Activation of LIM Kinases by Myotonic Dystrophy Kinase-related Cdc42-binding Kinase alpha *

Tomoyuki Sumi, Kunio Matsumoto, Akihiro Shibuya, and Toshikazu NakamuraDagger

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES

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 alpha  (MRCKalpha ), an effector protein kinase of Cdc42. In vitro, MRCKalpha phosphorylated the protein kinase domain of LIM kinases, and the site in LIMK2 phosphorylated by MRCKalpha proved to be threonine 505 within the activation segment. Expression of MRCKalpha induced phosphorylation of actin depolymerizing factor (ADF)/cofilin in cells, whereas MRCKalpha -induced ADF/cofilin phosphorylation was inhibited by the co-expression with the protein kinase-deficient form of LIM kinases. These results indicate that MRCKalpha 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
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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 alpha  (MRCKalpha ), a Cdc42-dependent protein kinase (23), activates both LIMK1 and LIMK2 downstream of Cdc42. Our results define a signal transduction pathway wherein MRCKalpha activates LIMK1 and LIMK2 downstream of Cdc42, which in turn regulates the actin cytoskeletal reorganization through phosphorylation and inactivation of cofilin.

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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 MRCKalpha (amino acids 1-1732), partial rat MRCKalpha 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-Delta C. To generate plasmids encoding MRCK-Delta C (amino acids 1-574), rat MRCKalpha 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-Delta 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.

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 beta -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).

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 beta -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 [gamma -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.

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).

    RESULTS AND DISCUSSION
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INTRODUCTION
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RESULTS AND DISCUSSION
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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.


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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 MRCKalpha (Fig. 2A). MRCK-Delta C is deleted with the C-terminal regulatory region containing GTPase-binding and PH domains. MRCK-Delta C/KD has a substituted amino acid involved in ATP binding in the protein kinase domain, and thus MRCK-Delta C/KD is likely to act in a kinase-deficient form. As shown in Fig. 2B (left panels), co-expression of MRCK-Delta C stimulated LIMK1 activity toward cofilin phosphorylation. Likewise, LIMK2 activity was stimulated by the co-expression with MRCK-Delta C (Fig. 2B, right panels). These stimulatory effects of MRCK-Delta C on LIMK1 and LIMK2 depended on protein kinase activity, because the kinase-dead form of MRCK-Delta C (MRCK-Delta C/KD) did not stimulate LIM kinases activity. We recently found that the dominant active form of ROCK (ROCK-Delta 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). MRCKalpha 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 MRCKalpha 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 MRCKalpha 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 MRCKalpha activates both LIMK1 and LIMK2 activity as an upstream protein kinase.


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Fig. 2.   Activation of LIMK2 activity by MRCKalpha . A, schematic diagram of MRCKalpha and its mutants. The structural domain of MRCKalpha is illustrated at the top; full-length MRCKalpha and two mutants are represented by the thick lines. The numbers indicate amino acid residues of the N and C termini of each mutant. The position of the point mutation is indicated by an asterisk. CR, cysteine-rich domain; PH, pleckstrin homology domain; GBD, GTPase-binding domain. B, activation of LIMK1 and LIMK2 kinase activity by dominant active MRCKalpha (MRCK-Delta C). COS-7 cells were co-expressed with either HA-tagged LIMK1 or LIMK2 and empty vector (Mock), Myc-tagged MRCK-Delta C, or its kinase-dead form (MRCK-Delta C/KD), respectively. C, activation of LIMK1 and LIMK2 by wild-type MRCKalpha . COS-7 cells were co-expressed with HA-tagged LIMK1 or LIMK2 and empty vector (Mock), Myc-tagged wild-type MRCKalpha , or wild-type ROCK, respectively. After transient expression, immunoprecipitated LIMK1 and LIMK2 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). The arrowheads indicate autophosphorylated LIMK1 and LIMK2. Cofilin phosphorylation was estimated using an image analyzer (BAS-2500, 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 means ± S.E. of three independent experiments.

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 MRCKalpha 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-Delta 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-Delta C. Thus, MRCKalpha -dependent LIMK2 activation is apparently mediated by the protein kinase domain of LIMK2.


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Fig. 3.   Activation and phosphorylation of LIMK2 protein kinase domain (PK) by MRCKalpha . A, activation of LIMK2 and PK toward cofilin phosphorylation by MRCKalpha . COS-7 cells were co-expressed with HA-tagged LIMK2 or its PK domain and empty vector (Mock) or Myc-tagged MRCK-Delta C, respectively. After transient expression, immunoprecipitated LIMK2 and PK were used, respectively, 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 immunoblots (bottom panel). The arrowhead indicates autophosphorylated full-length LIMK2. B, phosphorylation of the protein kinase domain of LIMK1 (1PK) and LIMK2 (2PK) by MRCKalpha . COS-7 cells were transfected with expression vector for Myc-tagged MRCK-Delta C or MRCK-Delta C/KD. After transient expression, immunoprecipitated MRCK-Delta C and MRCK-Delta C/KD were, respectively, used for in vitro kinase assay with GST, GST-1PK, or GST-2PK as substrate (top panel) and for anti-Myc immunoblots (bottom panel). The arrowhead indicates autophosphorylated MRCK-Delta C. C, requirement of Thr-505 for activation of LIMK2 by MRCKalpha . COS-7 cells were co-expressed with HA-tagged LIMK2 or its Thr-505 mutants and empty vector or Myc-tagged MRCK-Delta C, respectively. After transient expression, immunoprecipitated LIMK2 and its Thr-505 mutants were used, respectively, 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 immunoblots (lower panel). Protein kinase activity of wild-type LIMK2 in mock cells was taken as 1.0. Each value represents the means ± S.E. of three independent experiments. The arrowheads indicate autophosphorylated LIMK2.

To determine whether PKs are phosphorylated directly by MRCKalpha , 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-Delta C directly phosphorylated the protein kinase domain of LIMKs, whereas MRCK-Delta C/KD did not induce protein phosphorylation of GST-PKs. Because MRCKalpha shares a similar substrate specificity with Rho kinase/ROCK (23, 26) and ROCK-Delta 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 MRCKalpha . To address this possibility, we expressed MRCK-Delta C together with LIMK2T505V or LIMK2T505E and examined the potential of MRCK-Delta 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-Delta C together with phosphorylation of LIMK2. In contrast, neither activation of LIMK2T505V nor LIMK2T505V phosphorylation was evident by co-expression with MRCK-Delta 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-Delta C. These results suggest that MRCKalpha 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 MRCKalpha -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 MRCKalpha was expressed in cells, phosphorylated ADF/cofilin increased to 64% of the total ADF/cofilin (Fig. 4B). Because MRCKalpha does not directly phosphorylates ADF/cofilin (data not shown), this MRCKalpha -induced phosphorylation of ADF/cofilin may possibly be mediated via LIM kinases. Consistent with this notion, MRCKalpha -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 MRCKalpha was co-expressed with both LIMK1/KD and LIMK2/KD, MRCKalpha -induced ADF/cofilin phosphorylation was almost completely inhibited. These results strongly indicate that LIMK1 and LIMK2 function as downstream effectors of MRCKalpha and play a role in MRCKalpha -induced actin cytoskeletal reorganization through inactivation (phosphorylation) of ADF/cofilin.


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Fig. 4.   Two-dimensional immunoblots stained for ADF and cofilin. A, HeLa cells were expressed with empty vector (Mock) or HA-tagged LIMKs or LIMKs/KD. B, HeLa cells were co-expressed with empty vector (Mock) or Myc-tagged MRCKalpha and HA-tagged LIMKs/KD. After transient expression, cell lysates were electrophoresed on two-dimensional gels, and then ADF and cofilin were detected by immunoblotting and use of an anti-cofilin antibody. The total ADF and cofilin in the unphosphorylated form is shown in percentages. pADF/Cofilin, phosphorylated ADF and cofilin.

In the present study, we found that MRCKalpha , 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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger 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

    ABBREVIATIONS

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; MRCKalpha , myotonic dystrophy kinase-related Cdc42-binding kinase alpha ; PCR, polymerase chain reaction; KD, kinase-dead; FBS, fetal bovine serum; MEM, minimum essential medium; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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