MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration
2006; Springer Nature; Volume: 25; Issue: 4 Linguagem: Inglês
10.1038/sj.emboj.7600973
ISSN1460-2075
AutoresMiho Kobayashi, Michiru Nishita, Toshiaki Mishima, Kazumasa Ohashi, Kensaku Mizuno,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoArticle2 February 2006free access MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration Miho Kobayashi Miho Kobayashi Search for more papers by this author Michiru Nishita Michiru Nishita Search for more papers by this author Toshiaki Mishima Toshiaki Mishima Search for more papers by this author Kazumasa Ohashi Kazumasa Ohashi Search for more papers by this author Kensaku Mizuno Corresponding Author Kensaku Mizuno Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan Search for more papers by this author Miho Kobayashi Miho Kobayashi Search for more papers by this author Michiru Nishita Michiru Nishita Search for more papers by this author Toshiaki Mishima Toshiaki Mishima Search for more papers by this author Kazumasa Ohashi Kazumasa Ohashi Search for more papers by this author Kensaku Mizuno Corresponding Author Kensaku Mizuno Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan Search for more papers by this author Author Information Miho Kobayashi, Michiru Nishita, Toshiaki Mishima, Kazumasa Ohashi and Kensaku Mizuno 1 1Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan *Corresponding author. Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan. Tel.: +81 22 795 6676; Fax: +81 22 795 6678; E-mail: [email protected] The EMBO Journal (2006)25:713-726https://doi.org/10.1038/sj.emboj.7600973 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Vascular endothelial growth factor-A (VEGF-A) induces actin reorganization and migration of endothelial cells through a p38 mitogen-activated protein kinase (MAPK) pathway. LIM-kinase 1 (LIMK1) induces actin remodeling by phosphorylating and inactivating cofilin, an actin-depolymerizing factor. In this study, we demonstrate that activation of LIMK1 by MAPKAPK-2 (MK2; a downstream kinase of p38 MAPK) represents a novel signaling pathway in VEGF-A-induced cell migration. VEGF-A induced LIMK1 activation and cofilin phosphorylation, and this was inhibited by the p38 MAPK inhibitor SB203580. Although p38 phosphorylated LIMK1 at Ser-310, it failed to activate LIMK1 directly; however, MK2 activated LIMK1 by phosphorylation at Ser-323. Expression of a Ser-323-non-phosphorylatable mutant of LIMK1 suppressed VEGF-A-induced stress fiber formation and cell migration; however, expression of a Ser-323-phosphorylation-mimic mutant enhanced these processes. Knockdown of MK2 by siRNA suppressed VEGF-A-induced LIMK1 activation, stress fiber formation, and cell migration. Expression of kinase-dead LIMK1 suppressed VEGF-A-induced tubule formation. These findings suggest that MK2-mediated LIMK1 phosphorylation/activation plays an essential role in VEGF-A-induced actin reorganization, migration, and tubule formation of endothelial cells. Introduction Angiogenesis is a process whereby new blood vessels are generated from pre-existing ones. It is essential for a number of physiological and pathological processes, including embryonic development, tissue regeneration, wound healing, diabetic retinopathy, and tumor growth and metastasis (Risau, 1997). Vascular endothelial growth factor-A (VEGF-A, hereafter simply referred to as VEGF) is a potent stimulator of angiogenesis, and promotes migration, proliferation, and tubule formation of endothelial cells (Ferrara, 2004). Although VEGF-induced endothelial cell migration is an essential step for angiogenesis, the underlying signaling mechanisms are not well understood (Rousseau et al, 2000). VEGF exerts its angiogenic effects through the receptor tyrosine kinases, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1), which are expressed on the surface of endothelial cells (Zachary and Gliki, 2001). Activation of VEGFR-2 by VEGF leads to stimulation of various intracellular signaling cascades, including activation of the extracellular signal-regulated kinase (Erk) and p38 mitogen-activated protein kinase (MAPK) pathways (Zachary and Gliki, 2001). As the p38 MAPK inhibitor SB203580 inhibits VEGF-induced stress fiber formation and cell migration, the p38 MAPK pathway is believed to play a crucial role in endothelial cell migration by regulating actin cytoskeletal organization (Rousseau et al, 1997). p38 MAPK is activated by the upstream MAPK kinases, MKK3/MKK6 (Ono and Han, 2000). Activation of p38 MAPK by VEGF leads to the activation of MAPK-activated protein kinase-2 (MK2), which in turn stimulates phosphorylation of heat-shock protein 27 (Hsp27) (Guay et al, 1997; Rousseau et al, 1997; Landry and Huot, 1999). Hsp27 behaves as an actin filament cap-binding protein and, in its non-phosphorylated form, inhibits actin polymerization. MK2-catalyzed phosphorylation of Hsp27 allows its release from capped actin filaments, thereby stimulating actin polymerization (Landry and Huot, 1999). Thus, p38/MK2-mediated phosphorylation of Hsp27 is believed to be involved in VEGF-induced actin filament assembly and stress fiber formation, both of which are involved in endothelial cell migration (Rousseau et al, 2000). However, other signaling mechanisms for VEGF-induced and p38/MK2-mediated actin reorganization and cell migration remain to be elucidated. Cofilin plays an essential role in actin reorganization and cell migration by depolymerizing and severing actin filaments (Bamburg and Wiggan, 2002). The activity of cofilin is reversibly regulated by phosphorylation and dephosphorylation of Ser-3, with the phosphorylated form being inactive. LIM-kinase (LIMK) and TES-kinase are responsible for phosphorylation of this site, and thereby inactivate cofilin (Arber et al, 1998; Yang et al, 1998; Toshima et al, 2001). Cofilin phosphatases (termed Slingshot and chronophin) reactivate cofilin by dephosphorylating Ser-3 (Niwa et al, 2002; Nagata-Ohashi et al, 2004; Gohla et al, 2005). LIMK1 is activated through phosphorylation of Thr-508, in the kinase domain of LIMK1, by downstream kinases of the Rho family small GTPases, such as ROCK and PAK (Edwards et al, 1999; Maekawa et al, 1999; Ohashi et al, 2000). Thus, by phosphorylating cofilin, LIMK1 appears to play a critical role in stimulus-induced actin reorganization. LIMK1 is activated in response to various extracellular signals that stimulate cell migration, and stimulus-induced LIMK1 activation is required for chemokine-induced cell migration or LPA-induced tumor cell invasion (Nishita et al, 2002, 2005; Yoshioka et al, 2003). In contrast, overexpression of LIMK1 suppresses cell motility and polarized cell migration (Dawe et al, 2003; Endo et al, 2003). Thus, it seems likely that proper regulation of LIMK1 activity and cofilin phosphorylation is required for cell migration. In this study, we examined the role of LIMK1 in VEGF-induced actin reorganization, migration, and tubule formation of vascular endothelial cells. We report here that LIMK1 is activated in VEGF-stimulated endothelial cells, by a novel signaling pathway, composed of p38 MAPK–MK2–LIMK1, and that MK2 activates LIMK1 by phosphorylating Ser-323 in the extracatalytic domain of LIMK1. We also provide evidence that MK2-mediated LIMK1 phosphorylation/activation is critical for VEGF-induced stress fiber formation, cell migration, and tubule formation. Results VEGF induces LIMK1 activation and cofilin phosphorylation To examine whether LIMK1 is involved in VEGF-induced actin reorganization, we first analyzed changes in the kinase activity of LIMK1 and the level of Ser-3-phosphorylated cofilin (P-cofilin) in VEGF-stimulated human umbilical vein endothelial cells (HUVECs) and MSS31 endothelial cells. MSS31 cells have the properties of endothelial cells; they express PECAM-1, VE-cadherin, and VEGFR-1 and -2 (Supplementary Figure 1). We used 10 ng/ml VEGF for stimulation in this study. An in vitro kinase assay, using cofilin as a substrate, demonstrated that the kinase activity of LIMK1 increased 1.6- to 1.7-fold by 15 min after VEGF treatment, and then decreased by 30 min (Figure 1A). The 1.3-fold increase in LIMK1 activity was retained up to 6 h (Supplementary Figure 2). The level of P-cofilin (measured by immunoblots with anti-P-cofilin antibody) increased about two-fold by 15 min after VEGF treatment, and then decreased by 30 min (Figure 1B). Similar results were obtained by two-dimensional gel immunoblot analysis of P-cofilin levels, using an anti-cofilin antibody. P-cofilin represented 16% of cofilin in unstimulated cells. After VEGF stimulation, the relative abundance of P-cofilin reached a maximum (46%) by 15 min, and decreased to 24% by 30 min (Figure 1C). These results suggest that VEGF stimulates both LIMK1 activity and cofilin phosphorylation in a time-dependent manner, and that through cofilin phosphorylation, LIMK1 is involved in VEGF-induced actin reorganization. Figure 1.VEGF induces LIMK1 activation and cofilin phosphorylation. (A) LIMK1 activation. HUVEC and MSS31 cells were stimulated with VEGF. At the indicated time, cells were lysed and LIMK1 was immunoprecipitated (IP) with anti-LIMK1, and subjected to an in vitro kinase assay. Reaction mixtures were analyzed by autoradiography (32P-cofilin), Amido black staining (cofilin), and immunoblotting (IB) with anti-LIMK1 antibody. The bottom panel indicates the relative kinase activities of LIMK1, as measured by 32P incorporation into cofilin. Data are means±s.d. of five independent experiments. (B) Cofilin phosphorylation. HUVEC and MSS31 cells were stimulated with VEGF. Cell lysates were analyzed by immunoblotting with anti-P-cofilin and anti-cofilin antibodies. The bottom panel indicates the relative P-cofilin levels as means±s.d. of five independent experiments. (C) Two-dimensional gel analyses of P-cofilin levels. MSS31 cells were stimulated with VEGF, and cell lysates were analyzed by two-dimensional gel electrophoresis, followed by immunoblotting with anti-cofilin antibody. The right panel indicates the mean abundance of P-cofilin (means±s.d. of triplicate experiments), as the percentage of total cofilin. Download figure Download PowerPoint Kinase-negative LIMK1(D460A) suppresses VEGF-induced stress fiber formation We next examined whether LIMK1 is involved in VEGF-induced stress fiber formation. MSS31 cells were transfected with cyan fluorescence protein (CFP)-tagged wild-type LIMK1 (LIMK1(WT)-CFP) or the kinase-dead form LIMK1(D460A)-CFP, in which the catalytic Asp-460 is replaced by alanine. Cells were either untreated or stimulated with VEGF, and stained with rhodamine-phalloidin to visualize F-actin (Figure 2A). In the control MSS31 cells (transfected with vector alone), thick and transcytoplasmic actin stress fibers were induced in response to VEGF treatment. Expression of LIMK1(WT)-CFP caused a marked increase in the induction of thick stress fibers, even in the absence of VEGF. Following VEGF treatment, induction of thicker stress fibers increased further, in comparison to the surrounding LIMK1-non-expressing cells, indicating that LIMK1 has the potential to stimulate stress fiber formation. In contrast, expression of LIMK1(D460A)-CFP significantly suppressed VEGF-induced stress fiber formation. Figure 2B shows the data of quantitative analysis of the ratio of the cells with thick stress fibers. These results suggest that LIMK1 plays a critical role in VEGF-induced stress fiber formation in endothelial cells. Figure 2.Kinase-negative LIMK1(D460A) suppresses VEGF-induced stress fiber formation. (A) MSS31 cells were transfected with control vector (mock) or plasmid encoding LIMK1(WT)-CFP or LIMK1(D460A)-CFP. They were unstimulated or stimulated with VEGF for 15 min and then stained with rhodamine-phalloidin. Arrowheads indicate CFP fluorescence-positive cells (see Supplementary Figure 3A). Bar, 50 μm. (B) Quantitative analysis of data shown in (A). The percentages of the cells with thick stress fibers in the total CFP-positive cells are shown as means±s.d. of triplicate experiments. Download figure Download PowerPoint p38 MAPK mediates VEGF-induced LIMK1 activation, cofilin phosphorylation, and stress fiber formation Because p38 MAPK has been shown to mediate VEGF-induced actin reorganization (Rousseau et al, 1997), we examined the effects of SB203580, a specific inhibitor for p38 MAPK, on VEGF-induced LIMK1 activation and cofilin phosphorylation in MSS31 cells. Almost complete inhibition of both VEGF-induced LIMK1 activation and cofilin phosphorylation was achieved by pretreatment with 1 μM SB203580 (Figure 3A and B). This indicates that LIMK1 activation and cofilin phosphorylation are stimulated downstream of p38 MAPK. We also examined the effects of SB203580 on VEGF-induced stress fiber formation (Figure 3C). Pretreatment with SB203580, but not with vehicle alone, suppressed VEGF-induced stress fiber formation in the control MSS31 cells expressing CFP. In contrast, stress fibers induced by LIMK1(WT)-CFP expression were not affected by SB203580 treatment, either before or after VEGF stimulation, in comparison to the surrounding LIMK1-non-expessing cells. This result is a further indication that LIMK1 exerts its actions downstream of p38. Thus, p38 MAPK appears to mediate VEGF-induced LIMK1 activation, cofilin phosphorylation, and stress fiber formation. Figure 3.p38 MAPK mediates VEGF-induced LIMK1 activation, cofilin phosphorylation, and stress fiber formation. (A, B) SB203580 inhibits VEGF-induced LIMK1 activation and cofilin phosphorylation. MSS31 cells were pretreated with SB203580 (0, 1, and 5 μM) for 30 min and then stimulated with VEGF for 15 min. The levels of LIMK1 activity and P-cofilin were determined, as in Figure 1. Relative kinase activities of LIMK1 (A) and relative P-cofilin levels (B) are shown as means±s.d. of triplicate experiments. *P<0.001. (C) SB203580 inhibits stress fiber formation. MSS31 cells transfected with CFP or LIMK1(WT)-CFP were pretreated with 5 μM SB203580 or vehicle (DMSO) for 30 min and then stimulated with VEGF for 15 min. Cells were stained with rhodamine-phalloidin. Arrowheads indicate CFP-positive cells (see Supplementary Figure 3B). Bar, 50 μm. The bottom panel shows the data of quantitative analysis of triplicate experiments. (D) MKK6(DE) stimulates LIMK1 activity. MSS31 cells cotransfected with Myc-LIMK1, Flag-p38, and either HA-MKK6(DE) or HA-MKK6(AA) were treated with VEGF for 15 min, and Myc-LIMK1 was precipitated and subjected to an in vitro kinase assay. Kinase activities were quantified by autoradiography, and relative levels are indicated under the top panel. Expression of Myc-LIMK1 and HA-MKK6 mutants and activation of p38 (P-p38) were analyzed by immunoblotting, as indicated. (E) MKK6(AA) suppresses VEGF-induced stress fiber formation. MSS31 cells, transfected with HA-MKK6(DE) or HA-MKK6(AA), were stimulated with VEGF for 15 min and stained with rhodamine-phalloidin. Arrowheads indicate cells expressing MKK6(DE) or MKK6(AA), as measured by anti-HA staining (Supplementary Figure 3C). Bar, 50 μm. The bottom panel shows the data of quantitative analysis of triplicate experiments. Download figure Download PowerPoint To further examine the involvement of p38 MAPK in VEGF-induced LIMK1 activation, we tested the effects of expression of a constitutively active or a dominant-negative form of MKK6 in MSS31 cells. MKK6 is an MAPK kinase that phosphorylates and activates p38 MAPK. The constitutively active MKK6(DE) and dominant-negative MKK6(AA) mutants were constructed by replacing the two phosphorylation sites in MKK6 (Ser-207 and Thr-211) by Asp and Glu residues (DE), or two Ala residues (AA), respectively (Hanafusa et al, 1999). Coexpression of active MKK6(DE) significantly increased the kinase activity of LIMK1, in both unstimulated and VEGF-stimulated cells (Figure 3D). In contrast, coexpression of MKK6(AA) blocked VEGF-induced LIMK1 activation (Figure 3D). Immunoblot analysis with an anti-phospho-p38 (P-p38) antibody showed that p38 activity was enhanced by MKK6(DE) expression and/or VEGF treatment, but was suppressed by MKK6(AA) expression (Figure 3D). Expression of MKK6(DE) enhanced actin filament assembly in unstimulated cells, whereas expression of MKK6(AA) suppressed VEGF-induced stress fiber formation (Figure 3E). Taken together with the results of SB203580 treatment, these observations strongly suggest that an MKK6–p38 pathway mediates VEGF-induced LIMK1 activation, cofilin phosphorylation, and stress fiber formation. Phosphorylation of Thr-508 is not required for VEGF-induced and p38-mediated LIMK1 activation LIMK1 is activated by phosphorylation of Thr-508 by ROCK or PAK kinases. To examine whether Thr-508 phosphorylation is involved in VEGF-induced LIMK1 activation, we analyzed the VEGF-induced changes in the kinase activity of LIMK1(WT) and LIMK1(T508V), in which Thr-508 was replaced by a non-phosphorylatable valine. The basal kinase activity of LIMK1(T508V) is lower than that of LIMK1(WT) in unstimulated cells; however, VEGF stimulation resulted in an approximately 1.5-fold increase in the activity of both LIMK1(WT) and LIMK1(T508V) (Figure 4A). When either LIMK1(WT) or LIMK1(T508V) was coexpressed in 293T cells with active MKK6(DE) and p38, there was an approximately two-fold increase in activity (Figure 4B). These results suggest that both LIMK1(WT) and LIMK1(T508V) are activated by VEGF or active MKK6 to a similar extent, and that phosphorylation of Thr-508 is not required for VEGF-induced and p38-mediated LIMK1 activation. Coexpression with MKK6(DE) resulted in gel mobility shifts of LIMK1(WT) and LIMK1(T508V) (Figure 4B). These mobility shifts are the result of LIMK1 phosphorylation, as they were abolished by treatment with alkaline phosphatase (Figure 4C). Accordingly, LIMK1 is probably activated downstream of p38 MAPK, by phosphorylation of a site(s) other than Thr-508. Figure 4.Phosphorylation of Thr-508 is not required for VEGF-induced LIMK1 activation. (A) LIMK1(T508V) is activated by VEGF. MSS31 cells transfected with Myc-LIMK1(WT) or Myc-LIMK1(T508V) were stimulated with VEGF for 15 min. Relative kinase activities of these proteins were analyzed as in Figure 3D and are indicated under the top panel. (B) LIMK1(T508V) is activated by MKK6(DE). 293T cells were cotransfected with HA-MKK6(DE), Flag-p38, and either Myc-LIMK1(WT) or Myc-LIMK1(T508V). Myc-LIMK1 proteins were immunoprecipitated and subjected to an in vitro kinase reaction. Expression of Myc-LIMK1 and HA-MKK6(DE) and activation of p38 were analyzed by immunoblotting, as indicated. (C) Effects of alkaline phosphatase treatment on a gel mobility shift of LIMK1. Myc-LIMK1(WT) or Myc-LIMK1(T508V) cotransfected with HA-MKK6(DE) and Flag-p38 in 293T cells was precipitated with anti-Myc antibody, incubated with calf intestinal alkaline phosphatase (CIP), and then analyzed by immunoblotting with anti-Myc antibody. Download figure Download PowerPoint p38 MAPK phosphorylates, but fails to activate, LIMK1 To determine if LIMK1 is directly phosphorylated by p38, Myc-tagged LIMK1(T508V) and kinase-dead LIMK1(D460A) were incubated in vitro with [γ-32P]ATP in the absence or presence of recombinant active p38 (GST-p38) (Figure 5A). In the absence of p38, 32P was incorporated into LIMK1(T508V), but not LIMK1(D460A), indicating that LIMK1(T508V) underwent autophosphorylation. In the presence of active p38, 32P incorporation into LIMK1(D460A) and LIMK1(T508V) was increased, which suggests that p38 phosphorylates LIMK1 at a residue(s) other than Thr-508. However, there was no change in the kinase activity of LIMK1(WT) or LIMK1(T508V), after incubation with active p38 (Figure 5B). Thus, p38 has the potential to phosphorylate LIMK1, but is unable to activate LIMK1 by itself. A database search predicted that Ser-310 of LIMK1 was the most probable site for p38-catalyzed phosphorylation. In an in vitro kinase assay, active p38 enhanced phosphorylation of LIMK1(T508V), but not of LIMK1(S310A/T508V), in which Ser-310 and Thr-508 were replaced by alanine and valine, respectively (Figure 5C). These results indicate that Ser-310 of LIMK1 is the primary site of phosphorylation by p38. Figure 5.p38 MAPK phosphorylates, but does not activate, LIMK1. (A) p38 phosphorylates LIMK1. Myc-LIMK1(D460A) and Myc-LIMK1(T508V) expressed in 293T cells were precipitated with anti-Myc antibody and incubated with [γ-32P]ATP and active GST-p38. Reaction mixtures were separated by SDS–PAGE and analyzed by autoradiography. Relative values of 32P incorporation into Myc-LIMK1 mutants are indicated under the top panel. (B) LIMK1 is not activated by p38. Myc-LIMK1(WT) and Myc-LIMK1(T508V) expressed in 293T cells were immunoprecipitated, incubated with active GST-p38, and then subjected to an in vitro kinase assay. Relative kinase activities of LIMK1 are indicated under the top panel. (C) p38 phosphorylates Ser-310 of LIMK1. Myc-LIMK1(T508V) and Myc-LIMK1(S310A/T508V) expressed in 293T cells were precipitated with anti-Myc antibody and incubated with [γ-32P]ATP and active GST-p38. Reaction mixtures were separated by SDS–PAGE and analyzed by autoradiography. Relative values of 32P incorporation into Myc-LIMK1 mutants are indicated. Download figure Download PowerPoint MK2 activates LIMK1 by phosphorylation of Ser-323 MK2 was reported to mediate p38-induced stress fiber formation (Rousseau et al, 2000). As p38 did not directly activate LIMK1, we then asked whether MK2 is involved in p38-mediated LIMK1 phosphorylation and activation. When LIMK1(WT) and LIMK1(T508V) were incubated with [γ-32P]ATP and recombinant active MK2 (GST-MK2-Myc), 32P incorporation into LIMK1(WT) and LIMK1(T508V) was increased approximately two-fold (Figure 6A). The kinase activities of LIMK1(WT) and LIMK1(T508V) also increased to the same degree, after incubation with active MK2 (Figure 6A). These findings suggest that MK2 phosphorylates and activates LIMK1, and that phosphorylation of Thr-508 is not related to this reaction. Figure 6.MK2 activates LIMK1 by phosphorylation of Ser-323. (A) MK2 phosphorylates and activates LIMK1. Myc-LIMK1(WT) and Myc-LIMK1(T508V) expressed in 293T cells were precipitated with anti-Myc antibody and incubated with [γ-32P]ATP and active GST-MK2-Myc. Reaction mixtures were separated by SDS–PAGE and analyzed by autoradiography. Relative values of 32P incorporation into Myc-LIMK1 are indicated in the bottom left panel. After incubation with active MK2, Myc-LIMK1 immunoprecipitates were subjected to an in vitro kinase assay. Relative kinase activities are indicated in the bottom right panel. Data are means±s.d. of three independent experiments. (B) Alignment of sequences of putative MK2 phosphorylation sites in LIMK1 and HSP27, and the consensus sequence for MK2 substrates. An asterisk indicates the phosphorylation site. (C) MK2 activates LIMK1 by Ser-323 phosphorylation. Myc-LIMK1 mutants were expressed in 293T cells, immunoprecipitated, and incubated with [γ-32P]ATP and active GST-MK2-Myc, with or without active GST-p38. Both 32P incorporation into Myc-LIMK1 and LIMK1 activity were analyzed as in (A). Relative values of 32P incorporation and relative kinase activities are indicated in the bottom panels. (D) Activation of LIMK1 by Ser-323 phosphorylation in cultured cells. Myc-LIMK1 mutants were coexpressed with HA-MKK6(DE) plus Flag-p38 in 293T cells, immunoprecipitated, and subjected to an in vitro kinase assay. Relative kinase activities are indicated in the bottom panel as means±s.d. of five independent experiments. (E) Kinase activities of LIMK1 mutants. MSS31 cells transfected with Myc-LIMK1 mutants were stimulated with VEGF for 15 min. Myc-LIMK1 mutants were immunoprecipitated and subjected to an in vitro kinase assay. Relative kinase activities are shown in the bottom panel as means±s.d. of five experiments. Download figure Download PowerPoint MK2 phosphorylates several proteins, including HSP-27, at the serine residue in the consensus sequence motif, -Hyd-x-Arg-x-x-Ser-, where Hyd is a bulky hydrophobic residue (Stokoe et al, 1993). Human LIMK1 contains the corresponding sequence LGRSES323 in the region between a PDZ domain and a kinase domain, and the MK2 consensus motif is conserved between LIMK1 proteins from various species (Figure 6B). To determine if MK2 phosphorylates Ser-323 of LIMK1, and whether or not this phosphorylation is related to LIMK1 activation, we constructed the mutant LIMK1(S323A/T508V), in which Ser-323 and Thr-508 are replaced by alanine and valine, respectively. We also constructed the mutant LIMK1(S310A/S323A/T508V). After incubation with active MK2, we compared the phosphorylation level and kinase activity of these mutants to those of LIMK1(T508V). In cell-free assays, active MK2 phosphorylated LIMK1(T508V), but did not phosphorylate either (S323A/T508V) or (S310A/S323A/T508V) mutant (Figure 6C). Incubation with active MK2 together with active p38 resulted in a more highly phosphorylated LIMK1(T508V), a slightly phosphorylated LIMK1(S323A/T508V), and no increased phosphorylation of LIMK1(S310A/S323A/T508V) (Figure 6C). Similarly, active MK2 phosphorylated LIMK1(WT), but not LIMK1(S310A/S323A) (Supplementary Figure 4A). These results suggest that p38 and MK2 specifically phosphorylate LIMK1 at Ser-310 and Ser-323, respectively. Incubation with active MK2 increased the kinase activity of LIMK1(T508V), but did not alter the activity of either LIMK1(S323A/T508V) or LIMK1(S310A/S323A/T508V) (Figure 6C). Incubation with active MK2 and active p38 did not further increase the kinase activities of these mutants (Figure 6C). In a similar manner, active MK2 activated LIMK1(WT), but not LIMK1(S310A/S323A) (Supplementary Figure 4A). These findings suggest that in cell-free assays, MK2 stimulates the kinase activity of LIMK1 by phosphorylation of Ser-323. Although p38 phosphorylates LIMK1 at Ser-310, it fails to activate LIMK1. To examine whether Ser-323 phosphorylation is involved in MKK6/p38-mediated LIMK1 activation in cultured cells, LIMK1 mutants were coexpressed with MKK6(DE) and p38 in 293T cells, and their kinase activities were measured (Figure 6D). By coexpression with active MKK6(DE) and p38, the kinase activity of LIMK1(T508V) increased 1.7-fold, but the activities of S323A/T508V and S310A/S323A/T508V mutants did not alter. Similarly, coexpression with active MKK6(DE) and p38 increased the kinase activity of LIMK1(WT), but not the activity of LIMK1(S310A/S323A) (Supplementary Figure 4B). Thus, these results suggest that phosphorylation of Ser-323 is essential for p38-mediated LIMK1 activation in cultured cells. We also analyzed kinase activities of non-phosphorylatable LIMK1(S310A/S323A/T508V) and phosphorylation-mimic LIMK1(S310E/S323E/T508V) in MSS31 cells, before and after VEGF stimulation (Figure 6E). The activity of LIMK1(T508V) increased ∼1.8-fold, after stimulation with VEGF. In unstimulated cells, the kinase activity of LIMK1(S310A/S323A/T508V) was similar to that of LIMK1(T508V); however, unlike LIMK1(T508V), it was not activated by VEGF treatment. In unstimulated cells, LIMK1(S310E/S323E/T508V) showed an ∼2-fold increase in kinase activity, compared with LIMK1(T508V). After stimulation with VEGF, the kinase activity of LIMK1(S310E/S323E/T508V) did not increase further. In a similar experiment, VEGF stimulated the activity of LIMK1(WT), but not LIMK1(S310A/S323A) (Supplementary Figure 4C). These results support the suggestion that VEGF induces LIMK1 activation by phosphorylation of Ser-310 and Ser-323 residues. Phosphorylation of Ser-323 of LIMK1 is required for VEGF-induced cell migration and stress fiber formation It was reported that a p38–MK2 pathway plays a crucial role in the VEGF-induced endothelial cell migration and stress fiber formation (Rousseau et al, 2000). The finding that VEGF induced LIMK1 activation through the MK2-mediated phosphorylation of Ser-323 prompted us to investigate whether phosphorylation of Ser-323 is required for VEGF-induced cell migration. We examined the effects of expression of various LIMK1 mutants on VEGF-induced endothelial cell migration. MSS31 cells were cotransfected with LIMK1 mutants and YFP and subjected to a migration assay, using Transwell chambers. The number of YFP-positive cells that migrated across the membrane was determined 18 h after the addition of VEGF (Figure 7A). For control MSS31 cells expressing YFP alone, VEGF increased the number of migrating cells about two-fold. Expression of LIMK1(WT) increased the number of migrating cells in both unstimulated and VEGF-stimulated conditions. In contrast, expression of LIMK1(D460A) suppressed VEGF-induced migration. These results indicate that LIMK1 activity is crucial for VEGF-induced cell migration. Additionally, expression of LIMK1(T508V) increased cell migration in both unstimulated and VEGF-stimulated conditions, although the levels of enhancement were lower than those for the LIMK1(WT)-expressing cells. In contrast, for cells expressing S323A/T508V, S310A/S323A/T508V, or S310A/S323A mutant, VEGF-induced cell migration was suppressed. Expression of LIMK1(S310E/S323E/T508V) markedly increased the number of migrating cells in the absence of VEGF, but no further increase was observed with stimulation with VEGF. Taken together, these results suggest that LIMK1 activation, through MK2-catalyzed phosphorylation of Ser-323, plays an essential role in VEGF-induced endothelial cell migration. Immunoblot analysis demon
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