Inhibition of Nuclear Import of LIMK2 in Endothelial Cells by Protein Kinase C-dependent Phosphorylation at Ser-283*

LIM kinases (LIMKs) are mainly in the cytoplasm and regulate actin dynamics through cofilin phosphorylation. Recently, it has been reported that nuclear localization of LIMKs can mediate suppression of cyclin D1 expression. Using immunofluorescence monitoring of enhanced green fluorescent protein-tagged LIMK2 in combination with photobleaching techniques and leptomycin B treatment, we demonstrate that LIMK2 shuttles between the cytoplasm and the nucleus in endothelial cells. Sequence analysis predicted two PKC phosphorylation sites in LIMK2 but not in LIMK1. One site at Ser-283 is present between the PDZ and the kinase domain, and the other site at Thr-494 is within the kinase domain. Activation of PKC by phorbol ester treatment of endothelial cells stimulated LIMK2 phosphorylation at Ser-283 and inhibited nuclear import of LIMK2 and the PDZ kinase construct of LIMK2 (amino acids 142–638) but not of LIMK1. The PKC-δ isoform phosphorylated LIMK2 at Ser-283 in vitro. Mutational analysis indicated that LIMK2 phosphorylation at Ser-283 but not Thr-494 was functional. Serum stimulation of endothelial cells also inhibited nuclear import of PDZK-LIMK2 by protein kinase C-dependent phosphorylation of Ser-283. Our study shows that phorbol ester and serum stimulation of endothelial cells inhibit nuclear import of LIMK2 but not LIMK1. This effect was dependent on PKC-δ-mediated phosphorylation of Ser-283. Since phorbol ester enhanced cyclin D1 expression and subsequent G1-to-S-phase transition of endothelial cells, we suggest that the PKC-mediated exclusion of LIMK2 from the nucleus might be a mechanism to relieve suppression of cyclin D1 expression by LIMK2.

family of proteins, a member of the class of serine/threonine protein kinases, consists of LIMK1 and LIMK2 that specifically phosphorylate and inactivate cofilin, an actin-depolymerizing protein, thereby regulating actin cytoskeleton rearrangement (5,6). Other studies showed that cell cycle progression depends on the regulated activity of the LIMK-cofilin system (7,8). The kinase activity of LIMKs is regulated by members of the RhoG-TPase family, Rho, Rac, and CdC42, via their downstream protein kinases Rho kinase and p21-activated kinases 1 and 4. These kinases phosphorylate LIMK1 at Thr-508 in the activation loop of the kinase domain (9 -11). Rho kinase activates LIMK2 by phosphorylation at Thr-505 (12,13).
Several lines of evidence suggest that LIMKs also have a function in the nucleus. LIMK1 is predominantly localized in the cytoplasm but accumulates in the nucleus, when the cells are treated with the CRM1-dependent export inhibitor, leptomycin B (LMB) (14). In mouse tissues, various splice forms of LIMK2 have been reported that display an unique cellular localization (15,16). LIMK2a (full-length) and LIMK2b containing only one LIM domain are localized exclusively in the cytoplasm (17). In contrast, tLIMK2 (testis-specific LIMK2 splice form) lacking both LIM domains was found to be preferentially localized in the nucleus (18). The phenotype of the LIMK2 knock-out mouse showed a defect in spermatogenesis, suggesting a nuclear function of tLIMK2 in testis (18).
The kinase domain of LIMKs has a unique basic amino acid-rich motif between subdomains VII and VIII. The basic nature of this motif suggests that this may function as a nuclear localization signal (NLS). Moreover, LIMK1 has two export signal sequences within the PDZ domain (14). The NLS and NES might explain the nucleocytoplasmic shuttling of LIMK1. A recent study shows that the nuclear localization of LIMKs can mediate suppression of Rac/Cdc42-mediated cyclin D1 expression. This effect of LIMKs was independent of cofilin phosphorylation and regulation of actin dynamics (19).
In the present study, we addressed the question of whether LIMK2 shuttles between the nucleus and the cytoplasm in endothelial cells and explored possible mechanisms of regulation of LIMK2 nucleocytoplasmic shuttling. We found that LIMK2 shuttles between the cytoplasm and the nucleus in endothelial cells and discovered that PKC inhibits the nuclear import of LIMK2 by phosphorylating the enzyme at Ser-283. PKC-mediated exclusion of LIMK2 from the nucleus might relieve suppression of cyclin D1 expression, leading to G 1 phase cell cycle progression.
Construction of the Expression Plasmids-The pUC-SR␣-LIMK2 vector containing full-length cDNA of LIMK2 was kindly provided by Prof. Mizuno (Tohoku University, Sendai, Japan). The full-length coding sequence of LIMK2 was amplified by PCR with pUC-SR␣-LIMK2 as a template. The PCR-amplified product was cloned into EcoRI and SalI sites of pEGFP-C1 vector (Clontech) to obtain LIMK2 fused with EGFP. The full-length cDNA of LIMK1 was amplified by PCR from a cDNA pool of human umbilical vein endothelial cell total RNA. The following constructs of LIMK2 and LIMK1 were cloned into EcoRI and SalI sites of pEGFP-C1 vector: ⌬LIM1-LIMK2 (amino acids 69 -638), ⌬LIM2-LIMK2 (amino acids 72-124 deleted in full-length), PDZ kinase (amino acids 142-638, PDZK), and kinase domain (amino acids 315-638) of LIMK2 and PDZ kinase (amino acids 146 -647, PDZK) of LIMK1. The mutants of pEGFP-PDZ kinase (S283A, S283EE, T494A) were generated by the QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA) as per the manufacturer's instructions. To express recombinant His-tagged LIMK2 in P. pastoris, the PCR-amplified full-length LIMK2 was cloned into pPICZ expression vector by using EcoR1 and NotI sites. All the constructs were confirmed by DNA sequencing (Agowa GmbH Berlin, Germany).
Expression of Recombinant LIMK2 in P. pastoris-Expression plasmid pPICZ-LIMK2 was electroporated in P. pastoris strain GS115, and the clone with the highest protein expression was selected as per the manufacturer's instructions. The selected colony of transformed cells was grown in 300 ml of BMGY medium (1% (w/v) yeast extract, 2% (w/v) peptone, 100 mM potassium phosphate, pH 7.0,1.34% (w/v) yeast nitrogen base, 4 ϫ 10 Ϫ5 (w/v) biotin, and 1% (v/v) glycerol). After 30 -36 h of incubation at 28°C, the cells were pelleted at 1000 ϫ g and were resuspended in BMMY medium (BMGY medium in which the glycerol was replaced by 1% (v/v) methanol) to induce protein expression, and the cells were harvested 24 h later by centrifugation at 3000 ϫ g at 4°C for 10 min. The cells were washed once with ice-cold breaking buffer (50 mM sodium phosphate, pH 7.4, 10% glycerol) and resuspended in breaking buffer supplemented with protease inhibitor mixture. An equal volume of acid-washed chilled glass beads (0.5 mm in diameter) was added to the suspension, and cells were disrupted by vigorous vortexing 10 times for 1 min, with intervening 1 min of incubation on ice. Cell debris were removed by centrifugation at 2000 ϫ g at 4°C for 5 min followed by ultracentrifugation at 75,000 ϫ g for 10 min. The clear supernatant was bound to the pre-equilibrated Protino Ni 2000 prepacked (Macherey-Nagel) poly-histidine tag purification column at 4°C, and His-tagged LIMK2 was purified according to the manufacturer's instructions.
Cell Culture and Transfection-Human umbilical vein endothelial cells were obtained and cultured as described previously (20). Briefly, cells harvested from umbilical cords were plated onto collagen-coated (room temperature, 75 g/ml collagen G; Biochrom, Berlin, Germany) plastic culture flasks and were cultured at 5% CO 2 , and 37°C in complete endothelial growth medium (Promo Cell, Heidelberg, Germany). In all experiments, human umbilical vein endothelial cells up to third passage were used.
Transient transfection of endothelial cells was performed by electroporation as described previously (21) with modifications. Briefly, cells were grown up to 90% confluency, harvested by trypsin/EDTA (Sigma) treatment, and washed with phosphate-buffered saline. Cells (1.4 ϫ 10 6 cells/400 l) were suspended in electroporation buffer (20 mM Hepes, 137 mM NaCl, 5 mM KCl, 0.7 mM Na 2 HPO 4 , 6 mM D-glucose, pH 7.0). Plasmid (20 g in 30-60 l of electroporation buffer) was mixed with the cell suspension. The cells were incubated for 10 min (room temperature) and then transferred into a 4-mm gap electroporation cuvette. Cells were electroporated at 1000 microfarads and 210 V (Bio-Rad Gene Pulser), 400 l of complete endothelial growth medium (without antibiotics) was added, and cells were transferred to collagen-coated glass bottom Petri dishes that are designed for confocal microscopy (MatTek Corp., Ashland, MA). The cells were grown in fresh complete endothelial growth medium for 24 h to obtain subconfluency (washing and replenishment after 1 and 12 h). The transfection efficiency was 30 -60%.
Immunoprecipitation-Unstimulated and PMA-stimulated endothelial cells washed with ice-cold phosphate-buffered saline and lysed in an equal volume of 2ϫ immunoprecipitation lysis buffer (2% Nonidet P-40, 300 mM NaCl, 20 mM Tris (pH 7.5), 2 mM EGTA, 2 mM EDTA, 5 mM Na 3 VO 4 , Complete mini protease inhibitor, 1 tablet/5 ml (Roche Applied Science), phosphatase mixture 1:100, and 0.1% SDS) for 45 min on ice. The lysates were clarified by centrifugation at 16,000 ϫ g for 15 min, and then 40 l of 50% protein A-Sepharose slurry was added to the supernatants and incubated for 1 h at 4°C to preclear the supernatant. Protein A-Sepharose was prepared by incubating the beads in swelling buffer (20 mM NaH 2 PO 4 , 0.15 M NaCl, and 0.1% NaN 3 ) containing 2% bovine serum albumin to block unspecific binding. Precleared supernatants were incubated overnight with anti-LIMK2 antibody (1:50 dilution) followed by the addition of 80 l of 50% protein A-Sepharose slurry and incubation at 4°C for 1 h. The immunoprecipitates were collected by centrifugation at 16,000 ϫ g for 25 s and then washed three times with 1 ml of ice-cold 1ϫ immunoprecipitation lysis buffer. The immunoprecipitates were further processed for immunoblot analysis.
In Vitro Phosphorylation of LIMK2 by PKC-EGFP-LIMK2 and mutant S283A of EGFP-LIMK2 were immunoprecipitated from transfected endothelial cells. The immunoprecipitates were washed twice with PKC kinase buffer (20 mM HEPES (pH 7.4), 10 mM MgCl 2 , 100 M CaCl 2 ). LIMK2 immunoprecipitates or recombinant His-LIMK2 (0.5 g) were incubated with 0.3 units of different PKC isoforms/ml at 30°C in 100 l of buffer containing 100 M ATP, 0.03% Triton X-100, 100 g of phosphatidylserine/ml, and 20 g of diacylglycerol/ml. After incubation for 10 min, reactions were terminated by the addition of SDS-PAGE sample buffer and boiling the mixtures for 5 min. The phosphorylation of LIMK2 was analyzed after immunoblotting with antiphospho-Ser PKC substrate antibody.
Western Blot Analysis-Unstimulated and stimulated endothelial cells or immunoprecipitate samples were dissolved in an equal volume of 2ϫ Laemmli buffer. Equal amounts of proteins in the samples were subjected to SDS-PAGE and then transferred to nitrocellulose membrane at 200 mAmp for 60 min at 4°C using the Mini Trans-Blot electrophoresis cell (Bio-Rad). Membranes were blocked with 5% (w/v) nonfat milk and incubated with the respective primary and secondary antibodies. The dilutions of the primary antibodies were: anti-cofilin (1:12,000) and anti-phospho-cofilin, anti-LIMK2, anti-phospho-LIMK, anti-phospho-Ser PKC substrate antibodies and anti-cyclin D1 antibody (1:1000). The dilution of the secondary antibody (horseradish peroxidase-conjugated) was 1:5000. The membranes were developed with SuperSignal West Pico chemiluminescent substrate (Pierce) and exposed to Hyperfilm (Amersham Biosciences). The films were scanned into TIF format using a ScanJet 5300C (Hewlett-Packard Company, Palo Alto, CA). Densitometric analysis of the proteins was done using the public domain NIH ImageJ (version 1.32j) software. The calibration to an optical density scale of the scanner was done by using the Kodak step tablet (optical density 0.0 -3.0) as a reference image. The densitometric values of phosphorylated proteins were divided by the corresponding values of unphosphorylated proteins, respectively. Absorption of proteins in unstimulated control samples was set to 100%. Data are presented as mean Ϯ S.E. of three independent experiments.
S-phase Analysis of Endothelial Cells-Endothelial cells were growth-arrested in G 0 /G 1 phase by serum starvation (endothelial cell basal medium containing 0.4% serum) for 48 h. The 0-h value refers to the percentage of cells in S phase at this time point. The cells (0.1 ϫ 10 6 cells/well) were plated into a 6-well plate in endothelial cell complete medium (Promocell) or endothelial basal medium containing 5% serum or 200 nM PMA. The cells were harvested at various time points and fixed in ice-cold methanol at 4°C for 30 min. The cells were washed twice in 500 l of phosphate-buffered saline containing 1% FCS, and the final pellet was resuspended in buffer (20 g/ml propidium iodide, 10 g/ml RNase, 0.1% Triton-X-100 in phosphate-buffered saline) and incubated for 1 h. The cells were analyzed using a FACScan flow cytometer. Data were analyzed using the cellQuest TM software (BD Biosciences Immunocytometry Systems).
Confocal Microscopy-After 24 h of transfection, cells were transferred into phenol red-free OPTI-MEM ® I medium (Invitrogen) and kept for 1-2 h in an incubator. Unstimulated and PMA-stimulated cells were observed with a Zeiss LSM510 confocal laser-scanning microscope. The cells were kept under the microscope at 37°C. The argon laser (488 nM) was used as a light source for EGFP excitation. The microscope function was controlled by a light manager through the software LSM 510 META. For Z-stacking, the top and the bottom positions were selected, and 8 -15 slices were determined according to the pinhole size and scanning time. The area and the mean intensity of the EGFP were measured by LSM 510 META software. The measurements were carried out in three independent experiments with 20 cells randomly selected in each experiment. Mean Ϯ S.E. was calculated for each experiment.
FRAP and FLIP Analysis-Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) analysis was performed with the Zeiss LSM 510 confocal laser-scanning microscope as described previously (22). Briefly, an argon laser with a maximum output of 25 milliwatts was used at 25% capacity. During normal imaging, it was used at 4%. Each image was composed of an average of 4 -6 scans. After capture of four initial images, photobleaching was carried out at 100% laser transmission with 20 -40 iterations. For FRAP analysis, the entire nucleus was photobleached. The recovery of fluorescence intensity was measured in every 30 s in 12-bit fashion (4,096 gray tones). The average of the area of measurement and intensity was calculated, and the highest value of the four initial images before photobleaching was set to 100% intensity.
For FLIP experiments, the settings for image capture were the same as for the FRAP experiments, and images were recorded every 30 s. An area of cytoplasm was repeatedly bleached with 40 iterations at 100% laser transmission. The relative fluorescence in the nucleus of the bleached cell and an adjacent cell (unbleached control) was then measured.

LIMK2 Shuttles between the Cytoplasm and the Nucleus-To
investigate the intracellular distribution of LIMK2 in endothelial cells, cells were transiently transfected with an expression vector encoding full-length LIMK2 tagged with EGFP. As control, the empty vector EGFP-C1 encoding EGFP protein was transfected into the cells. The EGFP protein was diffusely distributed throughout the cytoplasm and the nucleus (Fig. 1A,  panel a), reflecting the unfacilitated diffusion of EGFP (ϳ27 kDa) through the nuclear pore. The EGFP-LIMK2 protein was mainly localized in the cytoplasm (Fig. 1A, panel b), and only a small fraction was in the nucleus (Fig. 1B).
To determine whether LIMK2 shuttles between the nucleus and cytoplasm in endothelial cells, EGFP-LIMK2-transfected endothelial cells were treated with LMB (10 ng/ml), which inhibits the CRM1-dependent export of proteins from the nucleus (23,24). After LMB treatment, EGFP-LIMK2 localized exclusively in the nucleus (Fig. 1A, panel d). The cytoplasmic distribution of EGFP was not affected by the LMB treatment (Fig. 1A, panel c). These results indicated that the cytoplasmic EGFP-LIMK2 continuously enters the nucleus and that CRM1 transports EGFP-LIMK2 actively and continuously out of the nucleus.
The nucleocytoplasmic shuttling of LIMK2 was directly visualized by photobleaching techniques in living endothelial cells. Endothelial cells were transfected with the EGFP-PDZ kinase domain construct of LIMK2 (EGFP-PDZK), which distributes equally between the nucleus and cytoplasm ( Fig. 2A). Analysis by FRAP and FLIP showed that EGFP-PDZK shuttled between nucleus and cytoplasm. Specifically, FRAP analysis after photobleaching of the nucleus showed that EGFP-PDZK entered the nucleus. The time for 50% recovery (1 ⁄2 ) of fluorescence was 205 s (supplementary Fig. 1). These results suggested that the NLSs within the kinase domain drive the EGFP-PDZK into the nucleus.
To study whether EGFP-PDZK was exported from the nucleus to the cytoplasm, the FLIP analysis was used. An area of the cytoplasm of a cell expressing EGFP-PDZK was photobleached, and fluorescence intensity in the nucleus of the photobleached cell was monitored. As control, the fluorescence intensity in the nucleus of an adjacent unbleached cell was measured. The level of nuclear fluorescence in the photobleached cell gradually decreased over time. In contrast, the nuclear fluorescence in the adjacent cell (unbleached) was unaffected (supplementary Fig. 2). This indicated that EGFP-PDZK was exported from the nucleus in endothelial cells.
LIM Domains Regulate the Cytoplasmic Localization of LIMK2, Not LIMK1-Previously, it has been reported that LIM domains interact with the kinase domain of LIMK1 (25). Since LIMK2 is found mainly in the cytosol of resting endothelial cells, the putative NLS of LIMK2 in the kinase domain might be masked by the interaction with the LIM domains. To investigate this, different constructs of LIMK2 were made with EGFP in which either one or both LIM domains were deleted. The nuclear localization of ⌬LIM1-LIMK2 (LIM1 domain deleted) was ϳ2 times higher than the wild type EGFP-LIMK2 (Fig. 2, A and B). This indicated that LIM1 plays a role in the cytoplasmic localization of LIMK2. In contrast, the ⌬LIM2-LIMK2 protein (LIM2 domain deleted) was localized mainly in the cytoplasm, similarly to the full-length EGFP-LIMK2 protein, suggesting that the LIM2 domain alone does not affect the subcellular localization of LIMK2. EGFP-PDZK of LIMK2 (both LIM domains deleted) was drastically increased in the nucleus (to 40% of the total protein; Fig. 2B). These results suggested that both LIM domains contribute to the cytoplasmic localization of LIMK2. Also, EGFP-LIMK1 was mainly present in the cytoplasm, and after LMB treatment, it was exclusively localized in the nucleus of endothelial cells (data not shown), COS cells, and Hela cells (27). However, in contrast to LIMK2, deletion of the two LIM domains of LIMK1 did not affect the subcellular distribution of LIMK1. The EGFP-PDZK of LIMK1 was still localized in the cytoplasm (Fig. 2, A and B). This indicated that the nucleocytoplasmic shuttling of LIMK1 and LIMK2 is regulated by different mechanisms.
LIM domains might either inhibit the nuclear import or accelerate the nuclear export of LIMK2. To distinguish between these two mechanisms, the kinetics of nuclear accumulation of EGFP-LIMK2-and EGFP-PDZK-LIMK2-transfected endothelial cells were compared after the addition of LMB to inhibit nuclear export. We found that the nuclear fluorescence of EGFP-PDZK-LIMK2 increased twice as rapidly (from 40 Ϯ 6 to 95 Ϯ 4%; mean Ϯ S.E.) as the nuclear fluorescence of EGFP-LIMK2 (from 6 Ϯ 2 to 35 Ϯ 7%; mean Ϯ S.E.) 20 min after LMB addition. These results indicated that the LIM domains reduced the rate of the nuclear import of LIMK2; they were, however, unable to inhibit nuclear accumulation of LIMK2 when cells were treated for 1 h with LMB (Fig. 1A, panel d).
Effect of PMA on the Subcellular Localization of LIMK2-Analysis of the amino acid sequence revealed two potential PKC phosphorylation sites in LIMK2; one site at Ser-283 is located between the PDZ and the kinase domain (Fig. 3A), and a second site is at Thr-494 in the kinase domain of LIMK2. There was no potential PKC phosphorylation site in LIMK1.
To explore whether PKC activation affects the nuclear localization of LIMK2, the subcellular distribution of EGFP-LIMK2, EGFP-PDZK, and EGFP kinase of LIMK2 in endothelial cells was studied before and after stimulation with PMA, a potent PKC activator. Within 30 min of stimulation with PMA, the amount of nuclear EGFP-LIMK2 (6 Ϯ 2%) decreased to barely detectable levels (1 Ϯ 0.5%, p Ͻ 0.05; Fig. 3B, panels a and e,  and 3C). Nuclear EGFP-PDZK of LIMK2 was shifted to the cytoplasm in PMA-treated cells (Fig. 3B, panels b and f, and 3C). There was no effect of PMA on the nuclear localization of EGFP kinase of LIMK2 (Fig. 3B, panels c and g). These results indicated that PKC activation inhibits the nuclear localization of LIMK2 and suggested that Ser-283 between the PDZ and kinase domain may be the target for PKC phosphorylation, whereas the second potential phosphorylation site, Thr-494 in the kinase domain, does not seem to be involved in the regulation of LIMK2 nucleocytoplasmic shuttling. The subcellular localization of EGFP-PDZK of LIMK1 was not affected by PMA stimulation, indicating that nucleocytoplasmic shuttling of LIMK1 is not regulated by PKC activation (Fig. 3B, panels d  and h).
To confirm that the PMA-induced exclusion of LIMK2 from the nucleus is regulated by PKC, two specific PKC inhibitors were used, Go6983 and Go6976 (28 -30). The PMA-stimulated translocation of EGFP-PDZK to the cytoplasm was completely blocked by these two PKC inhibitors (Fig. 3C).
PKC-dependent Phosphorylation of LIMK2 at Ser-283 in PMAstimulated Endothelial Cells-Phospho-specific antibodies are powerful tools to analyze protein phosphorylation. To investigate the potential PKC phosphorylation site in LIMK2, a specific anti-phospho-Ser PKC substrate antibody was used (31). This antibody binds with high affinity to the consensus peptide sequence (XXRRRS*LRRXX) for PKC phosphorylation present be-tween the PDZ and kinase domain of LIMK2 (Fig. 3A).
To analyze whether endogenous LIMK2 was phosphorylated by PKC, endothelial cells were untreated (control) or treated with PMA, and LIMK2 in endothelial cell lysates was immunoprecipitated with a specific anti-LIMK2 antibody and blotted with anti-phospho-Ser PKC substrate antibody. The phosphorylation of LIMK2 increased about 3.5-fold after PMA treatment (Fig. 4, A and B). Both PKC inhibitors Go6976 and Go6983 inhibited LIMK2 phosphorylation in control cells and PMA-stimulated cells (Fig. 4, A and B). Go6983 was more potent then Go6976. These results strongly suggested that PKC phosphorylates endogenous LIMK2 on Ser-283 in PMAstimulated endothelial cells.
To confirm the specificity of the anti-phospho-Ser PKC substrate antibody, a mutant of EGFP-PDZK was prepared in which Ser-283 was mutated to alanine. The mutant was transfected into endothelial cells, and cells were stimulated with PMA. The anti-phospho-Ser PKC substrate antibody detected a large increase of phosphorylation of wild type EGFP-PDZK after PMA stimulation (Fig. 4C). However, there was no signal on the immunoblots of the S283A mutant of EGFP-PDZK in PMA-stimulated cells or control cells (Fig. 4C). These results proved that Ser-283 in LIMK2 is phosphorylated in PMAstimulated cells. PKC-␦ Phosphorylates LIMK2 at Ser-283 in Vitro-To investigate whether PKC directly phosphorylates LIMK2 and to analyze which PKC isoform is involved, we performed in vitro kinase assays using recombinant LIMK2 and different isoforms of PKC. The result demonstrates that only PKC-␦ was able to phosphorylate LIMK2 (Fig. 4D, upper blot). To confirm that PKC-␦ phosphorylated LIMK2 at Ser-283, immunoprecipitates of transfected EGFP-LIMK2 and its mutant S283A were subjected to in vitro phosphorylation by PKC-␦. The results demonstrate that PKC-␦ phosphorylated wild type EGFP-LIMK2 but failed to phosphorylate the S283A mutant of EGFP-LIMK2 (Fig. 4D, lower blot). Together, the results proved that PKC-␦ phosphorylates LIMK2 at Ser-283.
PKC-mediated Phosphorylation of Ser-283, but Not Thr-494, Inhibits the Nuclear Translocation of LIMK2-To examine whether Ser-283 phosphorylation is responsible for the translocation of LIMK2 from the nucleus to the cytoplasm in PMAactivated cells, Ser-283 was modified to either alanine (S283A) or two glutamic acids (S283EE). Substitution of the phosphorylation site with two acidic amino acids (EE) mimics the phosphorylation of that site. LIMK2 constructs containing the S283EE mutation should be constitutively active, whereas constructs containing the S283A mutation should be inactive. After PMA stimulation of cells, wild type EGFP-PDZK was excluded from the nucleus, whereas the nuclear localization of the S283A mutant of EGFP-PDZK was not changed (Fig. 5, A  and B). In contrast, mutation of the second potential PKC phosphorylation site had no effect; the T494A mutant of EGFP-PDZK was translocated from the nucleus to the cytoplasm similar to the wild type protein after PMA treatment (Fig. 5, A  and B). In cells transfected with the active S283EE mutants of EGFP-PDZK-LIMK2 or EGFP-LIMK2, the protein was exclu-sively localized in the cytoplasm similar to the PMA-stimulated cells (Figs. 5A and 6). These results indicated that PKC regulates the nuclear transport of LIMK2 in PMA-activated endothelial cells through phosphorylation of Ser-283.
Serum Stimulation of Endothelial Cells Inhibits the Nuclear Localization of PDZK-LIMK2 by Protein Kinase C-dependent Phosphorylation of Ser-283-To provide evidence for the physiological relevance of the results, endothelial cells were stimulated with serum, which activates growth stimulatory signaling pathways such as PKC. FCS (10%) stimulation of endothelial cells, which had been serum-starved before for 15 h, inhibited the nuclear accumulation of EGFP-PDZK-LIMK2 similar to PMA. This effect was abolished by pretreatment of cells with Go6983 or by S283A mutation of EGFP-PDZK (Fig. 5, A and C). These results showed that exposure of endothelial cells to physiological growth factors present in serum-induced exclusion of LIMK2 from the nucleus, which was mediated by PKC phosphorylation of LIMK2 at Ser-283.
PKC-mediated LIMK2 Phosphorylation of Ser-283 Inhibits Nuclear Import of LIMK2-The exclusion of LIMK2 from the nucleus by PKC activation may be due to acceleration of nuclear export or inhibition of nuclear import. To distinguish between these two mechanisms, EGFP-LIMK2-transfected endothelial cells were treated with PMA, and LMB was then added to inhibit nuclear export. The kinetics of nuclear fluorescence of EGFP-LIMK2 after LMB addition showed a drastic inhibition of nuclear accumulation of LIMK2 after PMA pretreatment. Similar results were obtained in cells transfected with the active S283EE mutants of EGFP-LIMK2 (Fig. 6). These results indicated that phosphorylation of Ser-283 by PKC inhibits the nuclear import of LIMK2.

PKC Activation Stimulates Cyclin D1 Expression and Sphase Entry of Endothelial Cells-Since
it has been reported that nuclear localization of LIMKs can mediate suppression of cyclin D1 expression and inhibition of G 1 phase cell cycle progression (19), we asked whether protein kinase C activation affects cyclin D1 expression and G 1 -to-S-phase transition of endothelial cells. Stimulation of endothelial cells, which were arrested at the G 0 /G 1 phase of the cell cycle by prolonged serum starvation with PMA or serum, showed an accelerated S-phase entry 16 -24 h after treatment. Cyclin D1 expression known to stimulate G 1 phase cell cycle progression and S-phase entry was enhanced 8 h after PMA treatment and reduced by the PKC inhibitor Go6983 (Fig. 7). These results showed that PKC activation enhances cyclin D1 expression and subsequent G 1 phase progression of endothelial cells, probably through PKCmediated exclusion of LIMK2 from the nucleus, and relieved suppression of cyclin D1 expression.
PMA Activation of Endothelial Cells Does Not Stimulate LIMK-mediated Phosphorylation of Cofilin-PKC-induced phosphorylation of Ser-283 of LIMK2 might affect the kinase activity of LIMK2 and/or guide the enzyme to cofilin. Endothelial cells were stimulated with PMA, and the phosphorylation of LIMKs at Thr-505/Thr-508 (reflecting LIMK activation) and LIMK-mediated phosphorylation of cofilin were measured. PMA in contrast to thrombin did not stimulate Thr-505/Thr-508 phosphorylation of LIMK and cofilin phosphorylation in endothelial cells (Fig. 8 and data not shown). Therefore the PKC-mediated phosphorylation of LIMK2 affects the nucleocytoplasmic shuttling of LIMK2 but not its kinase activity toward cofilin. DISCUSSION In this study, we demonstrated a role of PKC in the regulation of nucleocytoplasmic shuttling of LIMK2 in endothelial cells. PKC phosphorylates LIMK2 at Ser-283, thereby inhibiting the translocation of LIMK2 from the cytoplasm to the nucleus.
Macromolecules larger than 40 -60 kDa are, in most cases, actively transported across the nuclear pore complex. The active nuclear import and export of proteins are mediated by specific amino acid sequences, NLSs and NESs, respectively (32), which are also present in LIMK1 (27). Previously, it has been demonstrated that LIMK1 has two NES at the C terminus of the PDZ domain and one NLS in the kinase domain. After LMB treatment, LIMK1 was predominantly localized in the nucleus, indicating that it shuttles between the nucleus and the cytoplasm (19,27). We observed that similar to LIMK1, LIMK2 was exclusively present in the nucleus of LMB-treated endothelial cells, indicating that LIMK2 also shuttles between the nucleus and the cytoplasm. The existence of functional NES and NLS in LIMK2 was further proved by FRAP and FLIP analysis of the EGFP-PDZ kinase domain of LIMK2 in endothelial cells.
Although both LIMK1 and LIMK2 shuttle between the nu- cleus and the cytoplasm, we showed a major difference of the two kinases in their regulation of shuttling. First, we found no PKC phosphorylation sites in LIMK1 in contrast to LIMK2, and we showed that PKC activation regulates the nucleocytoplasmic shuttling of LIMK2 but not LIMK1. Second, we demonstrated that the two LIM domains inhibit the nuclear import of LIMK2 but not LIMK1; the localization of the PDZ kinase (both LIM domains deleted) of LIMK2, but not LIMK1, was drastically increased in the nucleus. Studies from different groups demonstrated that LIM domains of LIMK1 interact with the kinase domain and that the LIM2 domain inhibits the kinase activity (26,33). Our results suggested that the LIM domains bind to the kinase domain of LIMK2 and LIMK1 differently, masking the NLS of LIMK2 but not LIMK1. In mouse testis, the main LIMK2 isoform is tLIMK2, which lacks both LIM domains and is present mainly in the nucleus (15,16). This is in agreement with our results showing that deletion of the two LIM domains drastically enhanced the nuclear localization of LIMK2.
We predicted two potential PKC phosphorylation sites in LIMK2, one at Ser-283 (between the PDZ and the kinase domain) and the other at Thr-494 (in the kinase domain, near to the predicted NLS). We showed that PMA-mediated activation of PKC inhibited the nuclear localization of the PDZ kinase construct of LIMK2, whereas no change in the subcellular localization of the kinase domain of LIMK2 was observed. This finding suggested that PKC regulates the nucleocytoplasmic shuttling of LIMK2, most likely through Ser-283 phosphorylation. Indeed we were able to show that this site of LIMK2 was phosphorylated by PKC in vitro as well as in intact endothelial cells as follows. (a) PKC phosphorylated recombinant LIMK2.
Of the various PKC isoforms tested, PKC-␦ was identified as the active enzyme. (b) PKC-␦ induced phosphorylation of wild type LIMK2 but not S283A mutated LIMK2. (c) PKC-mediated phosphorylation was demonstrated for the endogenous LIMK2 as well as for transfected LIMK2 constructs.
We demonstrated that Ser-283 but not Thr-494 was the functional relevant phosphorylation site of LIMK2. By introducing inactive as well as active mutants into the Ser-283 site of LIMK2, it was proven that phosphorylation of this site regulated nucleocytoplasmic shuttling of LIMK2. To distinguish between acceleration of nuclear export or inhibition of the nuclear import of LIMK2 by PKC, kinetic studies using the nuclear export inhibitor LMB were performed. We found a drastic inhibition of nuclear accumulation of LIMK2 after PMA pretreatment and of the active S283EE mutant of LIMK2. These results indicated that phosphorylation of Ser-283 by PKC inhibits the nuclear import of LIMK2.
What is the physiological relevance of PKC-mediated inhibition of LIMK2 translocation from the cytoplasm to the nucleus? Recently, it has been reported that nuclear localization of LIMKs suppressed Rac/Cdc42-mediated cyclin D1 expression. Importantly, the suppression of cyclin D1 expression by nuclear LIMKs was independent of the effect of LIMKs on cofilin phosphorylation and regulation of actin dynamics (19). Cyclin D1 plays a critical role in the progression of mammalian cells through the G 1 phase of the cell cycle (34,35), and cyclin D1 expression is induced by several growth stimulatory signaling pathways such as PKC (36,37). We observed that serum stimulation of endothelial cells also inhibited nuclear import LIMK2 by protein kinase C-mediated phosphorylation at Ser-283. Serum and PMA stimulated G 1 -to-S-phase transition of endothelial cells, which was preceded by protein kinase C-mediated stimulation of cyclin D1 expression. Together, these results led us to suggest that PKCmediated nuclear exclusion of LIMK2 might be a mechanism to relieve the suppression of cyclin D1 expression, thereby stimulating cell cycle progression.
How does PKC-mediated phosphorylation of LIMK2 regulate its nucleocytoplasmic shuttling? Many nucleocytoplasmic shuttling proteins such as diacylglycerol kinase , Ca 2ϩ /calmodulindependent protein kinase II, and cyclin B1 are phosphorylated near their NLS, thereby affecting their affinity to the importin protein complex (38 -40). Ser-283 is far away from the predicted NLS in the primary sequence of LIMK2, indicating that Ser-283 phosphorylation is not directly affecting the binding of NLS of LIMK2 to importin-␣. LIMK2 phosphorylated at Ser-283 might bind proteins such as 14-3-3, masking the NLS. This protein has been shown to bind to nucleocytoplasmic shuttling proteins after their phosphorylation and to mask their NLS, thereby keeping these proteins in the cytoplasm (41, 42).