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Genes to Cells (2004) 9, 591-600. doi:10.1111/j.1356-9597.2004.00745.x
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Caspase-mediated cleavage and activation of LIM-kinase 1 and its role in apoptotic membrane blebbing

Go Tomiyoshi{dagger}, Yuji Horita{dagger}, Michiru Nishita, Kazumasa Ohashi and Kensaku Mizuno*

Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Actin cytoskeletal reorganization plays a critical role in cell morphological changes, including membrane blebbing during apoptosis. LIM-kinase 1 (LIMK1) regulates actin cytoskeletal reorganization by phosphorylating and inactivating cofilin, an actin filament-depolymerizing and -severing protein. We now report that LIMK1 is cleaved and activated during anti-Fas antibody-induced apoptosis in Jurkat T cells. The cleavage and activation of LIMK1 were blocked by z-DEVD-fmk, an inhibitor for caspase-3 or related proteases, thus indicating that caspase-3-like proteases are responsible for LIMK1 cleavage. The caspase-mediated cleavage of LIMK1 occurs at Asp-240, a site at the N-terminal side of the protein kinase domain, which leads to the production of an N-terminally truncated, constitutively active LIMK1 fragment. Expression of an N-terminally truncated LIMK1 fragment, LIMK1(241–647), induced membrane blebbing in both Jurkat and HeLa cells, with an extent significantly higher than that of wild-type LIMK1. Down-regulation of endogenous LIMK1 expression by small interfering RNA (siRNA) reduced the Fas-induced membrane blebbing in Jurkat cells. These findings suggest that caspase-mediated cleavage and activation of LIMK1 play a role in the membrane bleb formation during apoptosis.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Apoptosis is important in many physiological and pathological processes, including development of multicellular organisms, preservation of tissue homeostasis and ablation of damaged or neoplastic cells (Ellis et al. 1991; Thompson 1995; Jacobson et al. 1997). In the execution step of apoptosis, characteristic cell morphological changes are induced, including membrane blebbing, cell shrinkage and nuclear condensation and fragmentation, thereafter cells are often fragmented into apoptotic bodies and engulfed by phagocytosis (Ellis et al. 1991; Thompson 1995; Jacobson et al. 1997). Whereas regulation of actin cytoskeletal reconstruction seems to be essential for such morphological changes during apoptosis (Cotter et al. 1992; Laster & Mackenzie 1996; Mills et al. 1998; Rao et al. 1999), the molecular mechanisms underlying these processes are not well understood.

Caspase family cysteine proteases are activated in apoptotic cells and play a critical role in the execution phase of apoptosis (Salvesen & Dixit 1997; Thornberry & Lazabnik 1998). Activation of caspases results in the proteolytic cleavage of a variety of cellular proteins, including actin cytoskeletal components and protein kinases involved in actin cytoskeletal reorganization, to induce cell morphological changes during apoptosis (Emoto et al. 1995; Rudel & Bokoch 1997; Lee et al. 1997; Takahashi et al. 1998; Levkau et al. 1998; Coleman et al. 2001; Sebbagh et al. 2001). Recent studies showed that caspase-mediated cleavage of Rho-associated kinase-I (ROCK-I) and p21-activated protein kinase-2 (PAK2), downstream effectors of Rho and Rac/Cdc42, respectively, produces constitutively active kinases and seems to be involved in the induction of membrane bleb formation in apoptotic cells (Rudel & Bokoch 1997; Lee et al. 1997; Coleman et al. 2001; Sebbagh et al. 2001).

LIM-kinases 1 and 2 (LIMK1 and LIMK2) are closely related serine/threonine kinases with structures composed of two LIM domains and a PDZ domain at the N terminus and a protein kinase domain at the C terminus (Mizuno et al. 1994; Okano et al. 1995). LIMKs specifically phosphorylate and inactivate cofilin, an actin-binding protein that stimulates depolymerization and severance of actin filaments (Bamburg 1999; Chen et al. 2000), thereby regulating actin cytoskeletal reorganization (Arber et al. 1998; Yang et al. 1998). LIMKs are activated by phosphorylation at Thr-508 by ROCK and PAK, thus indicating that Rho-ROCK and Rac/Cdc42-PAK signalling pathways induce activation of LIMKs, which in turn induces phosphorylation of cofilin to regulate actin reorganization (Edwards et al. 1999; Maekawa et al. 1999; Ohashi et al. 2000; Amano et al. 2001). Since over-expression of LIMKs induces changes in the actin cytoskeleton, including the accumulation of F-actin and the formation of stress fibers and membrane blebs in cultured cells (Arber et al. 1998; Yang et al. 1998; Maekawa et al. 1999; Amano et al. 2001), we assumed that LIMKs are involved in membrane bleb formation during apoptosis.

In this study, we show that LIMK1 is cleaved and activated during anti-Fas antibody-induced apoptosis in Jurkat T cells, in a manner dependent on caspase-3 activity. We also show that an N-terminally truncated LIMK1 fragment that is produced by caspase-mediated cleavage has higher kinase activity and stimulates membrane blebbing in Jurkat and HeLa cells. Down-regulation of LIMK1 expression by small interfering RNA (siRNA) reduced the Fas-induced membrane bleb formation in Jurkat cells. Our results suggest that caspase-mediated cleavage and activation of LIMK1 play a critical role in membrane bleb formation during apoptosis.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Cleavage and activation of LIMK1 during anti-Fas antibody-induced apoptosis in Jurkat cells

To determine if LIMK1 is cleaved during apoptosis, Jurkat cells were treated with anti-Fas monoclonal antibody and cycloheximide (CHX) for 0–4 h, and cell lysates were analysed by immunoprecipitation and immunoblotting with C-10 anti-LIMK1 antibody, which recognizes the C-terminal tail sequence of LIMK1. As shown in Fig. 1A, whereas only full-length LIMK1 with a molecular mass of about 70-kD was detected in non-stimulated Jurkat cells (at zero time), two cleavage fragments of LIMK1, a major 45-kD fragment and a minor 60-kD fragment, appeared after 2–4 h stimulation with anti-Fas antibody. Densitometric analysis revealed that about 40% of LIMK1 was cleaved to a 45-kD fragment after 4 h stimulation. Pretreatment of cells with benzyloxycarbonyl-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-fluoromethylketone (z-DEVD-fmk), an inhibitor for caspase-3 or related proteases, blocked the appearance of these LIMK1 fragments (Fig. 1B). These results suggest that LIMK1 is cleaved by caspase-3-like proteases during Fas-induced apoptosis in Jurkat cells. Similar pattern of cleavage of LIMK1 was observed when Jurkat cells were treated with tumour necrosis factor-{alpha} (data not shown).



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  		Figure 1  LIMK1 is cleaved and activated during Fas-induced apoptosis. (A) Cleavage of LIMK1. Jurkat cells were treated with anti-Fas antibody plus CHX. At 0–4 h after stimulation, cell lysates were immunoprecipitated and immunoblotted with C-10 anti-LIMK1 antibody. Arrows indicate the positions of LIMK1 and immunoglobulin heavy chain (IgH). Arrowheads indicate positions of LIMK1 fragments newly observed after Fas stimulation. (B) Inhibition of LIMK1 cleavage by z-DEVD-fmk. Jurkat cells were preincubated with or without z-DEVD-fmk, then stimulated with anti-Fas antibody plus CHX for 0–4 h. Cleavage of LIMK1 was analysed as in (A). (C) Activation of LIMK1. Jurkat cells were preincubated with or without z-DEVD-fmk, then stimulated with anti-Fas antibody plus CHX. At 0–4 h, LIMK1 was immunoprecipitated and subjected to in vitro kinase assay, using (His)6-cofilin as a substrate. Reaction mixtures were analysed by autoradiography (32P) and Amido black staining for cofilin. The right panel shows the relative kinase activity of LIMK1. Results are shown as the means ± S.E. of triplicate experiments. (D) Phase-contrast images of Jurkat cells treated with anti-Fas antibody plus CHX for 0–4 h. Time-dependent cell morphological changes are shown. Scale bar, 10 µm.

 
We also examined changes in the kinase activity of LIMK1 during Fas-induced apoptosis of Jurkat T cells. At 0–4 h after stimulation with anti-Fas antibody and CHX, LIMK1 was immunoprecipitated and subjected to in vitro kinase assay, using His6-cofilin as a substrate. As shown in Fig. 1C, the kinase activity of LIMK1 increased approximately two-fold at 4 h after anti-Fas antibody stimulation. Pretreatment of cells with z-DEVD-fmk almost completely inhibited Fas-induced activation of LIMK1 (Fig. 1C), which indicates that LIMK1 is activated by proteolytic cleavage mediated by caspase-3-like proteases after apoptotic stimulation. The time course of LIMK1 activation correlated well with that of membrane bleb formation of Jurkat cells after anti-Fas antibody stimulation (Fig. 1D).

LIMK1 is cleaved at Asp-240 by caspase-3 or related proteases

Since a short sequence motif DEID (amino acid residues 237–240), which is conserved in the PDZ domain of vertebrate LIMK1 but not of LIMK2, matches the optimal sequence of caspase-3 cleavage site (DEXD) (Thornberry et al. 1997), we assumed that LIMK1 is preferentially cleaved at the C-terminal side of Asp-240 (Fig. 2A). We therefore expressed Myc-tagged LIMK1(241–647) in 293T cells and compared its migration position on SDS-polyacrylamide gel electrophoresis (PAGE) with those of LIMK1 fragments produced in anti-Fas antibody-stimulated Jurkat cells. As shown in Fig. 2B, Myc-LIMK1(241–647) migrated to the position similar to that of the major 45-kD cleavage product of LIMK1. A slightly upper shift of Myc-LIMK1(241–647) is probably due to the existence of Myc epitope tag in this construct. Thus, Asp-240 seems to be the major cleavage site of LIMK1 after anti-Fas antibody stimulation. To further determine if caspase-3 is responsible for the apoptotic cleavage of LIMK1, 293T cells were co-transfected with caspase-3 and wild-type LIMK1 and cell lysates were analysed by immunoprecipitation and immunoblotting with C-10 anti-LIMK1 antibody. As shown in Fig. 2C (left panel), the 45-kD cleavage fragment of LIMK1 was detected in cells expressing caspase-3, but not in cells mock-transfected with control vector in place of the plasmid for caspase-3. The cleavage of LIMK1 by caspase-3 coexpression was inhibited by the pretreatment of cells with z-DEVD-fmk (data not shown). We also constructed the C-terminally HA-tagged LIMK1(D240A) mutant, in which the potential cleavage site residue Asp-240 was replaced by alanine. When HA-LIMK1 (D240A) was coexpressed with caspase-3 in 293T cells, no cleavage fragment of HA-LIMK1(D240A) was detected (Fig. 2C, right panel). Together these findings suggest that LIMK1 is cleaved at Asp-240 by caspase-3 or related proteases to produce an N-terminally truncated LIMK1 (241–647) fragment during apoptosis of Jurkat cells.



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  		Figure 2  LIMK1 is cleaved at Asp-240 by caspase-3-like proteases. (A) Schematic diagram of the structures of LIMK1 and Myc-LIMK1(241–647). LIM, LIM domain; PDZ, PDZ domain; kinase, protein kinase domain. The position of a potential cleavage site sequence (DEID) is indicated. (B) A 45-kD LIMK1 fragment produced by anti-Fas stimulation has a similar size to that of Myc-LIMK1(241–647). Jurkat cells were treated with anti-Fas antibody plus CHX for 0–4 h, then cell lysates were immunoprecipitated with C-10 anti-LIMK1 antibody, run on SDS-PAGE, together with 293T cell lysates transfected with plasmids encoding Myc-LIMK1(241–687), and immunoblotted with C-10 anti-LIMK1 antibody. IgH, immunoglobulin heavy chain. (C) Caspase-3 cleaves wild-type (WT) LIMK1 but not LIMK1(D240A). 293T cells were transfected with plasmids coding for wild-type LIMK1 or C-terminally HA-tagged LIMK1(D240A) with or without plasmids coding for caspase-3 and cultured for 16 h. Cell lysates were immunoprecipitated and immunoblotted with C-10 anti-LIMK1 polyclonal antibody or anti-HA monoclonal antibody, as indicated. Arrows indicate the positions of LIMK1 and immunoglobulin heavy chain (IgH). An arrowhead indicates the position of LIMK1 fragment newly observed by co-transfection with caspase-3.

 
Activation of LIMK1 by the cleavage at Asp-240

A number of protein kinases are activated after deletion of their extracatalyic regions (Johnson et al. 1996). To determine if the cleavage of LIMK1 at Asp-240 would induce activation of LIMK1, we expressed Myc-tagged full-length LIMK1 and its N-terminally truncated mutant, Myc-LIMK1(241–647), in 293T cells and compared their cofilin-phosphorylating activities, following immunoprecipitation with anti-Myc antibody. As shown in Fig. 3, Myc-LIMK1(241–647) exhibited about a 3.6-fold higher kinase activity, compared with that of the full-length Myc-LIMK1. Thus the cleavage of LIMK1 at Asp-240 leads to activation of LIMK1.



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  		Figure 3  An N-terminally truncated LIMK1(241–647) fragment exhibits higher kinase activity. (A) Myc-LIMK1 or Myc-LIMK1(241–647) were expressed in 293T cells, purified by immunoprecipitation with anti-Myc antibody, and subjected to in vitro kinase assay, using (His)6-cofilin as a substrate. Reaction mixtures were run on SDS-PAGE and analysed using autoradiography (32P) and Amido black staining for cofilin. Lower panel shows the immunoblot analysis of Myc-LIMK1 and Myc-LIMK1 (241–647) expression in cell lysates with 9E10 anti-Myc antibody. (B) Relative kinase activities are shown as the means ± S.E. of three independent experiments, with the activity of Myc-LIMK1 taken as 1.0.

 
LIMK1(241–647) induces membrane blebbing

We previously reported that over-expression of LIMK1 and LIMK2 in HeLa cells induces apoptotic-like membrane blebbing (Amano et al. 2001). To examine the role of caspase-mediated cleavage of LIMK1 in apoptotic membrane bleb formation, Jurkat cells were co-transfected with the plasmid coding for Myc-LIMK1 or Myc-LIMK1(241–647) together with the plasmid for yellow fluorescent protein (YFP) (to monitor the transfected cells) by electroporation and analysed the effects on cell morphology by phase-contrast microscopy. Cells were pretreated with z-DEVD-fmk to preclude the effect of electroporation-induced caspase activation. To quantify the effects, cells with or without membrane blebs were categorized into three classes (non-blebbing cells and weakly and severely blebbing cells), based on the cell morphologies, as shown in Fig. 4A. Whereas membrane blebs were barely detected in cells mock-transfected with control vector, expression of wild-type LIMK1 and LIMK1(241–647) significantly increased the ratio of membrane blebbing cells, to the extent of 25% and 49% of transfected (YFP-positive) cells, respectively (Fig. 4B). In particular, LIMK1(241–647) stimulated membrane bleb formation more abundantly and severely than did wild-type LIMK1, which suggests that caspase-mediated cleavage of LIMK1 plays an important role in membrane bleb formation in apoptotic Jurkat cells. A caspase inhibitor, z-DEVD-fmk, did not block the membrane blebbing induced by expression of LIMK1(241–647), indicating that an activated LIMK1 fragment has the potential to stimulate membrane blebbing, independent of the caspase activity, and LIMK1 functions downstream of caspase activation.



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  		Figure 4  Expression of LIMK1(241–647) induces membrane blebbing in Jurkat cells. (A) Three categories of cells with or without membrane blebbing. Jurkat cells were transfected with control vector plasmid, or plasmids for Myc-LIMK1 or Myc-LIMK1(241–647), together with plasmids for YFP (with a weight ratio 1:10 for YFP plasmid to LIMK1 plasmid), cultured for 8 h in the presence of z-DEVD-fmk, and fixed. Cell morphological changes in YFP-fluorescence positive cells were observed by phase-contrast microscopy. Cells were classified into three categories; non-blebbing cells, weakly blebbing cells (class 1), and severely blebbing cells (class 2). (B) Quantitative analysis of the effects of expression of Myc-LIMK1 and Myc-LIMK1(241–647) on the membrane bleb formation of Jurkat cells. The numbers of the class 1 and class 2 cells, in which membrane blebs were induced, were counted and the percentages of these cells in the YFP-positive cells were calculated. The results are shown as the means ± S.E. of triplicate experiments.

 
We also examined the effect of expression of LIMK1 or LIMK1(241–647) on the membrane bleb formation in HeLa cells. HeLa cells were transfected with plasmids for wild-type LIMK1, LIMK1(241–647) or control YFP and analysed for the formation of membrane blebs by phalloidin staining (Fig. 5A). As reported (Amano et al. 2001), higher magnification images of phalloidin staining revealed that F-actin-rich clumps in Fig. 5A correspond to the membrane blebs that were produced on the apical surfaces of HeLa cells. Whereas expression of wild-type LIMK1 induced membrane blebbing in 8% of the transfected cells, as reported (Amano et al. 2001), expression of LIMK1(241–647) induced it in 34% of the transfected cells (Fig. 5B). Thus, LIMK1(241–647) has potent activity to induce membrane blebbing in both Jurkat and HeLa cells.



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  		Figure 5  Expression of LIMK1(241–647) induces membrane blebbing in HeLa cells. (A) HeLa cells were transfected with Myc-LIMK1(WT) or Myc-LIMK1(241–647), and cultured for 16 h. Cells were costained with anti-Myc antibody and rhodamine-phalloidin for F-actin. Arrows indicate membrane blebbing cells. Scale bar, 20 µm. (B) Quantitative analysis of the effects of expression of control YFP, Myc-LIMK1 or Myc-LIMK1(241–647) on the membrane bleb formation of HeLa cells. The number of membrane blebbing cells was counted and the percentage of these cells in the YFP- or Myc-positive cells was calculated. The results are shown as the means ± S.E. of triplicate experiments.

 
Down-regulation of endogenous LIMK1 expression by siRNA reduces anti-Fas antibody-induced membrane blebbing

To further determine if LIMK1 is involved in membrane blebbing during apoptosis, expression of endogenous LIMK1 in Jurkat cells was suppressed by transfection of pSUPER siRNA expression plasmid (Brummelkamp et al. 2002), pSUPER-LK1, which directs the synthesis of siRNA targeting for LIMK1. An empty pSUPER vector and siRNA plasmid mutated in the LIMK1 target sequence, pSUPER-LK1mt, were also transfected as control experiments. Electroporation protocol resulted in more than 90% transfection efficiency, as monitored by co-transfection of a YFP plasmid. Immunoblot analysis revealed that transfection of cells with pSUPER-LK1 significantly reduced the level of endogenous LIMK1 expression, whereas pSUPER vector or mutated siRNA plasmid failed to suppress it (Fig. 6A). The level of the Ser-3-phosphorylated cofilin (P-cofilin) in cells transfected with pSUPER-LK1 was decreased to 55%, compared with that in cells transfected with pSUPER-LK1mt (Fig. 6A). Partial inhibition of cofilin phosphorylation suggests that cofilin kinases other than LIMK1 are also involved in cofilin phosphorylation in Jurkat cells. When Jurkat cells transfected with these plasmids were treated with anti-Fas antibody for 4 h, membrane blebbing was detected in 23% of cells transfected with pSUPER-LK1, whereas it was detected in 34% and 32% of cells transfected with a pSUPER vector and pSUPER-LK1mt, respectively (Fig. 6B). Thus, the down-regulation of endogenous LIMK1 expression partially inhibited Fas-induced membrane blebbing. These results suggest that LIMK1 is at least in part involved in the membrane bleb formation during Fas-induced apoptosis in Jurkat cells.



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  		Figure 6  Down-regulation of endogenous LIMK1 expression by siRNA suppresses Fas-induced membrane bleb formation. (A) Reduction of LIMK1 expression by siRNA. Jurkat cells were transfected with either an empty pSUPER vector, LIMK1 RNAi plasmid (pSUPER-LK1) or its mutated plasmid (pSUPER-LK1mt). After incubation for 60 h, LIMK1 expression was analysed by immunoprecipitation and immunoblotting with C-10 anti-LIMK1 antibody. Immunoblots of cell lysates with antibodies for ß-actin, Ser-3-phospho-cofilin (P-cofilin) and cofilin are also shown. Relative values of the densities of P-cofilin blots are indicated under the panel. (B) Effects of LIMK1 RNAi on Fas-induced membrane bleb formation. Jurkat cells transfected with control or LIMK1 RNAi plasmids were cultured for 60 h and then stimulated for 4 h with anti-Fas antibody and CHX. The number of membrane blebbing cells in at least 800 cells was counted. The results are shown as the means ± S.D. of triplicate experiments. *P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Apoptotic membrane blebbing requires actin filament assembly as well as actomyosin-based contractile force generation (Cotter et al. 1992; Laster & Mackenzie 1996; Mills et al. 1998; Rao et al. 1999). Since caspase-3-deficient cells fail to exhibit membrane blebs after apoptotic stimuli, caspase-3-mediated proteolytic cleavage seems to play a critical role for the apoptotic bleb formation (Zheng et al. 1998; Janicke et al. 1998). Previous studies revealed that ROCK-I activated by caspase-3 plays an essential role for membrane blebbing in apoptotic cells by stimulating actomyosin contractility through myosin light chain phosphorylation (Coleman et al. 2001; Sebbagh et al. 2001). In the present study we have shown that LIMK1 is cleaved and activated in Fas-stimulated apoptotic Jurkat cells. The cleavage product of LIMK1 is constitutively active and remarkably stimulates membrane blebbing when over-expressed in Jurkat cells, and siRNA-mediated suppression of LIMK1 expression reduced the extent of membrane bleb formation in Fas-stimulated Jurkat cells. These findings suggest that caspase-mediated activation of LIMK1 plays an important role in apoptotic membrane bleb formation, in collaboration with concomitantly activated ROCK-I. Activated LIMK1 is probably involved in membrane blebbing by promoting actin filament assembly through phosphorylating and inactivating cofilin and by stabilizing ROCK-induced actomyosin contractile structures. Apoptotic membrane bleb formation was only partially suppressed by LIMK1 knockdown by siRNA. It may be due to the functional redundancy of other cofilin kinases, including LIMK2, TESK1 and TESK2 (Amano et al. 2001; Toshima et al. 2001a, 2001b), since the level of P-cofilin was reduced only to half in LIMK1-knockdown cells.

We previously showed that LIMK1 is activated by ROCK via phosphorylation of Thr-508 within the activation loop in the kinase domain (Maekawa et al. 1999; Ohashi et al. 2000). Since ROCK-I is cleaved and activated in response to apoptotic stimuli, active ROCK-I may be involved in the activation of LIMK1 in apoptotic cells. However, pretreatment of Jurkat cells with Y-27632, a specific inhibitor for ROCK (Uehata et al. 1997), had no apparent effect on LIMK1 activation after Fas stimulation. Therefore, activation of LIMK1 in apoptotic cells is caused primarily by caspase-mediated proteolytic cleavage, rather than by active ROCK-I-mediated Thr-508 phosphorylation.

Caspase-3 has been shown to cleave at the C-terminal side of the conserved DEXD sequence motif (Thornberry et al. 1997; Thornberry & Lazabnik 1998). Vertebrate LIMK1 (but not LIMK2) contains the sequence DEID240 at the N-terminal side of the protein kinase domain. LIMK1 cleavage was inhibited in the presence of z-DEVD-fmk, suggesting that caspase-3 or related proteases are responsible for this event. Recombinant LIMK1(241–647) migrated on SDS-PAGE at the position similar to that of a major 45-kD LIMK1 fragment produced in Fas-induced apoptotic cells. Caspase-3, when expressed in 293T cells, cleaved wild-type LIMK1, but not LIMK1(D240A), in which a potential cleavage site Asp-240 is replaced by alanine. Taken together these findings suggest that apoptotic cleavage of LIMK1 primarily occurs at Asp-240 by a caspase-3-like protease. We previously showed that the LIMK1 mutants deleted with the N-terminal LIM and PDZ regions have higher kinase activity than does the full-length LIMK1 and that the excess amounts of the N-terminal LIM fragment of LIMK1 suppresses the kinase activity of the C-terminal kinase domain fragment of LIMK1 (Nagata et al. 1999). In accord with this, we have shown here that LIMK1(241–647), a candidate LIMK1 fragment generated by caspase-3, exhibits higher kinase activity than does full-length LIMK1. Thus, it is likely that caspase-3-mediated cleavage of LIMK1 during apoptosis leads to removal of the N-terminal autoinhibitory region of LIMK1 and thereby increases the kinase activity of LIMK1.

In conclusion, we obtained evidence that LIMK1 is cleaved at Asp-240 and activated by caspase-3-like protease and is involved in membrane blebbing during anti-Fas antibody-induced apoptosis in Jurkat T cells. These findings suggest a novel mechanism of LIMK1 activation and a novel physiological function of LIMK1.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cell culture and induction of apoptosis of Jurkat cells

Jurkat human leukaemia T cells were obtained from the Cell Resource Centre for Biomedical Research, Tohoku University (Sendai, Japan) and maintained in RPMI 1640 medium supplemented with 10% foetal calf serum (FCS). For inducing apoptosis, Jurkat cells were suspended in RPMI 1640 medium containing 25 mM HEPES (pH 7.4) and 10% FCS, incubated for 30 min at 37 °C, and stimulated with 200 ng/mL agonistic anti-Fas monoclonal antibody (clone CH11, Medical and Biological Laboratories, Nagoya, Japan) plus 100 µg/mL CHX (Sigma). In some experiments, cells were pretreated with 100 µM z-DEVD-fmk (Calbiochem) for 90 min before apoptotic stimulation. For kinase assay and immunoblot analysis, cells were lysed by adding an equal volume of cold 2 X lysis/kinase buffer (100 mM HEPES, pH 7.4, 300 mM NaCl, 1% Nonidet P-40, 10% glycerol, 2 mM MgCl2 2 mM MnCl2, 40 mM NaF, 2 mM Na3VO4, 2 mM dithiothreitol, 0.5 mM phenylmethylsulphonyl fluoride, 20 µg/mL of leupeptin, 20 µg/mL of pepstatin), then the suspension was incubated on ice for 30 min. After centrifugation, LIMK1 was immunoprecipitated with C-10 anti-LIMK1 antibody, raised against the C-terminal sequence of LIMK1 (Okano et al. 1995). Immunoprecipitates were washed three times with 1 X lysis/kinase buffer and subjected to an in vitro kinase assay and immunoblot analysis.

In vitro kinase assay

LIMK1 was immunoprecipitated with C-10 anti-LIMK1 or anti-Myc antibody. The precipitates were washed with 1 X lysis/kinase buffer and incubated for 1 h at 30 °C in 30 µL of 1X lysis/kinase buffer containing 50 µM ATP, 5 µCi of [{gamma}-32P]ATP (3000 Ci/mmol, Amersham Biosciences) and 2 µg of (His)6-cofilin, as described (Nishita et al. 2002). The reaction mixture was separated on SDS-PAGE on a 15% gel, transferred on to a polyvinylidene difluoride membrane, and subjected to autoradiography to measure 32P-labelled cofilin, using the BAS1800 Bio-Image Analyser (Fuji Film, Tokyo, Japan), and Amido black staining. Incorporation of 32P into cofilin linearly increased until 90 min after incubation.

Immunoblot analysis

Immunoblot analysis was done as described (Ohashi et al. 2000). Antibodies used are rabbit polyclonal antibodies to LIMK1 (C-10, Okanoet al. 1995), P-cofilin (Toshima et al. 2001a), and cofilin (Toshima et al. 2001a), and mouse monoclonal antibodies to Myc (9E10) and HA (12CA5) epitopes (Roche Diagnostics). Immunoreactive protein bands were visualized, using an enhanced chemiluminescence reagent (Amersham Biosciences).

Plasmid construction

Expression plasmids coding for Myc epitope (EQKLISEEDL)-tagged LIMK1 and its mutants were constructed, as described (Nishita et al. 2002). Expression plasmid coding for caspase-3 was generated by PCR amplification and subcloning into a pUCD2 expression vector. Plasmid coding for LIMK1(D240A)-HA was constructed using a site-directed mutagenesis kit (Stratagene). pSUPER vector for siRNA was provided by Dr R. Agami (Netherlands Cancer Institute) (Brummelkamp et al. 2002). To generate LIMK1 siRNA plasmid (pSUPER-LK1), pSUPER vector was ligated with annealed oligonucleotides, containing a 19-nucleotide (nt) sequence derived from human LIMK1 transcript (GAATGTGGTGGTGGCTGAC), a 9-nt spacer and the reverse complement of the same 19-nt sequence (Brummelkamp et al. 2002). To construct mutated LIMK1 siRNA plasmid (pSUPER-LK1mt), two bases in the 19-nt target recognition sequence were mutated (GAATGTTGTGGTGGCTGCC).

Transfection

For transfection, Jurkat cells (about 107 cells) were mixed with expression plasmids in 400 µL of electroporation medium (RPMI 1640 medium containing 20% FCS and 25 mM HEPES, pH 7.4) and electroporated at 280 V and 975 µF, using a Gene Pulser II (Bio-Rad). After electroporation, cells were cultured for 8 h in RPMI 1640 medium containing 10% FCS, 10 mM HEPES (pH 7.4), and 40 µM z-DEVD-fmk before examination. Cells were fixed with 4% formaldehyde and analysed, using phase-contrast and fluorescence microscopy. In the case of siRNA experiments, Jurkat cells were transfected with pSUPER plasmids by electroporation, incubated for 60 h in the absence of z-DEVD-fmk, then stimulated for 4 h with 200 ng/mL anti-Fas antibody plus 100 µg/mL CHX. 293T and HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% FCS and transfected with expression plasmids, using FuGENE 6 (Roche Diagnostics, Tokyo, Japan) and Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), respectively, according to the manufacturer's instructions.

Cell staining

HeLa cells transfected with plasmids were cultured for 12 h, fixed in 4% formaldehyde in phosphate-buffered saline (PBS) for 10 min, washed with PBS, and permeabilized in 0.2% Triton X-100 in PBS for 2 min. After blocking with 5 mg/mL bovine serum albumin in PBS, cells were stained with 9E10 anti-Myc antibody for 2 h at room temperature. After washing with PBS, the cells were incubated with FITC-conjugated anti-mouse IgG (Chemicon, Temecula, CA, USA) and rhodamine-conjugated phalloidin (Molecular Probes, Eugene, OR, USA) for 1 h at room temperature. Images were obtained using a Leica DMLB fluorescence microscope.


   Acknowledgements
 
This work was supported by a Grant-in-Aid for Creative Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and Uehara Memorial Foundation. We thank Dr R. Agami (Netherlands Cancer Institute) for providing pSUPER vector and M. Ohara for comments.


   Footnotes
 
Communicated by: Eisuke Nishida

{dagger}These authors contributed equally to this work. Back

* Correspondence: E-mail: kmizuno{at}biology.tohoku.ac.jp


   References
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 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 31 January 2004
Accepted: 26 March 2004




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