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Direct stimulation of receptor-controlled phospholipase D1 by phospho-cofilin

2007; Springer Nature; Volume: 26; Issue: 19 Linguagem: Inglês

10.1038/sj.emboj.7601852

1460-2075

Li Han, Matthias B. Stope, Maider López de Jesús, Paschal A. Oude Weernink, Martina Urban, Thomas Wieland, Dieter Rosskopf, Kensaku Mizuno, Karl H. Jakobs, Martina Schmidt,

Cardiomyopathy and Myosin Studies

Article13 September 2007free access Direct stimulation of receptor-controlled phospholipase D1 by phospho-cofilin Li Han Li Han Institut für Pharmakologie, Universitätsklinikum Essen, Essen, GermanyPresent address: Department of Infection Control, Chinese Military Institute of Disease Control & Prevention, Beijing 100071, China Search for more papers by this author Matthias B Stope Matthias B Stope Institut für Pharmakologie, Universitätsklinikum Essen, Essen, Germany Search for more papers by this author Maider López de Jesús Maider López de Jesús Institut für Pharmakologie, Universitätsklinikum Essen, Essen, Germany Search for more papers by this author Paschal A Oude Weernink Paschal A Oude Weernink Institut für Pharmakologie, Universitätsklinikum Essen, Essen, Germany Search for more papers by this author Martina Urban Martina Urban Institut für Pharmakologie, Universitätsklinikum Essen, Essen, Germany Search for more papers by this author Thomas Wieland Thomas Wieland Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Fakultät für Klinische Medizin Mannheim der Universität Heidelberg, Mannheim, Germany Search for more papers by this author Dieter Rosskopf Dieter Rosskopf Institut für Pharmakologie, Universitätsklinikum Essen, Essen, Germany Search for more papers by this author Kensaku Mizuno Kensaku Mizuno Department of Biomolecular Sciences, Graduate School of Life Sciences, Sendai, Miyagi, Japan Search for more papers by this author Karl H Jakobs Karl H Jakobs Institut für Pharmakologie, Universitätsklinikum Essen, Essen, Germany Search for more papers by this author Martina Schmidt Corresponding Author Martina Schmidt Institut für Pharmakologie, Universitätsklinikum Essen, Essen, Germany Department of Molecular Pharmacology, University of Groningen, Groningen, The Netherlands Search for more papers by this author Li Han Li Han Institut für Pharmakologie, Universitätsklinikum Essen, Essen, GermanyPresent address: Department of Infection Control, Chinese Military Institute of Disease Control & Prevention, Beijing 100071, China Search for more papers by this author Matthias B Stope Matthias B Stope Institut für Pharmakologie, Universitätsklinikum Essen, Essen, Germany Search for more papers by this author Maider López de Jesús Maider López de Jesús Institut für Pharmakologie, Universitätsklinikum Essen, Essen, Germany Search for more papers by this author Paschal A Oude Weernink Paschal A Oude Weernink Institut für Pharmakologie, Universitätsklinikum Essen, Essen, Germany Search for more papers by this author Martina Urban Martina Urban Institut für Pharmakologie, Universitätsklinikum Essen, Essen, Germany Search for more papers by this author Thomas Wieland Thomas Wieland Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Fakultät für Klinische Medizin Mannheim der Universität Heidelberg, Mannheim, Germany Search for more papers by this author Dieter Rosskopf Dieter Rosskopf Institut für Pharmakologie, Universitätsklinikum Essen, Essen, Germany Search for more papers by this author Kensaku Mizuno Kensaku Mizuno Department of Biomolecular Sciences, Graduate School of Life Sciences, Sendai, Miyagi, Japan Search for more papers by this author Karl H Jakobs Karl H Jakobs Institut für Pharmakologie, Universitätsklinikum Essen, Essen, Germany Search for more papers by this author Martina Schmidt Corresponding Author Martina Schmidt Institut für Pharmakologie, Universitätsklinikum Essen, Essen, Germany Department of Molecular Pharmacology, University of Groningen, Groningen, The Netherlands Search for more papers by this author Author Information Li Han1,‡, Matthias B Stope1,‡, Maider López de Jesús1,‡, Paschal A Oude Weernink1, Martina Urban1, Thomas Wieland2, Dieter Rosskopf1, Kensaku Mizuno3, Karl H Jakobs1 and Martina Schmidt 1,4 1Institut für Pharmakologie, Universitätsklinikum Essen, Essen, Germany 2Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Fakultät für Klinische Medizin Mannheim der Universität Heidelberg, Mannheim, Germany 3Department of Biomolecular Sciences, Graduate School of Life Sciences, Sendai, Miyagi, Japan 4Department of Molecular Pharmacology, University of Groningen, Groningen, The Netherlands ‡These authors contributed equally to this work *Corresponding author. Department of Molecular Pharmacology, University of Groningen, A. Deusinglaan 1, Groningen 9713 AV, The Netherlands. Tel.: +31 50 363 3322; Fax: +31 50 363 6908; E-mail: [email protected] The EMBO Journal (2007)26:4189-4202https://doi.org/10.1038/sj.emboj.7601852 Present address: Department of Infection Control, Chinese Military Institute of Disease Control & Prevention, Beijing 100071, China PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The activity state of cofilin, which controls actin dynamics, is driven by a phosphorylation–dephosphorylation cycle. Phosphorylation of cofilin by LIM-kinases results in its inactivation, a process supported by 14-3-3ζ and reversed by dephosphorylation by slingshot phosphatases. Here we report on a novel cellular function for the phosphorylation–dephosphorylation cycle of cofilin. We demonstrate that muscarinic receptor-mediated stimulation of phospholipase D1 (PLD1) is controlled by LIM-kinase, slingshot phosphatase as well as 14-3-3ζ, and requires phosphorylatable cofilin. Cofilin directly and specifically interacts with PLD1 and upon phosphorylation by LIM-kinase1, stimulates PLD1 activity, an effect mimicked by phosphorylation-mimic cofilin mutants. The interaction of cofilin with PLD1 is under receptor control and encompasses a PLD1-specific fragment (aa 585–712). Expression of this fragment suppresses receptor-induced cofilin–PLD1 interaction as well as PLD stimulation and actin stress fiber formation. These data indicate that till now designated inactive phospho-cofilin exhibits an active cellular function, and suggest that phospho-cofilin by its stimulatory effect on PLD1 may control a large variety of cellular functions. Introduction The dynamic nature of actin filament assembly/disassembly and its cellular organization is regulated by several actin-binding and -regulatory proteins, including the actin-depolymerizing factor (ADF)/cofilin family (Bamburg, 1999; Bamburg and Wiggan, 2002; Pollard and Borisy, 2003; DesMarais et al, 2005). Members of the ADF/cofilin family (hereafter referred to as cofilin) are now considered to be pivotal regulators of actin filament dynamic-dependent processes, including cell motility, neuronal pathfinding, membrane dynamics, establishment of cell polarity, cell division and apoptosis, and their dysfunction seems to contribute to the progression of diseases as diverse as cancer, Alzheimer's dementia and ischemic kidney disease (Bamburg and Wiggan, 2002; Chua et al, 2003; Wang et al, 2006). Cofilin binds to both G- and F-actin, but due to a higher affinity for ADP-bound subunits, the off-rate of actin monomers from the pointed end of actin filaments is increased; in addition, cofilin severs actin filaments and thus directly generates free actin barbed ends (Condeelis, 2001; DesMarais et al, 2005). The cofilin–actin interaction is tightly controlled by phosphocycling. Phosphorylation of cofilin at serine 3, located in the actin-binding domain, by the LIM (Lin-11/Isl-1/Mec-3) and the TES (testicular protein) kinases results in its inactivation, and the Unphosphorylatable S3A cofilin mutant as well as the phosphorylation-mimic cofilin mutants (S3D cofilin and S3E cofilin) have been widely used to define the role of cofilin in cellular responses (Bamburg, 1999; Huang et al, 2006). The two LIM-kinases, LIM-kinase1 and LIM-kinase2, are expressed in most tissues (Foletta et al, 2004; Acevedo et al, 2006) and are activated by Rho GTPases, through their effectors, Rho-kinase and p21-activated protein kinases (Edwards and Gill, 1999; Kaibuchi et al, 1999). Recently, interaction of LIM-kinase1 and cofilin with the scaffold protein 14-3-3ζ has been reported (Birkenfeld et al, 2003), leading to accumulation of inactive, phosphorylated cofilin (Gohla and Bokoch, 2002). Dephosphorylation of cofilin results in its reactivation, and is catalyzed by the cofilin-specific phosphatases of the slingshot family (Niwa et al, 2002; Kaji et al, 2003; Ohta et al, 2003) and chronophin (Gohla et al, 2005; Huang et al, 2006). The activity of the slingshot phosphatases is apparently also controlled by multiple signaling pathways, including Ca2+, cyclic AMP and phosphatidylinositol 3-kinase (for recent review see Huang et al, 2006). It has been also shown that the slingshot phosphatase 1L not only dephosphorylates cofilin but also LIM-kinase1, resulting in its inactivation, and that the phosphatase activity is regulated by F-actin and 14-3-3ζ (Nagata-Ohashi et al, 2004; Soosairajah et al, 2005). Phospholipase D (PLD) enzymes, PLD1 splice variants and PLD2, hydrolyze phosphatidylcholine of cell membranes to phosphatidic acid (PA), in response to stimuli, and are considered to be involved in a large variety of early and late cellular responses, including calcium mobilization, secretion, superoxide production, endocytosis, exocytosis, vesicle trafficking, glucose transport, mitogenesis and apoptosis (Exton, 2002). The rise in cellular PA, in particular by PLD1, has also been reported to induce stress fiber formation in suitable cell types (Ha and Exton, 1993; Cross et al, 1996; Kam and Exton, 2001; Porcelli et al, 2002; Komati et al, 2005). As PA-activatable phosphatidylinositol-4-phosphate 5-kinase (PIP 5-kinase) generates phosphatidylinositol 4,5-bisphosphate (PIP2) (reviewed in Oude Weernink et al, 2004a), known to act as a PLD cofactor and to associate with a plethora of actin-binding proteins that regulate actin dynamics (Exton, 2002; Yin and Janmey, 2003; Hilpela et al, 2004), such mechanisms may act in concert to promote actin cytoskeleton reorganization. PLD and PIP 5-kinase are now also recognized as effectors of Rho-dependent Rho-kinase (Schmidt et al, 1999; Oude Weernink et al, 2000, 2004b; Kam and Exton, 2001; Cummings et al, 2002; Yamazaki et al, 2002). Vice versa, components of the actin regulatory machinery have been found to affect PLD. PLD activity, primarily PLD2, is negatively regulated by β-actin and the actin-binding protein α-actinin (Park et al, 2000; Lee et al 2001). Meanwhile, it has been reported that G-actin inhibits PLD1 as well, but that F-actin has the opposite effect, suggesting that specifically PLD1 may act as an actin dynamic responsive cellular element (Kusner et al, 2002). Here we report on a novel molecular link between the actin cytoskeleton and PLD1. We demonstrate that receptor-induced and Rho/Rho-kinase-dependent activation of PLD1 is under control of LIM-kinase1 and its substrate, cofilin. Cofilin directly interacts with and stimulates in its phosphorylated state PLD1. Our data, thus, indicate for the first time that phospho-cofilin, until now considered to be biologically inactive, is an active cellular component, which by its signaling to PLD1 can regulate essential cellular functions known to be under control of PLD. Results Involvement of cofilin in muscarinic receptor signaling to PLD The Rho effector, Rho-kinase, mediates PLD stimulation by G-protein-coupled muscarinic acetylcholine receptors (mAChRs) in HEK-293 and N1E-115 neuroblastoma cells (Schmidt et al, 1999; Kam and Exton, 2001; Cummings et al, 2002). As PLD enzymes neither directly interact with, nor are phosphorylated by Rho-kinase (Schmidt et al, 1999), we considered the involvement of the Rho-kinase effector, LIM-kinase, in mAChR signaling to PLD. As shown in Figure 1A, overexpression of LIM-kinase1 in HEK-293 cells strongly increased PLD stimulation by the mAChR agonist, carbachol, whereas expression of kinase-deficient D460A LIM-kinase1 had the opposite effect. The carbachol-induced and LIM-kinase1-reinforced PLD stimulation was almost fully blunted by adenoviral expression of lipase-inactive K898R PLD1, whereas lipase-inactive K758R PLD2 had no effect. Similar data were obtained for mAChR regulation of PLD activity in N1E-115 neuroblastoma cells (data not shown). These data suggested that the receptor-induced PLD stimulation not only involves Rho-kinase, but probably also its substrate, LIM-kinase, and that the PLD isozyme responsible for the increased PLD activity is PLD1. Consequently, we studied whether LIM-kinase1 may phosphorylate PLD1. However, as shown in Figure 1B, purified recombinant LIM-kinase1 did neither phosphorylate purified GST-tagged PLD1 nor PLD2. LIM-kinase1 did also not bind to the PLD enzymes (data not shown). The purified LIM-kinase1 was active, as it phosphorylated, as expected, wild-type cofilin, but not its mutant, S3A cofilin (Figure 1B). Figure 1.Cofilin, the sole substrate of LIM-kinase, regulates stimulation of PLD1 by the M3 mAChR. HEK-293 cells were transfected with kinase-deficient LIM-kinase1 (D460A LIM-K1) or wild-type LIM-kinase1, either alone or with adenoviruses encoding LacZ, lipase-inactive K898R PLD1 or lipase-inactive K758R PLD2 (A), or transfected with empty vector (Control), wild-type cofilin and Unphosphorylatable S3A cofilin (C). After 48 h, stimulated [3H]PtdEtOH accumulation was determined in the presence of 1 mM carbachol (Carb) (A, C) or 100 nM PMA (C). Data shown are means±s.e. (n=3–4). The immunoblots demonstrate expression of LIM-kinase1, PLD enzymes and cofilin in cell lysates. (B) [γ-32P]ATP phosphorylation (32P autoradiography) of GST, GST-tagged PLD1, GST-tagged PLD2 (full-length each) and wild-type cofilin and S3A cofilin by LIM-kinase1. Data are representative of three to four similar experiments. *P<0.05. Download figure Download PowerPoint These data prompted us to study whether the LIM-kinase substrate, cofilin, may be involved in PLD stimulation. As illustrated in Figure 1C, overexpression of wild-type cofilin in HEK-293 cells greatly enhanced, by about 2.5-fold, stimulation of PLD by the mAChR agonist, carbachol, whereas expression of Unphosphorylatable S3A cofilin suppressed PLD stimulation by about 50%. In contrast, expression of wild-type and S3A cofilin did not alter PLD stimulation by the phorbol ester, phorbol 12-myristate 13-acetate (PMA), which is Rho- and Rho-kinase independent in HEK-293 cells (Voß et al, 1999), and which was also not affected by expression of LIM-kinase1 variants (data not shown). To confirm that cofilin mediates mAChR-induced PLD stimulation, human HEK-293 and mouse N1E-115 cells were transfected with siRNA pSUPER expression plasmids, which direct the synthesis of siRNAs targeting either human or mouse cofilin, respectively (Nishita et al, 2005; Kiuchi et al, 2007). As shown in Figure 2, these maneuvers greatly reduced the cellular content of cofilin in both cell types. Silencing of cellular cofilin in HEK-293 and N1E-115 neuroblastoma cells reduced the carbachol-induced PLD stimulation by about 60 and 80%, respectively (Figure 2). In contrast, knockdown of cofilin expression did not alter PLD stimulation by PMA in either cell type. Figure 2.Depletion of cellular cofilin reduces PLD stimulation by carbachol. HEK-293 cells were transfected with human cofilin siRNA pSUPER plasmid (siRNA hCofilin), or with the empty pSUPER vector (Control) (A). N1E-115 neuroblastoma cells were transfected with mouse cofilin siRNA pSUPER plasmid (siRNA mCofilin), or with the empty pSUPER vector (Control) (B). After 48 h, stimulated [3H]PtdEtOH accumulation was determined in the presence of 1 mM carbachol (Carb) or 100 nM PMA. Data shown are means±s.e. (n=3–4). The immunoblots demonstrate endogenous expression of cofilin in lysates of cells transfected with empty pSUPER vector (Control) or the indicated siRNA pSUPER plasmids. *P<0.05. Download figure Download PowerPoint These findings together suggested that cofilin, possibly in its phosphorylated state, may control the receptor stimulation of PLD. To substantiate this assumption, we first studied whether receptor activation leads to cofilin phosphorylation, and whether this phosphorylation is under control of a cofilin-specific phosphatase and the scaffold protein, 14-3-3ζ. As illustrated in Figure 3A, agonist activation of the mAChR induced a strong, but rather transient phosphorylation of cofilin in HEK-293 cells. The stimulatory effect of carbachol reached its maximum at 15 s and rapidly declined thereafter. Expression of slingshot phosphatase 1L completely abolished phosphorylation of cofilin, both in the basal state and after stimulation by carbachol (Figure 3B). In contrast, expression of 14-3-3ζ and inactive slingshot phosphatase 1L (not shown) strongly increased phosphorylation of cofilin in unstimulated cells, and there was no additional increase by the receptor activation. We then examined the effects of the slingshot and 14-3-3ζ on mAChR stimulation of PLD activity. Expression of slingshot phosphatase 1L significantly decreased PLD stimulation by carbachol, whereas inactive slingshot phosphatase 1L and 14-3-3ζ enhanced PLD stimulation by carbachol (Figure 3C, left panel). In contrast, PLD stimulation by epidermal growth factor (EGF), which similar to PLD stimulation by PMA, is independent of Rho and Rho-kinase in these cells (Voß et al, 1999), was not altered by expression of these proteins controlling the phosphorylation state of cofilin (Figure 3C, right panel). In line with a role of cofilin phosphorylation in PLD stimulation, expression of the phosphorylation-mimic S3D cofilin mutant strongly increased the carbachol-induced PLD stimulation, both in HEK-293 and N1E-115 neuroblastoma cells (Figure 4A). We next studied whether the expression of phosphorylation-mimic S3D cofilin could counteract the inhibitory effect of cofilin silencing on the PLD response. As shown in Figure 4B, expression of phosphorylation-mimic S3D mouse cofilin in cofilin-silenced HEK-293 cells considerably rescued PLD stimulation by carbachol. Figure 3.Regulation of the cellular phosphorylation state of cofilin and its impact on the PLD response. HEK-293 cells were stimulated without (−) or with (+) 1 mM carbachol for the indicated periods of time (A), or transfected with empty vector (Control), c-myc-tagged wild-type slingshot 1L, inactive slingshot 1L (C393S slingshot 1L) or VSV-G-tagged 14-3-3ζ, and stimulated with carbachol for 15 s (B). Phosphorylation of cofilin (P-Cofilin) and the total cellular content of cofilin were detected in cell lysates with anti-phospho-cofilin and anti-cofilin antibodies, respectively. Data are representative of three experiments. (C) Stimulated [3H]PtdEtOH accumulation was determined in the presence of 1 mM carbachol (Carb) or 100 ng/ml EGF. Data shown are means±s.e. (n=3–4). The immunoblots show expression of c-myc-tagged slingshot 1L and myc-tagged inactive slingshot 1L (C393S slingshot 1L), or VSV-G-tagged 14-3-3ζ. *P<0.05. Download figure Download PowerPoint Figure 4.Phosphorylation-mimic S3D cofilin potentiates PLD1 stimulation by carbachol and rescues PLD1 stimulation in cofilin-depleted cells. HEK-293 cells or N1E-115 cells were transfected with phosphorylation-mimic S3D cofilin, or with empty vector (Control) (A), or transfected with either phosphorylation-mimic S3D mouse cofilin, human cofilin siRNA pSUPER plasmid (siRNA hCofilin), or both constructs together (B). After 48 h, stimulated [3H]PtdEtOH accumulation was determined in the presence of 1 mM carbachol (Carb). Data shown are means±s.e. (n=3) (A), or are representative of three experiments (B). *P<0.05. Download figure Download PowerPoint Cofilin binds to and alters subcellular localization of PLD1 For analysis of cofilin–PLD interaction, studies were performed both in vitro with purified components and in intact cells. As illustrated in Figure 5A, purified wild-type His-tagged cofilin strongly bound to GST-tagged PLD1. In contrast, binding of Unphosphorylatable S3A cofilin to GST-tagged PLD1 was hardly detectable, similar as binding of wild-type or S3A cofilin to PLD2. Thus, cofilin can directly and specifically interact with PLD1 and this interaction apparently requires the phosphorylatable serine 3 of cofilin. To examine whether cofilin also interacts with PLD1 in intact cells, we transfected HEK-293 cells with PLD1 or PLD2 and cofilin mutants for immunofluorescence laser confocal microscopy analysis. As reported before in other cell types (Bamburg, 1999; Bamburg and Wiggan, 2002; Exton, 2002), we found that PLD1 localized to intracellular compartments and the plasma membrane, whereas PLD2 exclusively localized to the plasma membrane (Figure 5B, panels a and d); the cofilin mutants were found to be localized to the plasma membrane and intracellular compartments (Figure 5B, panels g and h). Coexpression of wild-type cofilin altered subcellular localization of PLD1 (Figure 5B, panel b). In cells coexpressing PLD1 and wild-type cofilin, PLD1 was found primarily at the plasma membrane. Coexpression of S3A cofilin and PLD1 caused only a minor subcellular redistribution of PLD1 (Figure 5B, panel c). In contrast to PLD1, the plasma membrane localization of PLD2 was not altered by coexpression of wild-type or S3A cofilin (Figure 5B, panels e and f). Thus, cofilin can specifically alter subcellular localization of PLD1. Figure 5.Direct cofilin–PLD1 interaction is reflected by cofilin-induced subcellular redistribution of PLD1, but not PLD2. (A) Immobilized GST, GST-tagged PLD1 and GST-tagged PLD2 were incubated with recombinant His6-tagged wild-type or unphosphorylatable S3A cofilin overnight at 4°C. Specifically bound proteins were separated by SDS–PAGE, transferred onto nitrocellulose membrane and detected by immunoblotting with anti-PLD and anti-His antibodies. Bar graph illustrates mean±s.e.m. (n=3), with the amount of wild-type cofilin bound to GST-PLD1 set to 1 (Control). (B) HEK-293 cells were transfected with PLD1 (a–c), PLD2 (d–f), HA-tagged wild-type cofilin (b, e, g) or with HA-tagged unphosphorylatable S3A cofilin (c, f, h), either alone (a, d, g, h) or with the indicated combinations. After 48 h, immunofluorescence laser confocal microscopy was performed as described in the Materials and methods section. Yellow color: merge of red (PLD) and green (cofilin) colors. Data are characteristic of three similar experiments. Scale bar, 10 μm. (C) HEK-293 cells were transfected with wild-type PLD1 or PLD2. After 48 h, the cells were treated for 15 s without (−) or with (+) 1 mM carbachol, followed by cell lysis and immunoprecipitation with anti-PLD antibodies. The PLD immunoprecipitates (IP) and total lysates were resolved by SDS–PAGE and probed with anti-phospho-cofilin (α-P-Cofilin) or anti-cofilin antibodies (α-cofilin) as indicated. The results shown are representative of 3–4 experiments. WB, Western blot. Download figure Download PowerPoint To substantiate that cofilin interacts with PLD1 also in intact cells, co-immunoprecipitation studies were performed. As illustrated in Figure 5C, left panel, cofilin and phospho-cofilin were co-immunoprecipitated with PLD1 from lysates of HEK-293 cells coexpressing PLD1 and cofilin, demonstrating their in vivo interaction. Most important, stimulation of the mAChR with carbachol for 15 s strongly enhanced the amount of cofilin and phospho-cofilin co-immunoprecipitated with PLD1. In contrast, cofilin and phospho-cofilin poorly co-immunoprecipitated with PLD2, and there was no effect of carbachol (Figure 5C, right panel). Phosphorylation of cofilin is essential for stimulation of PLD1 We then determined whether cofilin not only interacts with PLD1, but also controls its activity, and whether such regulation is dependent on the phosphorylation state of cofilin in vitro. For this, wild-type and Unphosphorylatable S3A cofilin were first treated with LIM-kinase1, in the absence and presence of MgATP, to allow for cofilin phosphorylation, and then the cofilin proteins were added to purified recombinant PLD enzymes for measurement of enzyme activity. Addition of purified recombinant wild-type or S3A cofilin pretreated with LIM-kinase1, in the absence or presence of MgATP had no effect on the activity of the PLD2 enzyme (Figure 6A, right bar graph). Furthermore, similar to buffer control, S3A cofilin was without effect on PLD1 activity. Wild-type cofilin pretreated with LIM-kinase1, but without MgATP, increased the activity of PLD1 only slightly, by about 50% (Figure 6A, left bar graph), whereas wild-type cofilin pretreated without LIM-kinase1 was without effect on PLD1 activity (data not shown). In contrast, wild-type cofilin phosphorylated by LIM-kinase1 (in the presence of MgATP; Figure 6A, 32P autoradiography) strongly enhanced PLD1 activity, by about three-fold (Figure 6A, left bar graph). To corroborate the hypothesis that it is the phosphorylated cofilin, which stimulates PLD1, purified recombinant phosphorylation-mimic cofilin mutants were added to purified recombinant PLD1. Similar to wild-type cofilin phosphorylated by LIM-kinase1, the phosphorylation-mimic S3D and S3E cofilin mutants strongly enhanced PLD1 activity, by about three-fold (Figure 6B). Thus, cofilin not only directly and specifically interacts with PLD1, but also strongly increases its activity, and this activation is dependent on the phosphorylation state of cofilin. Figure 6.Phospho-cofilin stimulates PLD1. (A) With LIM-kinase1, phosphorylated (+ATP) and unphosphorylated (−ATP) wild-type or unphosphorylatable S3A cofilin were incubated with GST-tagged PLD1 or GST-tagged PLD2 to measure PLD activity. The upper blots show purified GST-tagged LIM-kinase1, GST-tagged PLD1 and PLD2 and His6-tagged wild-type and S3A cofilin by Coomassie blue staining (CS), and immunoblotting with anti-GST and anti-His antibodies, respectively, as well as the specific phosphorylation of wild-type cofilin with [γ-32P]ATP (32P autoradiography) in the absence (−) and presence (+) of LIM-kinase1. (B) GST-tagged PLD1 was incubated without and with recombinant unphosphorylatable S3A cofilin, wild-type cofilin, phospho-mimetic S3D cofilin or phospho-mimetic S3E cofilin to measure PLD activity. Purified His6-tagged cofilin mutants are presented by Coomassie blue staining. The results shown are representative of 3–4 experiments. Download figure Download PowerPoint Identification of the PLD1 region responsible for interaction with cofilin To identify the PLD1 region involved in cofilin binding, we constructed GST-tagged PLD1 fragments (Figure 7A and B) and analyzed their ability to bind purified cofilin. The F-3 fragment of PLD1, encompassing the amino acids 585–712, was found to exclusively bind wild-type cofilin (Figure 7C). None of the PLD1 fragments analyzed, bound Unphosphorylatable S3A cofilin. Furthermore, there was no binding of cofilin to the corresponding PLD2 fragments (Lee et al, 2001; Chae et al, 2005) (Figure 7C). These data identify the PLD1-specific region encoded by the amino acids 585–712 to be important for the direct interaction with cofilin. To study whether cofilin phosphorylation alters binding to PLD1, binding of the phosphorylation-mimic S3D cofilin to the F-3 fragment of PLD1 was studied. As illustrated in Figure 7D, S3D cofilin bound to the F-3 fragment, but somewhat less than wild-type cofilin. Cellular cofilin phosphorylated by treatment of HEK-293 cells for 15 s with carbachol, also bound to the PLD1 F-3 fragment and to full-length PLD1, but again somewhat less than cofilin (data not shown). Thus, phosphorylation of cofilin strongly increases PLD1 activity, but not binding of cofilin to PLD1. Figure 7.Interaction of cofilin with the F-3 fragment of PLD1. (A) A schematic representation of the highly conserved regions in PLD1 and PLD2. PX, phox; PH, pleckstrin homology; CRI–CRIV, conserved regions I to IV. (B) Purified GST and GST-tagged fragments of PLD1 and PLD2, visualized by Coomassie blue staining. (C) Binding of purified recombinant wild-type or unphosphorylatable S3A cofilin to GST-tagged fragments of PLD1 or PLD2 was determined as described under Materials and methods. (D) Binding of purified recombinant wild-type or phospho-mimetic S3D cofilin to GST-tagged F-3 fragment of PLD1. The lower blot shows the loading control by Western blot (WB). The results shown are representative of 2–4 experiments. Download figure Download PowerPoint Inhibition of PLD stimulation and actin stress fiber formation by the cofilin-binding F-3 fragment As cofilin directly interacts with the F-3 fragment of PLD1, we used this fragment to interfere with the mAChR-induced cofilin-PLD1 interaction, as well as PLD stimulation and actin stress fiber formation. Expression of the F-3 fragment in HEK-293 cells strongly and specifically reduced PLD stimulation by carbachol, without affecting PLD stimulation by PMA (Figure 8A). In contrast, expression of the F-1 fragment of PLD1, that does not interact with cofilin, did not alter PLD stimulation by carbachol. The inhibitory effect of the F-3 fragment could be completely rescued by coexpression of wild-type cofilin (Figure 8A). These data indicate that the PLD1-specific F-3 fragment specifically interferes with the activation of PLD1, most probably by attenuating the mAChR-induced association of cofilin with PLD1. Indeed, the enhanced co-immunoprecipitation of cofilin with PLD1 induced by activation of the mAChR by carbachol was completely abolished by expression of the F-3 fragment (Figure 8B). Figure 8.The F