinase do not
recognize the nuclear LIM interactor that binds nuclear LIM domains
with high affinity (25). The predominant cytoplasmic localization of
LIM kinase (2, 4) and its ability to bind actin (10) indicate that it,
like many other extranuclear LIM domain containing proteins,
functionally associates with the cytoskeleton. Zyxin, cysteine-rich
protein and paxillin are localized along actin filament bundles and at
adhesion plaques (26, 27). The actin LIM protein is localized to the
cytoskeleton via its PDZ domain (28) while cysteine-rich protein 1 binds to To determine features that regulate LIM kinase we have measured the
activity of LIM kinase using transient transfection in COS-7 cells. An
in vivo assay of the biochemical activity of LIM kinase is
based upon the morphological extent of actin cable dissolution and
subsequent aggregation that results from inactivation of cofilin. Mutations were introduced into the LIM, PDZ, and kinase domains to
assess their contributions to the activity of LIM kinase that causes
actin accumulation in large uncleaved aggregates that were visualized
by fluorescently labeled phalloidin binding. Additionally, co-expression of an amino-terminal fragment that corresponds to a
naturally occurring splice variant with holo LIM kinase inhibited actin
aggregation. These studies indicate that the amino-terminal fragment
that contains the LIM and PDZ domains inhibits the catalytic activity
of the kinase domain. These studies also indicate that a threonine
residue in the catalytic loop, which is a major phosphorylation site in
other kinases, is necessary for catalytic activity and that the basic
insert in the activation loop contributes to biological activity. The
unique structural features of LIM kinase located both outside and
within the kinase domain thus control enzyme activity, subcellular
distribution, and substrate recognition necessary for regulation of
actin dynamics.
Materials--
COS-7 and HEK 293 cells were obtained from the
American Type Tissue Culture Collection, (Manassas, VA). Six-well
tissue culture plates and preferred glass coverslips were from Fisher
Scientific (Pittsburgh, PA) and cell culture media and serum were
purchased from FMC (Rockland, DE). Oligonucleotides used for
mutagenesis were made by Operon Technologies (Alameda, CA) and
molecular biology enzymes were purchased from New England Biolabs
(Beverly, MA). All polymerase chain reaction
(PCR)1 products were
amplified with Pfu polymerase. Cofilin was a kind gift from Laurent
Blanchoin and Tom Pollard (Salk Institute, La Jolla, CA).
RT-PCR Cloning and Library Screening--
RNA from A431 cells
was harvested using Tri-Reagent (Molecular Research Center Inc.,
Cincinnati, OH) and reverse transcriptase polymerase chain reactions
(RT-PCR) were carried out using the Superscript cDNA Cloning Kit
(Stratagene, La Jolla, CA) with random hexamers as the primers for the
cDNA synthesis reaction. LIM kinase and dLIM kinase were amplified
from the resulting cDNA library using primers with the sequences,
5'-GTCACTAAGCTTCATGAGGTTGACGCTACTTTGT-3' for the 5' end, and
5'-GTCACTAAGCTTCAGTCGGGGACCTCAGGGTGGGCAGG-3' for the 3' end. The PCR
products were amplified with Pfu polymerase, digested with
HindIII, and ligated into the HindIII sites in
the cloning vector, pBlueScriptII-KS (Stratagene). The sequence of the
full-length LIM kinase and dLIM kinase were verified by sequencing using Sequenase 2.0 (Amersham, Willinghamshire, United Kingdom).
To determine the expression pattern of the dLIM kinase splice variant,
18 libraries were screened using RT-PCR. Four libraries were obtained:
human fetal brain (a kind gift from Dimitri Krainc and R. Brent,
Harvard, Boston, MA), HeLa and SK-N-MC(CLONTECH, Palo Alto, CA), human placenta and A431 (Stratagene), and 14 libraries were made from cultured cells using the Superscript cDNA Cloning Kit. Oligonucleotide primers (5' primer
5'-CCCCTGAGCTCTCCGGCTTATACTC-3' and 3' primer
5'-CCTCCTTGAGGAACGTCCTCTGGGT-3') were made that would amplify the
region in LIM kinase that contains the deletion. PCR amplification of
the region around the alternate splice site would produce a
270-nucleotide (nt) fragment, while amplification of the identical
region in full-length LIM kinase would produce a 331-nt fragment. PCR
reactions were then run on a 1.5% agarose gel and stained with
ethidium bromide.
Site-directed Mutagenesis--
All LIM kinase mutations were
made by overlap extension (34, 35), except for the G177E/L178A and
T508EE, which replaces Thr508 with 2 Glu residues which
were made using the Quickchange mutagenesis kit (Stratagene). A
schematic representation of mutant LIM kinases is shown in Fig.
1. Kd1 and Kd3 constructs were made using
5' primers 5'-GACTCATCGAGTCGGACCTCATCCACGGGGAGGTGCTG-3' and
5'-GATCCTCGAGCCGGGCGCTGGCTCACTGGGCTCCCCG-3', respectively, and the 3'
primer 5'-GCGTCTAGATCAGTCGGGGACCTC-3'. The constructs were amplified
by PCR and ligated into the XhoI and NotI sites
of pcDNA-3M, a derivative of pcDNA-3 which contains a
luciferase start codon and a hemaglutinin epitope tag (HA-tag) (36).
Translation is initiated from the luciferase start codon and places an
in-frame HA-tag at the amino terminus of the constructs. All mutations
were verified by sequencing.
Cell Lines and Culture Conditions--
293 transformed human
kidney and COS-7 green monkey kidney cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum in a humidified 8% CO2 atmosphere at
37 °C. All constructs were cloned into pcDNA-3 (Invitrogen, Carlsbad, CA) except Kd1 and Kd3, which were cloned into pcDNA-3M. DNAs were transfected into COS-7 and 293 cells using calcium
phosphate-mediated transfection (37).
Production of Polyclonal Antibodies--
A peptide with the
sequence KETYRRGESGLPAHPEVPD corresponding to the carboxyl-terminal 18 residues and a peptide that corresponds to residues 255-271,
KEHDPHDTLGHGLGPETS, of human LIM kinase were conjugated to keyhole
lympet hemocyanin using gluteraldehyde (38). An amino-terminal lysine
was included on each peptide to facilitate coupling. The peptide
conjugates were used to immunize rabbits or chickens by Lampire
Laboratories (Ottsville, PA). Immunoprecipitations were done using the
rabbit antibody 5079 directed against the carboxyl-terminal peptide
except those involving dLIM kinase where antibody 5078, directed
against the internal peptide, was used. Western blotting was done with
the chicken antibody 625 directed to the internal peptide. The Kd3
construct was detected using an anti-HA monoclonal antibody (Berkeley
Antibody Co., Berkeley, CA).
Immunocytochemistry--
Cells used for immunocytochemistry were
plated onto preferred glass coverslips and harvested 60 h after
transfection. Cells were fixed with a 4% paraformaldehyde in 1 × phosphate-buffered saline for 25 min at room temperature. To determine
LIM kinase expression, cells were incubated 2-3 h with a 1:1,250
dilution of anti-LIM kinase rabbit antibody 5079 in buffer containing
0.1% Triton X-100, 0.024% saponin, and 2% bovine serum albumin. To visualize LIM kinase and to stain for actin filaments, cells were incubated for 2 h in buffer containing goat anti-rabbit Texas Red-conjugated secondary antibody and Oregon Green 488-labeled phalloidin (Molecular Probes, Eugene, OR). Cells were then equilibrated and mounted with Slow Fade mounting media. Pictures were taken with a
Zeiss Axiophot microscope with an attached Hamamatsu color chilled CCD
camera using × 40 and 60 objectives.
Immunoprecipitation and Kinase Assays--
Transiently
transfected 293 cells were grown for 72 h and harvested by washing
cells off the plate with phosphate-buffered saline and resuspending in
lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM
NaCl, 3% glycerol, 2% Triton X-100, 50 µM benzamidine,
2 µM aprotinin, 2 µM leupeptin, 1 mM phenymethlysulfonyl fluoride, 10 µg/ml pepstatin A, 10 µg/ml phenalthroline) and incubated on ice for 30 min. Cleared lysate
was presorbed with preimmune serum and protein A-Sepharose. LIM kinase
was then immunoprecipitated using the 5079 anti-LIM kinase antibody and
protein A-Sepharose and washed 4 times with lysis buffer.
Immunoprecipitates for kinase assays were washed and resuspended in
kinase assay buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 3% glycerol). An aliquot of immunoprecipitated LIM kinase was resuspended in SDS loading buffer and protein estimated by Western blotting using antibody 625 or anti-HA antibody. Protein was
detected with goat anti-chicken or anti-mouse secondary antibody conjugated to horseradish peroxidase and visualized with chemiluminescence.
Protein A-Sepharose beads containing immunoprecipitated LIM kinase were
resuspended in kinase reaction buffer and incubated for 150 min at
30 °C in kinase reaction buffer with 5 µM ATP and 10 µCi of [
Solution assays contained 200 µM
[ Effects of LIM Kinase on the Actin Cytoskeleton--
To
investigate the functional consequences of mutations in LIM kinase, an
in vivo assay was developed based on the effects of
transiently transfected LIM kinase on the actin cytoskeleton in COS-7
cells. Expression of LIM kinase was assessed using a rabbit polyclonal
anti-peptide antibody directed to the carboxyl terminus and effects on
the actin cytoskeleton were assessed using fluorescently labeled
phalloidin. Fig. 2 shows the range of
changes observed in the actin cytoskeleton; protein expression levels were based on the intensity of immunfluorescence and were qualitatively proportional to the extent of changes in the actin cytoskeleton indicated in the panels on the right. At low levels of LIM kinase expression there was a decrease in the extent of actin cables visualized and enhanced membrane staining compared with neighboring untransfected cells (Fig. 2, A and B).
Intermediate levels of LIM kinase resulted in loss of the cable-like
stress fibers and the appearance of multiple irregular lattice-like
actin filaments (Fig. 2, C and D). Higher levels of LIM
kinase expression resulted in accumulation of large masses of actin
near the cell periphery and diffuse cytoplasmic staining (Fig. 2,
E and F). The highest levels of LIM kinase
expression resulted in multiple discrete masses of actin (Fig. 2,
G and H). The extent of changes in the actin
cytoskeleton were scored using a 1+ to 4+
scale. The distribution of these actin cytoskeletal phenotypes in the
population of cells expressing LIM kinase are shown in Fig. 4. As
reported (2, 4), LIM kinase was expressed in the cytoplasm; at high
levels of aggregation the enzyme colocalized with the masses of actin.
With extensive disruption of the actin cytoskeleton, multiple nuclei
were often observed, consistent with resultant defects in nuclear
division.
A mutation of the predicted catalytic base that changes Asp to Asn
(D460N) is reported to abolish LIM kinase activity (6). Even high
levels of expression of a D460N mutant LIM kinase gave no change in the
actin cytoskeleton compared with untransfected cells (Fig. 2,
I and J). In vitro kinase assays
confirmed that D460N LIM kinase was devoid of catalytic activity (see
Fig. 10). The changes in the actin cytoskeleton were thus dependent on
the kinase activity of LIM kinase.
Effect of Mutations of the LIM and PDZ Domains on LIM
Kinase-induced Changes in the Actin Cytoskeleton--
To assess the
role of the LIM and PDZ domains on LIM kinase function, mutations were
introduced which were designed to disrupt each of these domains. The
structure of LIM domains is dependent on two Zn2+ atoms
that are coordinated tetrahedrally in amino- and carboxyl-terminal liganding modules (39). Metal binding is essential for protein structure and renaturation studies indicate that binding is sequential (40). The first conserved Cys residue of each LIM domain of LIM kinase
was mutated to Ser to disrupt metal coordination and LIM domain
structure (C25S for the first (LIM1) and C84S for the second (LIM2) LIM
domain). Mutations in the PDZ domain were based on the crystal
structure of the third PDZ domain of PSD-95 complexed to the carboxyl
terminus of the potassium channel (41). The signature sequence
Gly-Leu-Gly-Phe that constitutes the protein binding loop of PDZ
domains is 177Gly-Leu-Ser-Val in LIM kinase. To disrupt
target binding, the LIM kinase PDZ domain 177Gly-Leu was
mutated to Glu-Ala (G177E/L178A). These mutations are predicted to
disrupt both alignment of backbone interactions and the hydrophobic
pocket necessary for protein binding (41). Mutation of the
corresponding residues in the PDZ domain of Enigma, which also contains
a variant sequence at this position, abolished target
recognition.2
As shown in Fig. 3, A and
B, mutation of LIM1 did not affect the ability of LIM kinase
to induce actin aggregation. Aggregates of actin were observed
comparable to those observed with similar levels of WT LIM kinase (see
Fig. 2D). Mutation of LIM2 and the PDZ domain increased the
ability of LIM kinase to induce actin aggregation (Fig. 3,
C-F). At low levels of G177E/L178A LIM kinase expression,
phenotypic changes scored as 3+ to 4+ were seen
whereas comparable levels of holo LIM kinase expression gave actin
cytoskeleton changes scored as 1+ to 2+. As
shown in the left panels, mutations in the LIM and PDZ
domains did not affect subcellular localization as these mutant LIM
kinases remained outside the nucleus. These data indicate that LIM and PDZ domains are not necessary for biological responses to LIM kinase-induced actin aggregation; however, increased activity upon
mutational inactivation of the LIM2 and PDZ domains suggests these
suppress LIM kinase activity in the holoenzyme structure.
Although transfection of various LIM kinase mutants resulted in
equivalent average protein expression per dish (see Fig. 11), expression in individual cells within the population varied as shown in
Fig. 2. To assess the relative intrinsic activities of each form of the
LIM kinase, the distribution of induced changes in the actin
cytoskeleton in a population of cells expressing each mutant were
scored and presented as described by Arber and co-workers (9). Fig.
4 shows that actin cytoskeletal changes induced by WT-LIM kinase in COS-7 cells were primarily those indicated by 3+ and 2+ qualitative scores in Fig. 2.
Mutational inactivation of LIM2 and the PDZ domain shifted the
distribution toward the more severe actin aggregation phenotype
indicated as 4+ in Fig. 2H whereas the D460N LIM kinase had
little effect on the actin cytoskeleton relative to mock transfected
cells.
Expression of the Kinase Domain of LIM Kinase Is Sufficient to
Induce Actin Aggregation--
The observation that disruption of the
PDZ domain and the second LIM domain enhanced the in vivo
activity of LIM kinase suggested that the kinase domain was sufficient
to induce changes in the actin cytoskeleton. Two kinase constructs
containing the kinase domain of LIM kinase were made and expressed in
COS-7 cells (Fig. 1). Kd1 was composed of the conserved catalytic core
of the kinase domain from the glycine-rich loop to the end of LIM
kinase (residues 346-647). This construct, which includes all of the
conserved residues in the catalytic core, does not include the A Splice Variant of LIM Kinase Results in a Kinase-deleted
Form--
During cloning of the full-length LIM kinase transcript from
an A431 cell cDNA library, a splice variant was found that was identical to the LIM kinase sequence with the exception of a 61-base pair deletion upstream of the region coding for the kinase domain. The
deletion, from nt 977 to 1037 in the cDNA relative to the start
codon (Fig. 6A), is similar in
size to, but differs from, the deletion in the kinase core of LIM
kinase reported previously (2). Analysis of the genomic sequence (8)
showed that the deletion was the result of differential splicing at the
3' end of intron 7 which joins with the middle of exon 8 at nt 23116 instead of at the 5' end of exon 8, at nt 23055 as occurs with full-length LIM kinase (Fig. 6B). The deletion, located in
the sequence between the PDZ domain and the kinase domain, causes a
frameshift at amino acid 294 adding 12 amino acids not found in the
full-length protein and resulting in a premature truncation at amino
acid 305 (Fig. 6C). This truncation deletes the entire kinase domain, resulting in a catalytically inactive protein, termed
deleted LIM kinase (dLIM kinase). To determine if the amino-terminal splice variant dLIM kinase was found elsewhere, 18 cDNA libraries, either commercially available or made from cultured cells, were screened using RT-PCR and primers that would allow discrimination between dLIM kinase and LIM kinase. Fig. 6D shows that
full-length LIM kinase was found in all samples analyzed (upper
band), while dLIM kinase was found in 9 of the 18 analyzed
(lower band). In vitro transcription/translation
reactions showed that dLIM kinase encoded a 32-kDa protein (data not
shown). The truncated protein failed to induce actin bundling in COS-7
cells, and was expressed primarily in the cytoplasm with accumulation
in the perinuclear region (Fig. 6E).
The Kinase-inactive D460N LIM Kinase and the dLIM Kinase Splice
Variant Inhibit LIM Kinase Activity--
The effect of the
kinase-inactive D460N mutant on wild type LIM kinase activity was
investigated by co-transfecting expression constructs for both proteins
into COS-7 cells. As shown in Fig. 7,
A and B, D460N LIM kinase significantly blocked
the aggregation of actin induced by wild-type LIM kinase. The
inhibitory effects of D460N LIM kinase on the actin phenotypes induced
by WT LIM kinase are evident in the distributions shown in Fig. 4.
Although some effects of LIM kinase on the actin cytoskeleton
persisted, the D460N mutant largely inhibited the biological effects
not only of wild-type LIM kinase, but also of LIM kinase containing mutations in LIM1 (C25S), LIM2 (C84S), and the PDZ domain (G177E/L178A) (data not shown).
Hiraoka and co-workers (44) reported that when HA-tagged LIM kinase 1 and untagged wild-type protein were coexpressed in COS cells, the two
co-immunoprecipitated. Use of GST fusions of fragments of LIM kinase
indicated that the self-association of LIM kinase involved interaction
of the amino terminus with the carboxyl terminus. The inhibitory effect
of D460N on wild-type LIM kinase activity in vivo is
compatible with the dominant negative effect being due to the
postulated dimerization of the two proteins. Because D460N LIM kinase
inhibited the function of LIM kinase that contained mutations in the
LIM and PDZ domains it was uncertain which regions were responsible for
inhibition. To address this question, we coexpressed the amino-terminal
splice variant that lacks the kinase domain and the inactive kinase
fragment Kd1, with both wild type LIM kinase and the active kinase
fragment Kd3. dLIM kinase inhibited the actin aggregating effects of
LIM kinase whereas the inactive kinase fragment Kd1 did not (Fig. 7,
C-F). Moreover, dLIM kinase inhibited the
activity of the Kd3 kinase domain-only construct whereas Kd1 was
without effect (Fig. 7, I-L). In contrast the kinase
inactive D460N LIM kinase which inhibited holo LIM kinase did not
inhibit Kd3 (Fig. 7, G and H).
The finding that the amino-terminal splice variant dLIM kinase
inhibited the activity of Kd3 domain provides functional support for
the model of Hiraoka et al. (44) in which LIM kinase
monomers self-associates in an antiparallel fashion. The present data
indicates that it is the amino terminus which suppresses the kinase
activity of the dimeric enzyme. While Hiraoka and co-workers (44) found the LIM domains to be necessary for self-association, the present data
indicate that mutation of either LIM domain alone does not abolish
interaction as assessed by in vivo activity. An
amino-terminal fragment consisting of the 2 LIM domains alone also
inhibited LIM kinase activity (data not shown), suggesting that both
LIM domains function in this manner. The failure of D460N LIM kinase to
inhibit Kd3 suggests that self-association of holoenzyme monomers in
which both partners contribute amino- and carboxyl-terminal interaction
domains is stronger than association of fragments.
Mutations in the Activation Loop of the Catalytic Core Affect LIM
Kinase Activity--
Kinases are regulated by both modular domains and
phosphorylation (1, 45). Phosphorylation of a conserved Thr residue in
the activation or T loop of several kinases including
cAMP-dependent protein kinase, cyclin-dependent
kinase 2, and MAP kinase enhances catalytic efficiency (46, 47). MAP
kinase is also phosphorylated on Tyr in the sequence Thr-Glu-Tyr in the
activation loop and phosphorylation of both sites is required for
maximal activity (48, 49). The analogous activation loop Thr residue in
LIM kinase, Thr508, is adjacent to a Tyr residue,
Tyr507. Proschel et al. (6) reported that a GST
kinase domain LIM kinase fusion protein exhibited in vitro
autophosphorylation on Ser and Tyr residues with only trace amounts of
phosphate on Thr.
To evaluate the requirement for Thr508 and
Tyr507 as potential regulatory phosphorylation sites in LIM
kinase, Thr508 was mutated to Val (T508V) and Glu (T508E)
and Tyr507 was mutated to Phe (Y507F) and activities were
assayed. T508V abolished the ability of LIM kinase to induce changes in
the actin cytoskeleton in vivo (Figs. 4 and
8, A and B). This
loss of enzyme activity suggests that phosphorylation of
Thr508 in the activation loop is essential for enzymatic
activity. The T508V LIM kinase also interfered with the actin
cytoskeleton changes induced by WT LIM kinase (Fig. 4). Changing
Thr508 to Glu, which is reported to constitutively activate
protein kinase C by mimicking a phosphorylated Thr (50) also resulted in an enzyme that did not exhibit LIM kinase activity as assessed by
changes in the actin cytoskeleton (Figs. 4 and 8, C and
D).
To further investigate the potential effects of phosphorylation of
Thr508, this residue was mutated in the highly active
kinase-only construct Kd3. While Kd3 induced severe actin aggregation
(Fig. 9, panels A and
B), substitution of Glu for Thr at residue 508 failed to induce actin aggregation in COS-7 cells (Fig. 9, panels C
and D), comparable to lack of effects of the kinase inactive
Kd3-D46ON mutant (Fig. 9, panels G and H). In
contrast, substitution of 2 Glu residues whose charge may more closely
resemble a phosphate on Thr (Kd3-T508EE) resulted in full activity
comparable to that of Kd3 (Figs. 4 and 9, E and
F). These findings resemble studies of Mek1 where
substitution of an Asp or Glu residue for 1 serine phosphorylation site
partially activated the enzyme and substitution of 2 acidic residues
for both serine phosphorylation sites resulted in a fully active enzyme
(51). Together, these results suggest that phosphorylation of
Thr508 is required for LIM kinase activity. In contrast
changing T507F did not reduce LIM kinase activity on the actin
cytoskeleton (Fig. 8, panel E). Thus, Thr508 is
likely an essential phosphorylation site in LIM kinase whereas Tyr507 is not. The failure to autophosphorylate on Thr
in vitro (6) and the lack of kinase activity of bacterially
expressed Kd3 (data not shown) suggest Thr508 is
phosphorylated in trans by a distinct kinase.
The activation loop located between subdomains VII and VIII in the
catalytic core has been shown to influence substrate recognition in
MAPK (52). Both LIM kinases 1 and 2 contain a unique highly basic 11 amino acid insert in the activation loop that may function in
recognition of the unusual amino-terminal phosphorylation site on
cofilin. Alternatively, the basic residues may provide the substrate
determinants for Thr508 analogous to those of other
substrates for kinases such as cAMP-dependent protein
kinase and protein kinase C (53, 54). To investigate the function of
this insert, 2 mutants were constructed: in one, the kinase insert was
deleted (KI-del.) and in the other, the basic residues
502Arg-Lys-Lys were mutated to Gly-Ala-Ala (KI-mut) (Fig.
1). Both mutants failed to induce significant actin aggregation in
COS-7 cells (Fig. 10, A-D).
Activity was clearly reduced compared with the wild-type enzyme (Fig.
10, E and F) as even high levels of expression of
these mutant forms of LIM kinase resulted in no more than
2+ changes in the actin cytoskeleton. No change in
subcellular localization was noted by mutation or deletion of the
kinase insert region.
Mutations in LIM kinase Affect Phosphorylation of Cofilin in
Vitro--
Immunoisolated LIM kinase-catalyzed phosphorylation of
cofilin in vitro. As shown in Fig.
11A, both wild-type and Kd3
LIM kinase catalyzed phosphorylation of cofilin in vitro.
LIM kinase catalyzed phosphorylation of myelin basic protein to a low
stoichiometry whereas cAMP-dependent protein kinase and
protein kinase C efficiently catalyzed phosphorylation of MBP but not
cofilin (data not shown).
To assess the effects of phosphorylation of LIM kinase on kinase
activity, immunoprecipitates of wild-type and Kd3 LIM kinase were
treated with CIP to remove any phosphate groups and cofilin phosphorylation was measured. As shown in Fig. 11A, CIP
treatment abolished the activity of both forms of LIM kinase. Mutation
of Thr508 to either Val or Glu markedly reduced kinase
activity in vitro in agreement with loss of the effects of
these enzymes on the actin cytoskeleton in vivo (Fig.
11B and Table I). However,
substitution of 2 Glu residues for the single Thr (Kd3-T508EE) yielded
a fully active enzyme (Fig. 11B and Table I). Enzyme
activity in vitro thus paralleled effects in vivo
indicating that Thr508 is a phosphorylation site that is
essential for LIM kinase activity.
Mutations in the LIM and PDZ domains did not change the ability of LIM
kinase to catalyze cofilin phosphorylation in vitro (Fig.
11A and Table I). Similarly, mutations in the kinase insert retained significant activity (Fig. 11B). In more
quantitative solution assays of LIM kinase-catalyzed cofilin
phosphorylation the kinase insert mutant was also equal to that of the
WT enzyme (Table I). All forms of LIM kinase that phosphorylated
cofilin to detectable levels also exhibited self-phosphorylation (Fig. 11B). These results suggest that in vitro kinase
assays reflect the basal activity of LIM kinase. In the absence of the
kinase insert LIM kinase cannot be activated in vivo,
resulting in the observed marked reduction in actin aggregation
compared with WT LIM kinase. The insert in the activation loop along
with Thr508 is thus necessary for full in vivo
activity. The Km of LIM kinase for cofilin is 7.6 µM compatible with the intracellular concentration of
cofilin (Fig. 11C).
To demonstrate that the phosphorylation seen in vitro is due
to LIM kinase phosphorylation of cofilin at serine 3, a series of
cofilin mutants were assessed. Fig. 11D shows that wild-type cofilin, actophorin, and actophorin mutated at serine 84, but not
cofilin mutated at serine 3, were phosphorylated by LIM kinase in
vitro. This demonstrates that LIM kinase specifically
phosphorylates serine 3, in agreement with previously published data
(9, 10). Under saturating conditions 1 mol of phosphate was
incorporated per mol of cofilin. To demonstrate that the amino terminus
of LIM kinase specifically inhibits the kinase activity of LIM kinase, purified Kd3 LIM kinase was incubated without or with a GST fusion to
the dLIM kinase and in vitro kinase activity measured. As
seen in Fig. 11E, GST-dLIM kinase but not GST inhibited LIM
kinase catalyzed cofilin phosphorylation.
The actin cytoskeleton is a dynamic structure in which the rates
of polymerization and depolymerization of actin control cell motility,
cell division, and the formation of specialized structures. The
actin-binding protein, cofilin, is an important regulator of this
process, mediating cleavage and disassembly of actin filaments (14).
Phosphorylation of cofilin at Ser3 in the amino terminus
inactivates the actin cleaving/depolymerizing activity of this protein
(55). Phosphorylation and dephosphorylation of cofilin thus provides an
important control point for dynamic changes in the extent of actin
polymerization and consequent function. Arber et al. (9) and
Yang et al. (10) provided evidence that LIM kinase
phosphorylates and inactivates cofilin. In co-transfection and
microinjection experiments LIM kinase blocked the effects of cofilin,
but not those of a mutant cofilin containing an Ala replacement of the
Ser3 phosphorylation site. A kinase-inactive form of LIM
kinase lacked activity but blocked the effects of LIM kinase.
Importantly, both Arber et al. (9) and Yang et
al. (10) reported that the LIM kinase mediated phosphorylation of
cofilin and resultant cytoskeletal changes were enhanced by the
constitutively active V12 mutant of Rac and reduced by the dominant
negative N17 mutant of Rac. These findings support a model in which Rac
activates LIM kinase which phosphorylates and inactivates cofilin
resulting in decreased rates of actin depolymerization. The mechanisms
through which Rac regulates LIM kinase activity are unknown but do not
involve direct interactions between these two proteins (10).
The present studies confirm cofilin phosphorylation by LIM kinase and
demonstrate that in vivo LIM kinase causes changes in actin
cables with progressive accumulation of large aggregates of actin
consistent with loss of cofilin function. This biological "read
out" was used to assess the contribution of various regions of LIM
kinase to activity and to compare these results to cofilin phosphorylation in vitro.
While the structure of LIM kinase is unique in having 2 amino-terminal
LIM domains and a PDZ domain, it resembles other kinases such as Src
whose activity is regulated by modular protein domains located outside
the catalytic kinase core (56, 57). Both LIM and PDZ domains function
in protein-protein interactions as do the SH2 and SH3 domains in Src
(58-60). While the LIM and PDZ domains of LIM kinase likely recognize
proteins important in the overall biological function of this protein,
the present data indicate these domains also regulate the kinase
activity of LIM kinase. Mutation of LIM1 had no detectable effect on
LIM kinase activity but mutational inactivation of the second LIM
domain and of the PDZ domain enhanced biological activity of LIM kinase
in vivo, suggesting these domains directly or indirectly
restrict activity. The observation that a kinase-only construct Kd3
also exhibited enhanced biological activity supported this idea. The
kinase-inactive D460N LIM kinase acted in a dominant interfering manner
consistent with in vitro studies showing self-association
(44). The finding that a naturally occurring splice variant of LIM
kinase that consists of the amino terminus only (LIM plus PDZ domains)
inhibited not only the wild-type but also the kinase domain-only Kd3
mutant suggests that the self-association between the amino- and
carboxyl-terminal portions observed in vitro also occurs
in vivo. These data support a model of LIM kinase in which
the amino terminus interacts with the kinase domain to suppress
activity. In addition to the splice variant identified in this article,
one LIM kinase-1 and four LIM kinase 2 splice variants have been
identified (2, 6, 43). Three of these variants contain deletions that
disrupt the kinase domain while one contains only a kinase domain and is expressed only in testis. The ability of dLIM kinase to inhibit Kd3
suggests that these kinase inactive splice variants may, depending on
levels of expression, function as naturally occurring inhibitors of LIM kinase.
Several motifs within the kinase domain appear to control kinase
activity. The kinase domain contains both Thr and Tyr residues in the
activation loop as well as a distinct basic 11-amino acid insertion.
The present data indicate that Thr508 is an essential
phosphorylation site but Tyr507 is not. A Val substitution
at residue 508 abolished actin aggregating activity and a negatively
charged Glu that was predicted to partially mimic phosphorylated Thr
similarly resulted in loss of activity. However, placement of 2 Glu
residues in this position resulted in a fully active enzyme both
in vivo and in vitro. Phosphorylation of Thr in
the activation loop of several Ser/Thr kinases is essential for
catalytic activity (61). In the MAPK signal transduction pathway, both
MAPK kinase (Mek) and MAPK are regulated by dual phosphorylations in
the activation loop. Mek1 is activated by phosphorylation of 2 nearby
Ser residues and replacement of both with Asp is necessary for full
activity (51). Mek activation of MAPK requires phosphorylation of both
a Tyr and Thr residue in the activation loop (49). However, A kinase
and CDK are phosphorylated on a single Thr residue in this loop (45,
46). While additional phosphorylations may regulate LIM kinase, that of
Thr508 is essential. The requirement for 2 Glu residues for
activity may be due to interactions with the adjacent basic insertion
region. Because bacterially expressed LIM kinase did not exhibit
self-phosphorylation on Thr (and had very low catalytic activity) (6)
phosphorylation of Thr508 is predicted to occur in
trans. The sequence of the Thr508 site in LIM
kinase, 504Lys-Lys-Arg-Tyr-Thr-Val-Val, clearly differs
from the phosphorylation site in cofilin (2, 11) making
autophosphorylation unlikely. LIM kinase contains the determinants in
the active site that are characteristic of protein kinases which are
activated by phosphorylation (61). Thus, upstream signals may be
transmitted to LIM kinase both via the amino-terminal LIM and PDZ
domains and via regulation of a kinase that phosphorylates LIM kinase
at Thr508 to activate the enzyme. Protein kinases that are
regulated by Rac (for review, see Ref. 62) are candidates to be
involved in the signal transduction pathway linking Rac to LIM kinase
activation and consequent cytoskeleton changes.
The basic insert in the activation loop which precedes
Thr508 likely provides important substrate determinants for
phosphorylation at this site. Deletion or mutation of basic residues in
this insertion largely abolished LIM kinase activity in
vivo. However, the KI deletion did not decrease LIM
kinase-catalyzed phosphorylation of cofilin in vitro. These
results suggest that while the insert is not required for basal LIM
kinase activity, it is necessary for activation of the enzyme in
vivo.
LIM kinase thus contains a number of regulatory features consistent
with a pivotal role in control of dynamics of the actin cytoskeleton.
Relief of inhibitory constraints imposed by amino-terminal LIM and PDZ
regulatory domains and phosphorylation of an activation loop Thr are
consistent with the general structural features that regulate the
activity of other protein kinases and are proposed to be the targets
for receipt of upstream signaling information.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actinin via its first LIM domain (29). Deletion of muscle
LIM protein (cysteine-rich protein 3 or muscle LIM protein), which
localizes to actin filaments via LIM domains, results in disruption of
cardiomyocyte architecture and dilated cardiomyopathy (30). Some PDZ
domains that bind to the consensus Ser/Thr-X-Val/Leu/Ile at
the carboxyl terminus of target proteins (31) also bind to the
cytoskeleton via interaction with
-actinin-2 (28). Other PDZ domain
containing proteins also bind to actin via F-actin-binding domains (32, 33). LIM kinase thus contains structural features of cytoskeletal regulatory proteins and kinase activity specific for cofilin.
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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Fig. 1.
Mutations in LIM kinase. The indicated
mutations in LIM kinase were prepared and inserted into the pcDNA 3 expression vector as described under "Experimental Procedures."
Point mutations are in italics with letters corresponding to
the one-letter amino acid code. The black box in dLIM kinase
represents 12 amino acids not present in wild-type LIM kinase. The
black bar within the kinase domain represents the 11-amino
acid insertion.
-32P]ATP (4500 Ci/mmol), 2 mM
MnCl2, and 5 mM MgCl2 with 25 µM cofilin. Reactions were stopped by addition of SDS
sample buffer and loading on a 14% SDS-polyacrylamide gel
electrophoresis. Gels were dried and exposed to X-Omat AR film (Kodak,
Rochester, NY) for 1 h at room temperature with an intensifying
screen. Films were scanned with a Molecular Dynamics Personal
Densitometer SI and analysis was done using Image Quart Vd 1.1 (Molecular Dynamics). Phosphatase treatment of immunoprecipitated LIM
kinase was carried out at 30 °C for 25 min with and without calf
intestinal alkaline phosphatase (CIP) (New England Biolabs), in kinase
reaction buffer. CIP-treated and mock treated LIM kinase was washed 4 times in kinase reaction buffer and kinase reactions were run as described.
-32P]ATP (specific activity 0.5 µCi/nmol) varying
amounts of cofilin, 2 mM MnCl2, 5 mM MgCl2, 50 mM HEPES (pH 7.4), 150 mM NaCl, and various forms of LIM kinase expressed by
transient transfection of HEK 293 cells. Reactions were incubated for
the indicated times at 30 °C and terminated by spotting an aliquot
onto Whatman 3MM filter disks that were placed in cold 10%
trichloroacetic acid. Fillers were sequentially washed in
cold/heated/cold 5% trichloroacetic acid, dried in 70% ethanol, and
counted. Background values using untransfected HEK 293 cell lysates
were subtracted and kinase activity was adjusted for LIM kinase
expression quantitated by chemiluminescence imaging using Molecular
Dynamics (Bio-Rad) hardware and software. SDS-polyacrylamide gel
electrophoresis analysis indicated that >90% of radioactivity incorporated was into cofilin and that cofilin phorphorylation was not
detected using untransfected lysates. Triplicate data points are shown
for assays that were repeated.
RESULTS
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ABSTRACT
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DISCUSSION
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Fig. 2.
LIM kinase induces an actin aggregation
phenotype that is dependent on kinase activity. COS-7 cells were
transfected with 1 µg of either WT LIM kinase (A-H) or
mutant kinase-inactive LIM kinase D460N (I-J). Cells were
transfected, harvested, and stained with anti-LIM kinase antibodies
(A, C, E, G, and I) or phalloidin (B, D, F,
H, and J). The extent of changes in the actin
cytoskeleton are indicated by the scale on the right and the
intensity of LIM kinase staining is indicated on the left.
The scale bar equals 20 µm, for all figures. The same
magnification was used in panels A, B, G-J, and in
panels C-F.
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Fig. 3.
The second LIM domain and the PDZ domain
negatively regulate the activity of LIM kinase. COS-7 cells were
transfected with 1 µg of either LIM kinase C25S (A and
B), LIM kinase C84S (C and D), or LIM
kinase G177E/L178A (E and F). Cells were stained
for LIM kinase (left panels) and actin (right
panels), and graded on expression level (left) and
actin aggregation (right).
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Fig. 4.
Distribution of actin cytoskeletal phenotypes
in cells expressing various LIM kinase mutants. COS-7 cells were
transfected with the indicated LIM kinase mutants and stained for LIM
kinase and actin. Transfected cells were identified and the actin
phenotype scored in 100 cells without knowledge of the form of LIM
kinase. , 1+;
, 2+;
, 3+;
, 4+, as defined in the legend to Fig. 2.
A
helix, which runs down the back of the large and small lobes of
cAMP-dependent protein kinase and is important for
stability and catalytic activity of that enzyme (42). The second
construct, Kd3, which is composed of the entire kinase domain, included
the
A helix (residues 302-647). This construct corresponds to a
naturally occurring splice variant of LIM kinase that is found in
testis (43). Even high levels of expression of Kd1 failed to induce
changes in the actin cytoskeleton (Fig.
5, A and B). The
punctate perinuclear staining suggested that Kd1 may not be folded and
processed normally. In contrast, Kd3 was extremely active, inducing a
strong aggregation phenotype (Fig. 5, C and D;
Fig. 4). The complete kinase domain with the
A helix is thus
required for biological effects on the actin cytoskeleton. The enhanced
activity of Kd3 supports the idea that the amino terminus of LIM kinase
suppresses activity of the holenzyme.
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Fig. 5.
The kinase domain-only construct Kd3 has high
actin aggregating activity. COS-7 cells were transfected with 1 µg of either Kd1 (A and B) or Kd3 (C
and D) and stained for LIM kinase (left panels)
and actin (right panels) as described under "Experimental
Procedures." Expression and aggregation were graded as
indicated.
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Fig. 6.
A splice variant of LIM kinase results in
deletion of the kinase domain. A, sequence of the
splice junction of the variant dLIM kinase. The deleted sequence is
underlined and the intron branch site is double
underlined. 32 nt not included are indicated by dots.
B, schematic of the intron and exon splicing of the
full-length and dLIM kinase constructs. While holo LIM kinase joins the
3' end of exon 7 to the 5' end of exon 8, dLIM kinase joins it to the
middle of exon 8. C, sequence of the carboxyl terminus of
the splice variant that terminates at Val293 of LIM kinase.
Twelve residues added prior to the stop codon are
underlined. D, comparative expression of
wild-type and variant LIM kinase in various tissues and cell lines as
determined by RT-PCR. The full-length LIM kinase PCR product is 331 nt
whereas the dLIM kinase PCR product is 270 nt. All cell lines were of
human origin except for B82 and CV-1. Fetal brain library was made by
Dimitri Krainc (DK). Libraries purchased from Stratagene(ST),
CLONTECH(CL) are indicated, except SK-N-MC
(lane 5) which was also from CLONTECH.
E, immunochemical detection of the splice variant dLIM
kinase expressed in COS-7 cells using the internal peptide antibody
5078 (left panel) and lack of effect on the actin
cytoskeleton as determined by phalloidin staining (right
panel). The sequence data are available from GenBank under
accession number AF134379.
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Fig. 7.
The kinase inactive mutant D460N and the dLIM
kinase splice variant act in a dominant interfering manner. COS-7
cells were transfected with 1 µg of each DNA coding for LIM kinase
D460N (A, B, G, and H), wild-type LIM kinase
(A-F), Kd3 (G-L), Kd1 (C, D, I, and
J), and dLIMK (E, F, K, and L) except
for panels G and H which were transfected with 4 µg of D460N LIM kinase. Cells were stained for LIM kinase with the
5079 antibody, to detect changes in bundling activity (left
panels) and actin (right panels). In panel K
dLIM kinase was detected with 5078 internal peptide antibody and Kd3
with the carboxyl-terminal 5079 antibody.
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Fig. 8.
Thr508 is necessary for LIM
kinase effects on the actin cytoskeleton. COS-7 cells were
transfected with 1 µg of T508V LIM kinase (A and
B), T508E LIM kinase (C and D), or
Y507F LIM kinase (E and F). Cells were stained
for LIM kinase (left panels) and actin (right
panels).
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Fig. 9.
Two glutamic acid residues in the activation
loop activate LIM kinase. COS-7 cells were transfected with Kd3
(A and B), Kd3 containing a single Glu
replacement of Thr (T508E) (C and D), Kd3
containing 2 Glu residues in this area (T508EE) (E and
F) or the kinase-inactive mutation D460N in Kd3
(G and H). Cells were stained for LIM kinase
(left panels) and actin (right panels).
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Fig. 10.
The basic insert sequence affects the actin
aggregating activity of LIM kinase. COS-7 cells were transfected
with 1 µg of either kinase insert-deleted LIM kinase (A
and B), the 3 amino acid kinase insertion mutation
(C and D), or Kd3 containing the kinase insert
mutation (E and F). Cells were stained for LIM
kinase (left panels) and actin (right
panels).
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Fig. 11.
In vitro phosphorylation of
cofilin by wild-type and mutant LIM kinase. All DNAs were
transfected into HEK 293 cells and lysates were used in in
vitro kinase assays and Western blotting as described under
"Experimental Procedures." A, left: LIM
kinase inactivation by dephosphorylation. Immunoprecipitated LIM kinase
and Kd3 were incubated without or with CIP, washed, and in
vitro kinase activity assayed using cofilin and
[ -32P]ATP as substrates. An autoradiogram of
32P-labeled cofilin (upper panel) and a Western
blot of immunoprecipitated LIM kinase and Kd3 (lower panel)
are shown. A, right: effects of mutations in the amino
terminus of LIM kinase on phosphorylation of cofilin.
Immunoprecipitated LIM kinase mutants were assayed for in
vitro kinase activity (upper panel) and blotted for
protein amount (lower panel). B, effects of
mutations within the kinase domain on activity. Various forms of LIM
kinase were immunoprecipitated and assayed for phosphorylation of
cofilin (upper panel). Western blotting was used to adjust
the amount of enzyme used in enzyme assays (lower two
panels). C, determination of Km for
cofilin. The indicated concentrations of cofilin were added to solution
assays containing Kd3 and incubated for 12 min at 30 °C.
Incorporation of 32P into cofilin was quantitated as
described under "Experimental Procedures." D,
phosphorylation of Ser3 of cofilin. Lysates of 293 cells
transfected with WT LIM kinase were used in kinase assays containing WT
and mutant cofilin (serine 3 to alanine, S3A) and WT and mutant
actophorin (serine 84 to alanine, S84A). Two µg of substrate were
used in each reaction. E, effect of dLIM kinase on LIM
kinase activity. Reactions contained soluble WT LIM kinase alone or
with a 5-fold molar excess of GST dLIM kinase or GST.
Cofilin phosphorylation by various forms of soluble LIM kinase
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Laurent Blanchoin for cofilin and helpful discussion.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK13149.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF134379.
¶ To whom correspondence should be addressed: University of California San Diego, School of Medicine, 9500 Gilman Dr. 0650, La Jolla, CA 92093-0650. Tel.: 619-534-4310; Fax: 619-534-8193; E-mail: ggill{at}ucsd.edu.
2 P. M. Guy, D. A. Kenny, and G. N. Gill, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: PCR, polymerase chain reaction; nt, nucleotide; RT-PCR, reverse transcriptase-PCR; LIM1, LIM domain 1; LIM2, LIM domain 2; CIP, calf intestinal alkaline phosphatase; GST, glutathione S-transferase; MAPK, mitogen-activated protein kinase.
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