Interplay between components of a novel LIM kinase–slingshot phosphatase complex regulates cofilin
2005; Springer Nature; Volume: 24; Issue: 3 Linguagem: Inglês
10.1038/sj.emboj.7600543
ISSN1460-2075
AutoresJuliana Soosairajah, Sankar Maiti, O’Neil Wiggan, Patrick D. Sarmiere, Nathalie Moussi, Boris Šarčević, Rashmi Sampath, James R. Bamburg, Ora Bernard,
Tópico(s)Signaling Pathways in Disease
ResumoArticle20 January 2005free access Interplay between components of a novel LIM kinase–slingshot phosphatase complex regulates cofilin Juliana Soosairajah Juliana Soosairajah The Walter & Eliza Hall Institute of Medical Research, Victoria, Australia Search for more papers by this author Sankar Maiti Sankar Maiti Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA Search for more papers by this author O'Neil Wiggan O'Neil Wiggan Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA Search for more papers by this author Patrick Sarmiere Patrick Sarmiere Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA Search for more papers by this author Nathalie Moussi Nathalie Moussi The Walter & Eliza Hall Institute of Medical Research, Victoria, Australia Search for more papers by this author Boris Sarcevic Boris Sarcevic Cancer Research Program, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia Search for more papers by this author Rashmi Sampath Rashmi Sampath Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA Search for more papers by this author James R Bamburg Corresponding Author James R Bamburg Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA Search for more papers by this author Ora Bernard Corresponding Author Ora Bernard The Walter & Eliza Hall Institute of Medical Research, Victoria, Australia Search for more papers by this author Juliana Soosairajah Juliana Soosairajah The Walter & Eliza Hall Institute of Medical Research, Victoria, Australia Search for more papers by this author Sankar Maiti Sankar Maiti Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA Search for more papers by this author O'Neil Wiggan O'Neil Wiggan Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA Search for more papers by this author Patrick Sarmiere Patrick Sarmiere Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA Search for more papers by this author Nathalie Moussi Nathalie Moussi The Walter & Eliza Hall Institute of Medical Research, Victoria, Australia Search for more papers by this author Boris Sarcevic Boris Sarcevic Cancer Research Program, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia Search for more papers by this author Rashmi Sampath Rashmi Sampath Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA Search for more papers by this author James R Bamburg Corresponding Author James R Bamburg Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA Search for more papers by this author Ora Bernard Corresponding Author Ora Bernard The Walter & Eliza Hall Institute of Medical Research, Victoria, Australia Search for more papers by this author Author Information Juliana Soosairajah1, Sankar Maiti2, O'Neil Wiggan2, Patrick Sarmiere2, Nathalie Moussi1, Boris Sarcevic3, Rashmi Sampath2, James R Bamburg 2 and Ora Bernard 1 1The Walter & Eliza Hall Institute of Medical Research, Victoria, Australia 2Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA 3Cancer Research Program, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia ‡These authors contributed equally to the results presented in this manuscript ‡Senior authors contributed equally to this work *Corresponding authors: Department of Biochemistry and Molecular Biology, Colorado State University, 235 MRB, Fort Collins, CO 80523-1870, USA. Tel.: +1 970 491 6096; Fax: +1 970 491 0494; E-mail: [email protected] and Eliza Hall Medical Research, Parkville 3052, Victoria, Australia. Tel.: +61 39 345 2555; E-mail: [email protected] The EMBO Journal (2005)24:473-486https://doi.org/10.1038/sj.emboj.7600543 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Slingshot (SSH) phosphatases and LIM kinases (LIMK) regulate actin dynamics via a reversible phosphorylation (inactivation) of serine 3 in actin-depolymerizing factor (ADF) and cofilin. Here we demonstrate that a multi-protein complex consisting of SSH-1L, LIMK1, actin, and the scaffolding protein, 14-3-3ζ, is involved, along with the kinase, PAK4, in the regulation of ADF/cofilin activity. Endogenous LIMK1 and SSH-1L interact in vitro and co-localize in vivo, and this interaction results in dephosphorylation and downregulation of LIMK1 activity. We also show that the phosphatase activity of purified SSH-1L is F-actin dependent and is negatively regulated via phosphorylation by PAK4. 14-3-3ζ binds to phosphorylated slingshot, decreases the amount of slingshot that co-sediments with F-actin, but does not alter slingshot activity. Here we define a novel ADF/cofilin phosphoregulatory complex and suggest a new mechanism for the regulation of ADF/cofilin activity in mediating changes to the actin cytoskeleton. Introduction Intrinsic and extrinsic signals regulate the dynamic nature of actin filament formation through an abundance of actin-binding and regulatory molecules. Filament-capping proteins, filament-destabilizing proteins, and monomer sequestering molecules can influence the formation, elongation, and destabilization of actin filaments (Pollard and Borisy, 2003). Regulation of these individual pathways ultimately function to cause changes in the actin cytoskeleton. Proteins of the actin-depolymerizing factor (ADF)/cofilin (AC) family dynamically regulate the actin network by increasing the off-rate of actin monomers from the pointed end of actin filaments and by filament severing (reviewed in Bamburg and Wiggan, 2002). Phosphorylation of serine 3 (ser 3) by the LIM or TES kinases results in AC inactivation, whereas dephosphorylation by SSH phosphatases results in their reactivation. Critical to our understanding of the integration of signalling pathways regulating actin function is an understanding of the mechanisms influencing AC activity. The two LIM kinases, LIMK1 and LIMK2, are expressed in most tissues (Ikebe et al, 1998; Foletta et al, 2004) and negatively regulate the actin-dynamizing activities of AC. LIMK are regulated by the Rho GTPases (Arber et al, 1998; Yang et al, 1998) that, through their effectors Rho-kinase (ROCK) and p21-activated kinases (PAK1 and 4), activate LIMK1 and 2 by phosphorylation at Thr 508 and 505, respectively (Edwards and Gill, 1999; Maekawa et al, 1999; Ohashi et al, 2000; Sumi et al, 2001). LIMK1 associates with 14-3-3 scaffold proteins (Birkenfeld et al, 2003), which could affect its activity, localization, and proximity to other effector molecules (Fu et al, 2000). Overexpression of 14-3-3ζ in vivo promotes the accumulation of the inactive, phosphorylated form of AC in cells (Gohla and Bokoch, 2002). However, it is unclear whether this involves protection from dephosphorylation or increased phosphorylation. Reactivation of AC proteins occurs by dephosphorylation (Morgan et al, 1993), brought about by a class of phosphatases, named slingshot (SSH) (Niwa et al, 2002). SSH phosphatases contain conserved A and B domains with unknown functions, a protein phosphatase domain (PTP) containing the catalytic sequence of HCxxGxxR, and in some family members, a C-terminal F-actin-binding region. Currently, three human and mouse isoforms, SSH-1, 2, and 3, have been identified, each with long and short variants (Niwa et al, 2002; Ohta et al, 2003). Each member has somewhat different tissue expression patterns and phosphatase activity (Ohta et al, 2003), but their regulation remains to be determined. Here we demonstrate that the regulation of AC activity occurs most likely through dynamic associations between actin, 14-3-3ζ, slingshot, PAK4, and LIMK1. Our results show compartmentalization of SSH-1L and LIMK1, and SSH-1L and 14-3-3ζ in neuronal growth cones and to lamella and ruffles in constitutively active rac1-expressing fibroblasts. We demonstrate that purified hSSH-1L, LIMK1, and 14-3-3ζ interact in vitro and that F-actin is required for purified hSSH-1L to dephosphorylate phosphoAC in vitro. In addition, hSSH-1L can dephosphorylate active LIMK1 on Thr 508, and it is thus a LIMK1 phosphatase, as well as a pAC phosphatase. Furthermore, phosphorylation of hSSH-1L by PAK4 negatively regulates its activity, providing a reciprocal control of LIMK1 and SSH-1L by PAK4. Results SSH-1L can co-localize with LIMK1 and 14-3-3 ζ in discrete regions within neuronal and non-neuronal cells Endogenous LIMK1 is localized to the growth cones of E18 rat hippocampal neurons, regions also enriched in endogenous SSH-1L (Figure 1Aa–d). 14-3-3 is also enriched in growth cones, where it shows substantial overlap to regions of high SSH-1L staining (Figure 1Ba–d). As observed with LIMK1, SSH-1L is not exclusively co-localized with 14-3-3 in these cells. However, these data suggest a potential dynamic and regulated process promoting the association of SSH with other regulators of AC proteins. Figure 1.Localization of LIMK1, hSSH-1L and 14-3-3 in neuronal growth cones and Saos-2 cells. (A) Endogenous LIMK1 and endogenous SSH-1L localization in the growth cone of an E18 rat hippocampal neuron. (a) Phase, and epi-fluorescence images of same growth cone labelled with (b) rat anti-LIMK1 and (c) rabbit anti-hSSH-1L (Supplementary Figure 1). (d) Overlay of fluorescence images. Bar in Aa for rows A and B=5 μm. (B) Endogenous SSH-1L and 14-3-3 in the growth cone of an E18 hippocampal neuron. (a) Phase, and epi-fluorescence images of same growth cone labelled with (b) 14-3-3 pan antibody and (c) rabbit anti-hSSH-1L. (d) Overlay of fluorescence images. (C) Saos-2 cells immunostained for endogenous (a, g, j) or overexpressed (d) SSH-1L, LIMK1 (b, e, h, k) and an overlay (c) showing little co-localization. (d–f) Saos-2 cells overexpressing hSSH-1L which now co-localizes with LIMK1 at focal adhesion (arrow) (d) as seen in the overlay (f). (g–l) Saos-2 cell expressing the constitutively active V12rac. Overlay in (i) shows co-localization to a cortical band (arrow in h) that contains F-actin (not shown) and to membrane ruffles (arrows in k) seen at a higher focal plane of a different cell (j–l). Bars=10 μm in a–f and 5 μm in j–l. Download figure Download PowerPoint In the osteosarcoma cell line, Saos-2, LIMK1 is localized to focal adhesions (Figure 1Cb–c), whereas SSH-1L staining is more diffuse. However, overexpressed hSSH-1L does co-localize with LIMK1 at focal adhesions (Figure 1Cd–f). Saos-2 cells expressing constitutively active rac (racV12) lose focal adhesions and both LIMK1 and SSH-1L associate with a circumferential band of actin in the lamella (Figure 1Cg–i), and are found in membrane ruffles (Figure 1Cj–l). Thus, LIMK1 and SSH-1L localize to the same cellular domains in response to rac signalling. SSH interacts with several proteins involved in the regulation of actin dynamics To determine which proteins associate with the different forms of hSSH, we overexpressed His/myc-hSSH (1L, 1S, 2S and 3S) in HEK293 cells, bound, washed, and eluted the hSSH isoforms and their associated proteins from nickel resin at neutral pH (Figure 2A). In addition to the expected SSH isoforms detected on a Western blot, endogenous LIMK1 and 14-3-3 were in all eluates (Figure 2A). Actin was present in all samples except those from cells expressing hSSH-1S, which also had the least 14-3-3 binding. When resin washes were at pH>8, no endogenous LIMK1 remained associated with any of the bound hSSH isoforms and actin and 14-3-3 remained associated with only the hSSH-1L (Figure 2B), suggesting that it has the highest affinity for these proteins. Figure 2.Identification of proteins associated with overexpressed His-myc hSSH-1l, 1S, 2S, and 3S in HEK 293 cells. Lysates from 10 cm dishes of HEK293 cells infected 48 h earlier with adenoviruses expressing His-myc hSSH-1L, 1S, 2S, and 3S were passed over nickel resin, washed with lysis buffer and eluted with lysis buffer containing 250 mM imidazole. (A) Immunoblots of proteins eluted at pH 8.3. (C) Domain organization of SSH-1L and LIMK1, showing the nomenclature of the parental fragments used in this study. Most of these fragments were tagged with His, myc, Flag (F), GST, GFP or HA when used for expression experiments. Download figure Download PowerPoint Association between endogenous LIMK1 and SSH Most cell lines examined expressed both LIMK1 and SSH-1L (data not shown). Mouse embryo fibroblasts (MEFs) expressed both proteins at comparable levels and therefore were used to study their association. MEF lysate was incubated with anti-LIMK1 mouse mAb and the immunocomplexes were subjected to Western blot analysis with rabbit anti-SSH-1L antibodies. Association between endogenous LIMK1 and SSH was observed (Figure 3A). Figure 3.Analysis of LIMK1 and SSH interaction. (A) Interaction between endogenous LIMK1 and endogenous SSH-1L. MEF lysates immunoprecipitated with mouse anti-LIMK1 mAb were subjected to Western blotting with anti-SSH antibodies (top panel). The filter was reprobed with rat anti-LIMK1 mAb to show that LIMK1 was present in the IP and in the lysate (bottom panel). As negative control, the lysate was immunoprecipitated with IgG2. (B) Flag-tagged full-length (F-LIMK1) and the kinase domain of LIMK1 (F-kinase) but not F-LIM1&2 and F-PDZ domains interact with the full-length myc-SSH-1L. F-kinase bound 10 × more myc-SSH-1L protein than full-length LIMK1. The filter was reprobed with rabbit anti-myc polyclonal antibodies (middle panel) to demonstrate that equal amounts of myc-SSH were immunoprecipitated, whereas the bottom panel shows expression of the different LIMK1 proteins in lysates prepared from transfected COS-7 cells. (C) F-LIMK1 interacts with GST-SSH-t, the N-terminal A+B domain, and the phosphatase domain, but not with GST alone. (D) GST-SSH-t interacts with GFP-tagged active (GFP-LIMK1) or inactive full-length and kinase domains of LIMK1 but not with GFP alone. GFP-LIMK1M1 represents kinase-dead LIMK1 (D470A), GFP-K is GFP-tagged kinase domain, and GFP-Ks is the GFP-tagged dominant-negative splice variant kinase domain of LIMK1. Download figure Download PowerPoint The kinase domain of LIMK1 and the N-terminal A and B and phosphatase domains of hSSH1L are required for their association To identify the region in the LIMK1 protein responsible for its co-immunoprecipitation with SSH, Flag-tagged LIM, PDZ and kinase domains (F-LIM, F-PDZ, and F-kinase), and myc-tagged hSSH-1L (myc-hSSH-1L), cDNA constructs (see Figure 2C for nomenclature of untagged fragments) were transfected separately into COS-7 cells. After 48 h, the lysates were combined and the myc-tagged proteins immunoprecipitated with anti-myc antibodies. Samples were analysed by Western blotting for Flag. The kinase domain, and not the LIM or PDZ domains, mediates the interaction with SSH-1L (Figure 3B). To identify the region in hSSH-1L responsible for the interaction with LIMK1, extracts of mammalian cells expressing GST-SSH-A+B, the phosphatase domain (GST-Pase), and the C-terminal truncated GST-SSH-t were incubated with extracts of mammalian cells expressing F-LIMK1 (Figure 3C). The GST-fusion proteins were pulled down and subjected to Western blot analysis. F-LIMK1 was associated with GST-SSH-A+B, GST-SSH-Pase, and GST-SSH-t, but not with GST alone. Thus, SSH-1L associates with LIMK1 via its A+B and the phosphatase domains. The catalytic activities of LIMK1 and SSH are not essential for their interaction To determine whether the activity of the LIMK1 kinase domain is important for its interaction with slingshot, GFP-tagged full-length and kinase domains of active and inactive LIMK1 and GST-SSH-t were expressed either separately or together in 293T cells. After 48 h, the GST-tagged proteins were pulled down and the protein complexes analysed on Western blots. Both wild-type and kinase-dead LIMK1 bound to GST-SSH-t (Figure 3D) and GST-SSH-t (CS) (data not shown), indicating that active kinase and phosphatase are not necessary for this association. hSSH-1L directly interacts with the kinase domain of LIMK1 As actin and 14-3-3 proteins associate with SSH-1L (Figure 2), we sought to determine whether LIMK1 and hSSH-1L interact directly. We therefore purified the His/myc-hSSH-1L from overexpressing HEK293 cells. Most hSSH-1L-associated proteins are removed from the nickel resin by washing with 2.5 M urea. The urea was removed and the purified hSSH-1L eluted (Figure 4A). GST-LIMK1 was purified from overexpressing HEK293 cells on glutathione Sepharose and washed to remove contaminating proteins prior to elution. Neither GST-LIMK1 nor hSSH-1L contained actin or 14-3-3 (Figure 4A). Purified GST-LIMK1 and His-hSSH-1L were incubated in vitro. Purified hSSH-1L was pulled down on glutathione beads only in the presence of GST-LIMK1 and GST-LIMK1 was pulled down on nickel beads only in the presence of His-hSSH-1L (Figure 4B). To demonstrate that the kinase domain mediated this direct binding, the kinase domain of bacterially expressed His-LIMK1 was incubated with bacterially expressed GST-SSH-t (C-terminal deleted) coupled to glutathione Sepharose. The His-kinase domain interacted with GST-hSSH-t but not with GST alone (Figure 4C), suggesting a direct interaction between SSH and the LIMK1 kinase domain. Furthermore, since these domains were bacterially expressed, it is unlikely this interaction requires specific post-translational modifications or is bridged by actin. Figure 4.Purified LIMK1 and hSSH-1L interact directly in vitro through the kinase domain of LIMK1 and the N-terminal half of hSSH-1L. (A) Silver-stained gel (left) and immunoblots of purified LIMK1 (right) (lane 1) and hSSH-1L (lane 2). In lane 3 the 2.5 M urea wash prior to elution of hSSH-1L was omitted. Note that the SSH-1L and LIMK1 preparations used for in vitro binding (lanes 1 and 2) are devoid of both actin and 14-3-3 immunoreactivity. Both showed minor contaminating bands by SDS–PAGE that are probably degradation products. (B) Nickel resin and glutathione-Sepharose pulldown assays of purified His-myc hSSH-1L and GST-LIMK1. The His-myc hSSH-1L is pulled down with glutathione beads only in the presence of GST-LIMK1, and the GST-LIMK1 is pulled down on nickel beads only in the presence of His-myc hSSH-1L. (C) Immunoblots of pulldown experiments showing direct interaction between bacterially expressed LIMK1 kinase domain (His-kinase) and truncated SSH (GST-SSH-t) proteins. Download figure Download PowerPoint Slingshot protein is a LIMK1 phosphatase The direct interaction between LIMK1 and SSH suggested that some functional co-regulation might be involved. To test if SSH alters LIMK1 activity, we compared the ability of wild-type and phosphatase-inactive SSH-1L (CS; C393S) to dephosphorylate LIMK1 and affect its kinase activity. LIMK1 is activated by phosphorylation of Thr 508, followed by autophosphorylation on serines; therefore, LIMK1 activity positively correlates with its level of phosphorylation. To demonstrate the direct effect of hSSH-1L on phospho-LIMK1 (p-LIMK1), purified GST-LIMK1, bound to glutathione beads, was autophosphorylated with [γ32P]ATP followed by washing. [32P]GST-LIMK1 was incubated with either active or inactive SSH-1L for 10–100 min. The amount of [32P] released by the phosphatase was measured before proteins were subjected to Western blotting. The amount of [32P] associated with the LIMK1 band was determined from PhosphorImager analysis. All samples were normalized for the amounts of LIMK1 and hSSH-1L, as determined by densitometric analysis of the immunoblotted SSH and LIMK1 proteins. Reduced levels of [32P]LIMK1 and increased levels of free [32P] were observed when p-LIMK1 was incubated in the presence of active SSH-1L. In contrast, in the presence of catalytically inactive SSH-1L (CS), there was no change in either the level of p-LIMK1 or free [32P], further suggesting that LIMK1 is a substrate for SSH-1L phosphatase (Figure 5A). Figure 5.Slingshot is a LIMK1 phosphatase. Active and inactive myc-SSH-1L proteins were immunopurified with anti-myc mAb and GST-LIMK1 was affinity-purified with glutathione Sepharose. The purified proteins were incubated in the presence of 5 μCi [γ32P]ATP for 30 min, followed by Western blotting and autoradiography. (A) The level of GST-LIMK1 phosphorylation is greatly decreased only in the presence of active myc-SSH-1L (top panel). The levels of GST-LIMK1 (middle panel) and myc-SSH (bottom panel) were analysed by Western blotting. (B) Time course of in vitro phosphatase assay with [32P]LIMK1 as substrate. The relative level of phosphorylated LIMK1 was calculated from the ratio of p-LIMK1/LIMK1 values determined by densitometry in the presence of active SSH. The level of [32P] released during the assay was measured and plotted. The level of [32P]LIMK1 after 60 min incubation with myc-SSH-1L (CS) is shown as an open square and the level of [32P] released as an open diamond. (C) Comparison of the activities of full-length and C-terminal truncated mammalian and bacterial SSH expressed proteins. Each point was calculated taking into consideration the amounts of protein present in the assay. (D) The effect of bacterially and mammalian expressed GST-SSH-t on the phosphorylation of HA-LIMK1 and HA-LIMK2 by in vitro kinase assay. Bacterially expressed SSH-t(B) is more active than SSH-t(M) expressed in mammalian cells (top panel). Note that SSH-t expressed in mammalian cells is phosphorylated, probably by a kinase that is present in the protein complex. After phosphorImaging, the filter was probed with anti-HA mAb (middle panel) and with rabbit anti-GST antibodies (bottom panel) to demonstrate the levels of HA-LIMK1&2 and the SSH proteins, respectively. Download figure Download PowerPoint Similar experiments were performed with C-terminal truncated hSSH-1L (SSH-t, aa 1–533). Affinity-purified [32P]LIMK1 (0.1 μg) was used as substrate for bacterially and mammalian expressed GST-SSH-1L or GST-SSH-1L-t, using expressed GST as a control. In the presence of both mammalian and bacterially expressed GST-SSH-t, the phosphorylation of LIMK1 was greatly reduced, indicating that the C-terminal domain of SSH-1L is not necessary for its phosphatase activity towards LIMK1. In addition, bacterially expressed GST-SSH-t was more active than its mammalian expressed counterpart (Figure 5B and C). This was not true for the bacterial SSH-1L, because it requires F-actin for activity (see below). LIMK1 and 2 have high homology and they both interact with SSH (Figure 3 and data not shown). To test whether this interaction leads to the dephosphorylation of LIMK2, lysates from cells expressing HA-LIMK1 and HA-LIMK2 were immunoprecipitated with anti-HA antibodies subjected to the in vitro phosphatase assay. In the presence of GST-SSH-t purified from mammalian and bacterial cells, the levels of p-LIMK1 and p-LIMK2 decreased. GST-SSH-t purified from bacteria was much more active than that purified from mammalian cells. Significantly, the level of p-LIMK1 decreased by twice as much as the level of p-LIMK2 when normalized to the amount of LIMK loaded (Figure 5D), suggesting that LIMK1 is a better SSH-1 substrate than LIMK2. Slingshot downregulates the activity of LIMK1 towards cofilin To identify the phosphoamino acids of LIMK1 dephosphorylated by SSH, we first tested the ability of SSH to dephosphorylate LIMK1s, the splice variant of LIMK1 lacking kinase activity, which can be phosphorylated on Thr 508 by PAK or ROCK but does not undergo autophosphorylation on serine residues (Arber et al, 1998 and unpublished observation). Cell lysates of 293T cells expressing GST-LIMK1s were incubated in the presence of mammalian GST-SSH-t or GST-SSH-t (CS) for 30 min, followed by Western blot analysis with an antibody that recognizes phospho-Thr-508 (Supplementary Figure 1). The level of p-LIMK1 decreased after incubation with SSH-t but not with SSH-t (CS), indicating that SSH can dephosphorylate LIMK1 on Thr 508 (Figure 6A). To test if SSH can dephosphorylate the autophosphorylated serine residues, constitutively active LIMK1-EE508 was immunoprecipitated from HEK293 cells and was autophosphorylated in the in vitro kinase assay. The labeled protein was incubated for 1 h with bacterially expressed GST-SSH-t, followed by Western blotting and phosphorImager analysis. In the presence of GST-SSH-t, the levels of p-LIMK1 and p-LIMK-EE508 were reduced by five- and three-fold, respectively (Figure 6B), indicating that SSH can dephosphorylate LIMK1 on autophosphorylation sites. Figure 6.SSH-t downregulates the activity of LIMK1 towards cofilin by dephosphorylating Thr 508 and other phosphorylated serine residues. (A) Lysates of GST-LIMK1s expressing 293T cells were subjected to Western blotting before and after incubation with mammalian expressed purified GST-SSH-t or GST-SSH-t (CS). The filter was probed with anti-pLIMK1 antibody, stripped and reprobed with rat anti-LIMK1 mAbs. Incubation with SSH-t, but not with SSH-t (CS), resulted in decreased level of p-LIMK1. The numbers at the bottom represent the ratio between p-GST-LIMK1 and GST-LIMK1 as determined by densitometry. (B) Constitutively active LIMK1-EE508 and GST-LIMK1 were immunoprecipitated or pulled down from overexpressing mammalian cells and subjected to in vitro kinase assay. In the presence of bacterially expressed GST-SSH-t, the levels of p-LIMK1 and p-LIMK1-EE508 proteins were reduced by five- and three-fold, respectively. (C) Lysate of F-LIMK1-expressing cells was incubated with glutathione-bound GST-SSH-t, GST-SSH-t (CS) or GST expressed in mammalian cells. After centrifugation, the supernatants were precipitated with anti-Flag beads followed by in vitro phosphatase assay with [32P]cofilin as a substrate. In the presence of GST-SSH-t, the levels of p-cofilin and p-LIMK1 were greatly reduced. Download figure Download PowerPoint Next we examined whether the dephosphorylation of LIMK1 by SSH resulted in downregulation of its kinase activity towards cofilin. F-LIMK1, GST-SSH-t, and GST-SSH-t (CS) were expressed separately in 293T cells. The cell lysate of F-LIMK1-transfected cells was incubated for 40 min with glutathione-bound GST-SSH-t, GST-SSH-t (CS) or GST alone. The supernatant was separated and subjected to immunoprecipitation with anti-Flag beads, followed by the in vitro kinase assay in the presence of chicken cofilin. The activity of LIMK1 toward cofilin was reduced five-fold after incubation with GST-SSH-t, but decreased only slightly after incubation with GST-SSH-t (CS) or GST alone (Figure 6C), indicating that dephosphorylation of LIMK1 by SSH results in downregulation of its activity. Similarly, the level of [32P]LIMK1 was also reduced five-fold after incubation with SSH-t, but only slightly after incubation with SSH-t (CS) or GST (Figure 6C), indicating that dephosphorylation of LIMK1 by SSH-t results in downregulation of its activity towards cofilin. The activity of hSSH-1L is regulated by F-actin and by phosphorylation His/myc-hSSH-1L is active when expressed in cells, dephosphorylating completely the endogenous pool of ADF/cofilin, whereas the CS mutant is inactive (Supplementary Figure 2). When overexpressed hSSH-1L and its CS mutant were purified on nickel resin, they both bound actin and 14-3-3 in equal amounts (Supplementary Figure 3A). However, only hSSH-1L, but not its CS mutant, was able to dephosphorylate pAC in vitro (data not shown; Niwa et al, 2002). When the nickel resin containing the bound hSSH-1L was washed free of contaminating proteins with 2.5 M urea before elution, the purified hSSH-1L lost its pADF phosphatase activity, which was restored upon addition of actin (Supplementary Figure 3B). This restoration of activity depended upon the continuous presence of F-actin (Supplementary Figure 3C). Purified hSSH-1L, as well as 1S, 2S, and 3S, stained positively for phosphate (Figure 7A). Phosphorylation is primarily on serine as shown by 2D phosphoamino-acid analysis of overexpressed GST-SSH-t (Figure 7B). Interestingly, bacterially expressed GST-SSH-t was also primarily phosphorylated on serine by PAK4 in vitro (Figure 7B). The majority of the phosphate could be removed from the hSSH-1L isoform by incubation with λ-phosphatase for 1 h (Figure 7A). Even after dephosphorylation, the purified hSSH-1L requires F-actin for activity (Figure 7C). In the presence of F-actin, the dephosphorylated form of hSSH-1L has greater phosphoADF phosphatase activity than the phosphorylated hSSH-1L fraction (Figure 7C and D). Figure 7.hSSH-1L is a phosphorylated on serine residues and phosphorylation inhibits its activity toward pADF and is necessary for binding 14-3-3ζ. (A) Coomassie Blue stained nickel-resin purified hSSH isoforms isolated from overexpressing HEK 293 cells (left panels) are phosphorylated as shown by staining with the Pro-Q Diamond phosphoprotein stain (right two panels). Treatment of hSSH-1L with bacterial λ-phosphatase for 60 min markedly reduced the phosphoprotein staining (far right panel) although the total protein loaded remains constant (Coomassie Blue panel). The images of the phosphoprotein stain have been inverted for easier visualization of the bands. (B) Phosphoamino analysis of G
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