JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M109218200 on March 26, 2002

J. Biol. Chem., Vol. 277, Issue 22, 20026-20032, May 31, 2002
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
277/22/20026    most recent
M109218200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kanazawa, H.
Right arrow Articles by Imai, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kanazawa, H.
Right arrow Articles by Imai, Y.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Macrophage/Microglia-specific Protein Iba1 Enhances Membrane Ruffling and Rac Activation via Phospholipase C-gamma -dependent Pathway*

Hiroko Kanazawa, Keiko Ohsawa, Yo Sasaki, Shinichi KohsakaDagger, and Yoshinori Imai

From the Department of Neurochemistry, National Institute of Neuroscience, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan

Received for publication, September 24, 2001, and in revised form, March 22, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Iba1 is a macrophage/microglia-specific calcium-binding protein that is involved in RacGTPase-dependent membrane ruffling and phagocytosis. In this study, we introduced Iba1 into Swiss 3T3 fibroblasts and demonstrated the enhancement of platelet-derived growth factor (PDGF)-induced membrane ruffling and chemotaxis. Wortmannin treatment did not completely suppressed this enhanced membrane ruffling in Iba1-expressing cells, whereas it did in Iba1-nonexpressing cells, suggesting that the enhancement is mediated through a phosphatidylinositol 3-kinase (PI3K)-independent signaling pathway. Porcine aorta endothelial cells transfected with expression constructs of Iba1 and PDGF receptor add-back mutants were used to analyze the signaling pathway responsible for the Iba1-induced enhancement of membrane ruffling. In the absence of Iba1 expression, PDGF did not induced membrane ruffling in cells expressing the Tyr-1021 receptor mutant, which is capable of activating phospholipase C-gamma (PLC-gamma ) but not PI3K. In contrast, in the presence of Iba1 expression, membrane ruffling was formed in cells expressing the Tyr-1021 mutant. In addition, Rac was shown to be activated during membrane ruffling in cells expressing Iba1 and the Tyr-1021 mutant. Furthermore, dominant negative forms of PLC-gamma completely suppressed PDGF-induced Iba1-dependent membrane ruffling and Rac activation. These results indicate the existence of a novel signaling pathway where PLC-gamma activates Rac in a manner dependent on Iba1.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell motility is a dynamic process driven by structurally and functionally coordinated reorganization of the actin cytoskeleton (1, 2). Among various types of cells, macrophages are extremely motile to migrate rapidly to sites of infection or inflammation, suggesting that highly integrated systems should exist to regulate the actin cytoskeleton in macrophages (3, 4). In addition to circulating monocytes/macrophages, there are many types of tissue-resident macrophages, including Langerhans cells, Kupffer cells, dendritic cells, splenocytes, and microglia. In response to various pathological phenomena, microglia are activated to exhibit drastic changes in shape and the abilities to become locomotive and to phagocytose (5, 6). These cellular reactions are also profoundly underlaid by dynamic remodeling of the actin cytoskeleton.

The Rho family GTPases, Cdc42, Rac, and Rho, are known to be molecular switches that organize remodeling of the actin cytoskeleton (7). Among them, in fibroblasts, Rac is activated by receptor tyrosine kinases such as platelet-derived growth factor receptor (PDGFR),1 leading to the formation of lamellipodia and membrane ruffles (8). Dominant active RacV12 induces remarkable membrane ruffling, and dominant negative RacN17 completely inhibits peptide growth factor-induced membrane ruffling; therefore, Rac is recognized to be an essential component in this type of membrane ruffling (8). Some studies describe signaling molecules capable of interacting with Rac; however, the processes by which receptor tyrosine kinases activate Rac are not fully understood.

Previously, we identified a calcium-binding protein, Iba1, which is restrictedly expressed in macrophages/microglia (9), and showed that the expression of Iba1 is up-regulated in activated microglia following facial nerve axotomy (10). In our recent study, Iba1 was further characterized by using a microglial cell line MG5 (11) and loss of function Iba1 mutants, and it was demonstrated that mutant Iba1 effectively suppresses the membrane ruffling produced by stimulation with macrophage colony-stimulating factor (M-CSF) or by expression of dominant active RacV12 (12). These observations suggested that Iba1 was involved in the molecular basis of membrane ruffling of macrophages/microglia and interacted with the signaling of Rac, which is a key molecule in controlling membrane ruffling also in macrophages (13). Iba1 is therefore considered to be one of the candidate molecules underlying the extremely motile property of macrophages/microglia.

In this study, to address this hypothesis, we introduced Iba1 in Swiss 3T3 fibroblasts, porcine aorta endothelial (PAE) cells, and Chinese hamster ovary (CHO) cells, none of which expresses endogenous Iba1, and examined the formation of membrane ruffles, chemotaxis, and profiles of intracellular signaling molecules, including PDGFR, phosphatidylinositol-3 kinase (PI3K), phospholipase C-gamma (PLC-gamma ), and Rac.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Transfection-- Swiss 3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). Mouse iba1 cDNA (9) was inserted into the tetracycline-regulated expression vector pTet-Splice (Invitrogen) to construct pTet-iba1. The cells were transfected with pTet-iba1, transactivator pTet-tTAK, and pSV2-neo by calcium phosphate coprecipitation, and stably transfected clones were isolated by selection with 400 µg/ml G418 (Invitrogen).

PAE cells (14), kindly provided by Dr. C.-H. Heldin (Ludwig Institute for Cancer Research, Sweden) and Dr. Koutaro Yokote (Chiba University, Japan) were cultured in Ham's F12 medium (Invitrogen) supplemented with 10% FCS. pLXSN plasmids carrying wild type (WT) and a series of mutant human beta -PDGFRs (15) were kindly provided by Dr. A. Kazlauskas (Schepens Eye Research Institute, Harvard Medical School, Boston, MA). F5 mutant PDGFR, which was constructed by the substitution of phenylalanines for five tyrosine residues that are required for the binding of PI3K, RasGAP, SHP-2, and PLC-gamma 1, is unable to associate with any of these proteins. Add-back mutants of PDGFR were generated by restoring tyrosine residues at individual binding sites for each of the receptor-associated proteins (15). PAE cells were transfected with the tetracycline-regulated Iba1-expressing system and cloned as described above. Subsequently, Iba1-expressing cells were transfected with WT PDGFR or the add-back series of PDGFR mutants by the FuGENE6 transfection reagent (Roche Molecular Biochemicals, Germany) and selected by 5 µg/ml of blasticidin S (Funakoshi, Japan).

PAE transfectants were incubated in Ham's F12 containing 0.5% FCS for 8 h before microinjection of 0.6 µg/µl pFLAG-CMV2 carrying WT and Y771F/Y783F plc-gamma 1 cDNAs, which were kindly provided by Dr. P.-G. Suh (16) (Pohang University of Science and Technology, Korea). Injected PAE cells were maintained for 3 h at 37 °C to induce protein expression. Expression plasmids for glutathione S-transferase (GST)-PLC-gamma 1-2SH2 and GST-PI3-K SH2 (N) (17) were kindly provided by Dr. T. Takenawa and Dr. K. Fukami (Institute of Medical Science, University of Tokyo, Japan). The purified GST fusion proteins were microinjected into the cytosol and incubated for 10 min at 37 °C. The cells were then stimulated with PDGF (50 ng/ml) for 5 min.

CHO cells were maintained in RPMI 1640/Ham's F12/DMEM (2:1:1) medium supplemented with 10% FCS. CHO cells were transiently transfected using LipofectAMINE Plus reagent (Invitrogen) with pLXSN carrying WT PDGFR, pFLAG-CMV2 carrying WT or mutant plc-gamma 1, and pEGFP-C1 (CLONTECH, Palo Alto, CA) carrying WT-iba1 or mutant iba1-(1-115) (12).

A microglial cell line, MG5, was maintained as described previously (12).

Western Blotting-- Cells were lysed in radioimmune precipitation buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 1% Triton-X100, 1% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 20 µM leupeptin, 1 mM sodium vanadate, and 10 µM pepstatin. The lysate was clarified by centrifugation, normalized for protein concentration, and subjected to SDS-PAGE. Separated proteins were electrotransferred to an Immobilon (Millipore, MA) membrane, which was then blocked with 25 mM Tris-HCl, pH 7.5, containing 125 mM NaCl, 0.1% Tween 20, and 4% skim milk. The membrane was incubated with anti-Iba1 antibody (1 µg/ml) (12), then subsequently with a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (1:1500 dilution) (Bio-Rad, CA), and visualized using an ECL Western blotting detection system (Amersham Biosciences, Inc., UK).

Phalloidin Staining and Immunocytochemistry-- Swiss 3T3 or PAE transfectants (1 × 104) were plated on a 13-mm poly-D-lysine (Sigma Chemical Co., St. Louis, MO)-coated glass coverslip, cultured for 2 days, then serum-starved for 13 h in DMEM without FCS (Swiss 3T3) or in Ham's F12 with 0.5% FCS (PAE) in the presence or absence of 0.5 µg/ml tetracycline. The cells were stimulated with human PDGF-BB (UBI, NY), bradykinin (Sigma), or lysophosphatidic acid (LPA) (Sigma), then fixed for 60 min at room temperature with 3% paraformaldehyde in phosphate-buffered saline (PBS). The cells were treated for 5 min with PBS containing 1 mg/ml sodium borohydride, permeabilized for 20 min with PBS containing 0.1% Triton-X100, and blocked for 2 h with PBS containing 3% normal goat serum and 3% bovine serum albumin (BSA) (blocking buffer). The cells were incubated for 13 h at 4 °C in blocking buffer containing 8 µg/ml rabbit anti-Iba1 antibody, then washed with PBS and incubated for 2 h in blocking buffer containing 5 µg/ml fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibody (BioSource, Camarillo, CA) and 6 units/ml Texas Red-conjugated phalloidin (Molecular Probes, Eugene, OR). The cells were then observed with fluorescence microscope AX70 (Olympus, Japan) or confocal laser scanning microscope CLSM2010 (Amersham Biosciences, Inc.).

Chemotaxis Assay-- Cell migration was assayed by a modified Boyden chamber method (18) using a 96-well chemotaxis chamber (Neuro Probe, Cabin John, MD). The lower wells that were filled with 25 µl of DMEM containing 1% BSA and PDGF were overlaid with an 8-µm pore polycarbonate filter (Neuro Probe) pre-coated with 100 µg/ml type I collagen (Cohesion, Palo Alto, CA). Above the membrane, the upper wells were set and loaded with 50 µl of DMEM with 1% BSA containing 3 × 105 trypsin-dispersed cells per milliliter. The chamber was then incubated at 37 °C for 8 h. The cells on the upper side of the filter were scraped off, and the cells that had migrated to the lower side of the filter were fixed and stained in PBS containing 12% formaldehyde, 10% ethanol, and 0.05% crystal violet. Cell numbers were counted in four fields per well. Values are means of triplicate experiments.

Determination of Activated Rac-- The PAK pull-down assay was performed mainly as described previously (12, 19). Cells (106) were lysed in 500 µl of Hepes-buffered saline (25 mM Hepes-NaOH, pH 7.3, 150 mM NaCl, 5 mM MgCl2, 0.5 mM EGTA, 20 mM beta -glycerophosphate, 0.5% Triton-X100, 4% glycerol, 10 mM NaF, 2 mM sodium vanadate, 5 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 10 µM pepstatin, and 0.5 mM dithiothreitol). The lysates were cleared by centrifugation, incubated with GST-PAK fusion protein (12, 19) and glutathione-Sepharose 4B (Amersham Biosciences, Inc.) for 30 min at 4 °C, and subsequently washed in Hepes-buffered saline. Bound activated Rac was visualized by Western blotting with an anti-Rac1 antibody (0.5 µg/ml) and HRP-conjugated anti-mouse goat IgG (1:1500 dilution) using the ECL system.

Immunoprecipitation-- Serum-starved quiescent PAE and M-CSF-starved MG5 cells were stimulated with 50 ng/ml PDGF and 100 ng/ml mouse M-CSF (R&D Systems, Minneapolis, MN), respectively. The cells were lysed in radioimmune precipitation buffer at 4 °C for 20 min. Insoluble material was removed by centrifugation, and the cell lysates were normalized for protein concentration before immunoprecipitation. The lysates were incubated with 1.7 µg/ml anti-PLC-gamma 1 (UBI) or anti-PLC-gamma 2 (Santa Cruz Biotechnology, Santa Cruz, CA) antibody and with protein G-Sepharose beads (Amersham Biosciences, Inc.). The precipitated proteins were then subjected to Western blotting with an anti-phosphotyrosine antibody, 4G10 (Seikagaku, Japan), and HRP-conjugated anti-mouse goat IgG (Amersham Biosciences, Inc.) using the ECL system.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Enhancement of PDGF-induced Membrane Ruffling in Iba1-expressing Swiss 3T3 Cells-- In this study, to analyze the functions of intact Iba1, we transfected a tetracycline-inducible Iba1-expression construct into Swiss 3T3, a fibroblast cell line expressing no endogenous Iba1. As a result, we established five clones of stable transfectants exhibiting inducible Iba1 expression. Immunoblotting with the anti-Iba1 antibody demonstrated that the expression of Iba1 was tightly inhibited under the presence of tetracycline whereas 13 or 22 h after the removal of tetracycline, strong expression of Iba1 was induced (Fig. 1A).


View larger version (81K):
[in this window]
[in a new window]
  		Fig. 1.   Enhanced membrane ruffling of Iba1-expressing Swiss 3T3 cells. A, inducible expression of Iba1 in Swiss 3T3 transfectants. Swiss 3T3 transfectants, after tetracycline removal for indicated times, were lysed and subjected to Western blot analysis with anti-Iba1 antibody. The arrowhead indicates the position of Iba1. B, phalloidin staining of Swiss 3T3 transfectants. Serum-starved transfectants cultured in the presence or absence of tetracycline were stimulated with 100 ng/ml bradykinin, 100 ng/ml LPA, or 3 ng/ml PDGF for 10 min. The cells were then fixed and stained with Texas Red-conjugated phalloidin. Scale bar, 50 µm. C, double staining of Swiss 3T3 transfectants. Iba1-expressing Swiss 3T3 transfectants were stimulated with 3 ng/ml PDGF for 10 min. The cells were fixed and then doubly stained with Texas Red-phalloidin and anti-Iba1 antibody. Scale bar, 50 µm.

In Swiss 3T3 cells, bradykinin, LPA, and PDGF are known to specifically activate Cdc42, Rho, and Rac, respectively, and lead the cells to form filopodia, stress fibers, and membrane ruffles (7). To examine the effects of Iba1 on these structures, the Iba1-inducible cells were serum-starved, stimulated with bradykinin, LPA, and PDGF, and stained with phalloidin to visualize the actin cytoskeleton. When Iba1 expression was suppressed in the presence of tetracycline, the cells formed filopodia, stress fibers, and membrane ruffles in response to bradykinin, LPA, and PDGF, respectively (Fig. 1B), as reported for parent Swiss 3T3 cells (8, 20-22). When Iba1 expression was induced by tetracycline removal, the cells also formed filopodia and stress fibers indistinguishable from those shown in the absence of Iba1 expression after stimulation with bradykinin and LPA (Fig. 1B). By contrast, in response to PDGF, the Iba1-expressing cells formed apparently enhanced membrane ruffles in comparison with the Iba1-nonexpressing cells (Fig. 1B). When the cells were doubly stained with phalloidin and the anti-Iba1 antibody after PDGF stimulation, Iba1 was shown to be localized at the sites of membrane ruffles, together with F-actin (Fig. 1C), but Iba1 did not colocalize with F-actin in filopodia or stress fibers induced by bradykinin or LPA stimulation (data not shown). All other clones of the transfectants exhibited similar enhanced membrane ruffling (data not shown), indicating that Iba1 definitely enhances PDGF-dependent membrane ruffling in Swiss 3T3 transfectants.

Enhanced Chemotaxis of Iba1-expressing Swiss 3T3 Cells-- Because membrane ruffling is considered to be related to cell motility (23), we determined the chemotaxis of Iba1-expressing cells by the Boyden chamber method (18) using PDGF as a chemoattractant. As shown in Fig. 2, Swiss 3T3 parent cells and the Iba1-nonexpressing transfectants showed similar motile responses toward PDGF in a dose-dependent manner, whereas the Iba1-expressing cells exhibited about a 2-fold increase in chemotactic response. Similar results were obtained in all clones of Iba1 transfectants. Tetracycline itself had no effect on PDGF-induced migration in Swiss 3T3 cells (data not shown). These results indicate that Iba1 is also able to enhance the chemotaxis of Swiss 3T3 cells.


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 2.   Enhanced chemotaxis of Iba1-expressing Swiss 3T3 cells. Swiss 3T3 cells (open triangles) and Iba1-transfectans cultured in the presence (open circles) or absence (closed squares) of tetracycline were examined for chemotaxis against PDGF by Boyden chamber. Values are means of triplicate experiments.

PI3K-independent Membrane Ruffling of Iba1-expressing Swiss 3T3 Cells-- The PI3K signaling pathway is reported to be necessary for PDGF-induced membrane ruffling of Swiss 3T3 cells (24). To investigate whether this pathway is also required for the Iba1-dependent enhancement of membrane ruffling, the effect of PI3K inhibitors, wortmannin and LY294002, on PDGF-induced membrane ruffling was examined in both Iba1-nonexpressing and -expressing cells. Without treatment with the inhibitors, both transfectants formed membrane ruffles as a result of PDGF stimulation, but the extent of ruffle formation was greater in Iba1-expressing cells than in Iba1-nonexpressing cells (Fig. 3). With the wortmannin treatment, membrane ruffling of Iba1-nonexpressing transfectants was completely abolished, indicating that the formation of membrane ruffles of Iba1-nonexpressing Swiss 3T3 cells depends totally on the PI3K signaling pathway. By contrast, the Iba1-expressing cells formed obvious membrane ruffles even after wortmannin treatment, indicating that membrane ruffling of Iba1-expressing cells does depend on a certain signaling pathway in addition to PI3K. The same results were obtained using another PI3K inhibitor, LY294002 (Fig. 3). These observations led us to speculate that the enhanced membrane ruffling associated with Iba1 is transduced by a PI3K-independent pathway.


View larger version (84K):
[in this window]
[in a new window]
  		Fig. 3.   Effects of PI3K inhibitors on membrane ruffling of Iba1-expressing Swiss 3T3 cells. Serum-starved Swiss 3T3 transfectants cultured in the presence or absence of tetracycline were pretreated with or without 100 nM wortmannin or 50 µM LY294002 for 30 min and then stimulated with 3 ng/ml PDGF for 10 min. The cells were fixed and stained with Texas Red-phalloidin. Scale bar, 50 µm.

Involvement of PLC-gamma in Membrane Ruffling of Iba1-expressing PAE Cells-- To elucidate the possibility that Iba1 is involved in the PI3K-independent signaling pathway, we utilized the add-back mutants of PDGFR, which were transfected into PAE cells lacking endogenous PDGFR (14). PDGFR associates with various signaling molecules via its autophosphorylated tyrosines. PI3K selectively targets tyrosine at amino acid positions 740 (Tyr-740) and Tyr-751, whereas RasGAP, SHP-2, and PLC-gamma recognize Tyr-771, Tyr-1009, and Tyr-1021, respectively. These signaling molecules are unable to bind to the PDGFR F5 mutant, in which all of the five tyrosines were replaced by phenylalanines (15). PAE cells, which did not express Iba1, were co-transfected with the Iba1-inducible construct and PDGFR mutant-expression vectors. Similar expression levels were seen for all the PDGFRs in the stable transfectants, as measured by fluorescence-activate cell sorting analysis (data not shown). Without stimulation with PDGF, the morphology of the cells was identical in the presence and absence of Iba1 expression (data not shown). When the Iba1-nonexpressing cells were stimulated with PDGF, obvious membrane ruffles were formed in the cells co-transfected with WT PDGFR receptor or Y740/51 mutant, which is capable of PI3K activation, in agreement with a previous report (25). In contrast, cells expressing F5 or Tyr-1021 receptor did not respond to PDGF (Fig. 4). These observations indicate the necessity of PI3K signaling for PDGF-induced membrane ruffling in the absence of Iba1. On the other hand, after the induction of Iba1, apparent membrane ruffles were formed in the cells transfected with Tyr-1021 mutant, capable of activation of PLC-gamma . Membrane ruffling was also detected in the Iba1-expressing cells with the WT or Y740/51 mutant, as in the Iba1-nonexpressing cells (Fig. 4). Cells expressing Tyr-771, Tyr-1009, or kinase inactive receptor (Arg-635) did not show PDGF-induced membrane ruffling regardless of Iba1 expression (data not shown). These observations strongly suggest that PLC-gamma is the key signaling molecule in Iba1-dependent and wortmannin-resistant membrane ruffling.


View larger version (92K):
[in this window]
[in a new window]
  		Fig. 4.   PLC-gamma -dependent membrane ruffling of PDGF-stimulated PAE cells expressing Iba1 and WT or a series of PDGFR mutants. WT-, Y740/51-, Tyr-1021-, and F5-PDGFR-expressing PAE Iba1 transfectants cultured in the presence or absence of tetracycline were each stimulated with 50 ng/ml PDGF for 5 min, then fixed and stained with Texas-Red phalloidin. Scale bar, 50 µm.

Requirement of Iba1 in PLC-gamma -dependent Rac Activation-- Iba1 was recently demonstrated to function together with Rac in the membrane ruffling of microglia, and Rac was shown to be activated during their membrane ruffling (12). To investigate whether Rac is also activated in Iba1- and PLC-gamma -dependent membrane ruffling, the activation of Rac was monitored by pull-down assay with the Cdc42/Rac interactive binding domain of PAK (19) using PAE transfectants. In the absence of Iba1, PDGF stimulation efficiently converted Rac into the GTP-bound form in the cells expressing WT and the Y740/51 mutant but not in the cells expressing the Tyr-1021 (Fig. 5A), Tyr-771, or Tyr-1009 (data not shown) mutant. All lysates contained equal amounts of total Rac. These results indicate that Rac was activated through the PI3K-dependent pathway in the absence of Iba1. However, in the presence of Iba1, in addition to WT- or Y740/51-expressing cells, Tyr-1021-expressing cells also showed Rac activation in response to PDGF (Fig. 5B). These observations indicate the existence of an Iba1- and PLC-gamma -dependent Rac-activating pathway that triggers the formation of membrane ruffles.


View larger version (31K):
[in this window]
[in a new window]
  		Fig. 5.   PLC-gamma -dependent Rac activation in Iba1-expressing PAE cells. PAE Iba1 transfectants expressing WT or PDGFR mutants cultured in the presence (A) or absence (B) of tetracycline were stimulated with or without 50 ng/ml PDGF for 2 min. After stimulation, the cells were lysed and GTP-bound active Rac was precipitated with GST-PAK-bound glutathione-Sepharose-4B beads. GTP-bound Rac (upper panels) and total Rac in the cell lysates (lower panels) were identified by Western blotting with an anti-Rac antibody. Arrowheads indicate positions of Rac. Highly intense bands migrating around 29 kDa correspond to GST-PAK protein.

Inhibitory Effects of PLC-gamma Mutants on Iba1-dependent Membrane Ruffling and Rac Activation-- To confirm the involvement of PLC-gamma in Iba1-dependent and PI3K-independent Rac activation, we investigated the effects of PLC-gamma mutants that act as dominant negative forms against endogenous PLC-gamma . PLC-gamma 1-Y771F/Y783F had phenylalanines substituted for tyrosines 771 and 783 and obtained the ability to specifically suppress the activity of endogenous PLC-gamma 1 (16). FLAG-tagged PLC-gamma 1 WT or Y771F/Y783F mutant was expressed in the PAE cells expressing both PDGFR Tyr-1021 and Iba1. Subsequently, the cells were stimulated with PDGF and stained with phalloidin. Typical membrane ruffles were formed in WT PLC-gamma -expressing cells that were located by the marker signal of co-expressed EGFP (Fig. 6A, upper panel). By contrast, PDGF-induced membrane ruffling was inhibited in PLC-gamma 1-Y771F/Y783F-expressing cells (Fig. 6A, lower panel). When the PLC-gamma 1-expressing cells were located with an anti-FLAG antibody, the cells that were recognized corresponded perfectly to the EGFP-expressing cells (data not shown). GST-PLC-gamma 1-2SH2 is a fusion protein of GST and a PLC-gamma 1 fragment containing two SH2 domains but no catalytic domain and is able to specifically inhibit PLC-gamma 1 signaling, whereas GST-PI3K SH2 (N) contains the PI3K N-terminal SH2 domain but does not suppress PLC-gamma signaling (17). Into the PAE cells expressing both PDGFR Tyr-1021 and Iba1, we microinjected GST-PLC-gamma 1-2SH2 or GST-PI3K SH2(N) fusion protein together with FITC-conjugated dextran to mark the cells that were injected. The cells injected with GST-PLC-gamma 1-2SH2 fusion protein were stimulated with PDGF and stained with phalloidin. PDGF-induced membrane ruffling was inhibited in the GST-PLC-gamma 1-2SH2- injected cells, whereas the cells injected with GST-PI3K SH2 (N) were not inhibited.


View larger version (48K):
[in this window]
[in a new window]
  		Fig. 6.   Inhibitory effects of PLC-gamma mutants on membrane ruffling and Rac activation. A, serum-starved Iba1- and Tyr-1021 PDGFR-expressing PAE cells were microinjected into the nucleus with pFLAG-CMV2 carrying WT or Y771F/Y783F PLC-gamma 1 cDNA together with pEGFP-C1, and incubated for 3 h to induce protein expression. B, serum-starved Iba1- and Tyr-1021 PDGFR-expressing PAE cells were microinjected into the cytosol with GST-PLC-gamma 1-2SH2 or GST-PI3K SH2 (N) fusion proteins together with FITC-dextran and incubated for 10 min. Both types of cells were stimulated with 50 ng/ml PDGF for 5 min, fixed, and stained with Texas Red-phalloidin (A and B, left panels). Injected cells were located by fluorescence of epidermal growth factor protein and FITC-dextran (A and B, mid panels). Scale bar, 20 µm. C, CHO cells expressing PDGFR, and WT (W) or mutant (m) of Iba1 and FLAG-tagged PLC-gamma 1 were stimulated with 50 ng/ml PDGF for 2 min, lysed, and examined by GST-PAK pull-down assay, as shown in Fig. 5. Rac1, Iba1, PLC-gamma 1, and PDGFR in cell lysates were identified by Western blotting with anti-Rac1, anti-Iba1, anti-FLAG, and anti-PDGFR antibodies, respectively.

Next we investigated whether PLC-gamma 1-Y771F/Y783F mutant blocked Iba1-dependent Rac activation. We induced the transient expression of WT PDGFR, and WT or mutant Iba1 and PLC-gamma 1 in CHO cells, which lacked PDGFR expression (26), and then examined Rac activity by GST-PAK pull-down assay. Under the conditions we used, in CHO cells expressing PDGFR only, Rac was not activated by PDGF stimulation (Fig. 6B, lanes 1 and 2), and membrane ruffles were not formed (data not shown). In the cells expressing both Iba1 and PDGFR, Rac was activated (lanes 3 and 4) and membrane ruffles were formed (data not shown) in response to PDGF, indicating that CHO cells also contain the pathway exerting Iba1-dependent Rac activation. In cells expressing Iba1 and PDGFR, expression of the PLC-gamma 1-Y771F/Y783F mutant completely suppressed Rac activation in response to PDGF (lanes 13 and 14). These results indicate the specific involvement of PLC-gamma in Iba1-dependent Rac activation.

When the cells expressing PDGFR and PLC-gamma 1 were stimulated with PDGF, Rac activation was induced (lanes 7 and 8), whereas this Rac activation was inhibited by additional expression of mutant Iba1-(1-115), which effectively suppresses the membrane ruffling of MG5 cells (12) (lanes 11 and 12). PDGF did not cause Rac activation in the cells expressing PDGFR and mutant PLC-gamma 1-Y771F/Y783F or mutant Iba1-(1-115) (lanes 5, 6, 9, and 10). These results suggest a functional link between PLC-gamma and Iba1 in membrane ruffling and Rac activation.

Ligand-induced Tyrosine Phosphorylation of PLC-gamma -- During the ligand-induced activation process, PLC-gamma is known to be phosphorylated by receptor tyrosine kinases (27); thus we next analyzed the tyrosine phosphorylation of PLC-gamma in response to growth factors stimulating membrane ruffling. In mammals, two types of PLC-gamma are known, PLC-gamma 1 and PLC-gamma 2. As shown in Fig. 7, PAE cells selectively express PLC-gamma 1, whereas the microglial cell line MG5 predominantly expresses PLC-gamma 2. The PAE cells expressing Iba1 and WT PDGFR were stimulated with PDGF, immunoprecipitated with an anti-PLC-gamma 1 antibody, and immunoblotted with an anti-phosphotyrosine antibody, 4G10. Phosphorylation of PLC-gamma 1 was detected 30 s after PDGF stimulation, and after 2 min, the signal was greatly intensified (Fig. 7A). When MG5 cells were stimulated with M-CSF, phosphorylation of PLC-gamma 1 was, by contrast, undetectable even after 2 min. On the other hand, phosphorylation of PLC-gamma 2 was clearly detected in MG5 cells after M-CSF stimulation (Fig. 7B). These observations indicate that, in response to peptide growth factors, PLC-gamma 1 and - gamma 2 are phosphorylated in these Iba1-expressing cells, including macrophages/microglia, and exert their activity during membrane ruffling.


View larger version (35K):
[in this window]
[in a new window]
  		Fig. 7.   Ligand-dependent tyrosine phosphorylation of PLC-gamma . Serum-starved WT PDGFR-expressing PAE and M-CSF-starved MG5 cells were stimulated for the indicated times with or without 50 ng/ml PDGF and 100 ng/ml M-CSF, respectively. The cells were lysed and immunoprecipitated with an anti-PLC-gamma 1 (A) or anti-PLC-gamma 2 (B) antibody. Samples were subjected to Western blotting with an anti-phosphotyrosine antibody, 4G10 (upper panels) and sequentially reprobed with anti-PLC-gamma 1 (A) and anti-PLC-gamma 2 (B) antibodies (lower panels). Arrowheads indicate positions of PLC-gamma 1 and PLC-gamma 2.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In our previous report (12), Iba1 was revealed to be a macrophage/microglia-specific EF hand protein responsible for M-CSF- and Rac-induced membrane ruffling. In this study, to analyze the functions of Iba1 in more detail, we introduced an Iba1 expression construct into Swiss 3T3, PAE, and CHO cells, stimulated the cells with PDGF, and examined signaling profiles leading to Rac activation and subsequent membrane ruffling. As a result, we elucidated a novel signaling pathway where, in the presence of Iba1, Rac is activated in a manner dependent on PLC-gamma .

In response to PDGF, Iba1-expressing Swiss 3T3 cells exhibited enhanced membrane ruffling and increased chemotactic activity in comparison with Iba1-nonexpressing Swiss 3T3 cells (Figs. 1B and 2). Recent experiments have suggested that PI3K plays a role in receptor tyrosine kinase-mediated membrane ruffling and chemotaxis in many types of cells (24, 25, 28-30). Among them, in Swiss 3T3 and PAE cells, PI3K is reported to be exclusively responsible for Rac activation (24, 25, 31, 32). Indeed, treatment with wortmannin or LY294002 effectively suppressed the PDGF-induced membrane ruffling of Iba1-nonexpressing Swiss 3T3 cells (Fig. 3). In addition, in the absence of Iba1, PDGF could not induce Rac activation and membrane ruffling in PAE cells expressing PDGFR mutants incapable of activating of PI3K (Fig. 4 and 5A). By contrast, membrane ruffling of Iba1-expressing Swiss 3T3 cells was not significantly inhibited by wortmannin or LY294002 (Fig. 3). Furthermore, in the presence of Iba1, PDGF stimulation actually induced Rac activation and membrane ruffling in PAE cells expressing the PDGFR mutant that is capable of activating PLC-gamma but not PI3K (Figs. 4 and 5). These findings indicate the existence of an Iba1-dependent, PI3K-independent pathway leading to Rac activation and membrane ruffling, and suggest the involvement of PLC-gamma in this pathway. In fact, dominant negative forms of PLC-gamma clearly inhibited Iba1-dependent membrane ruffling (Fig. 6, A and B) and Rac activation (Fig. 6C). PLC-gamma was further shown to be phosphorylated during membrane ruffling (Fig. 7). These observations indicate that PLC-gamma is specifically involved in Iba1-dependent Rac activation and membrane ruffling and that Iba1 is the molecule responsible for connecting the signaling pathways of Rac and PLC-gamma .

Recent studies have provided increased evidence for PI3K-independent Rac activation. Wortmannin-treated macrophages still induced ruffling at the dorsal surface after M-CSF stimulation (33). N-Formyl-Met-Leu-Phe induced wortmannin- and LY294002-resistant Rac activation in neutrophils (34-36). In our studies, pretreatment with wortmannin did not block M-CSF-induced membrane ruffling of microglial cell line MG5 and primarily cultured microglia (data not shown). These observations strongly suggest the existence of a PI3K-independent pathway leading to Rac activation and membrane ruffling. Kundra et al. (37) showed that the mutant PDGFR, which lacks the binding site for PLC-gamma , could not transduce chemotactic signals when expressed in TRMP cells. Expression of another PDGFR mutant that induces increased PLC-gamma activation by PDGF showed increased chemotactic activity in PAE cells (31). It is probable that PI3K and/or PLC-gamma are required for chemotaxis by PDGFR. Hashimoto et al. (38) reported that both PLC-gamma and Rac are involved in B cell antigen receptor-induced activation of MAP kinases, suggesting that PLC-gamma and Rac are able to work cooperatively in a common signaling process. However, to date, the molecules that link PLC-gamma and Rac pathways have been completely unknown. Iba1 or unknown Iba1-related molecules should, at least in part, underlie PI3K-independent, PLC-gamma -dependent pathways that induce Rac activation.

CHO cells expressing PDGFR did not respond to PDGF in Rac activation; however, overexpression of PLC-gamma induced Rac activation in response to PDGF (Fig. 6C). Furthermore, once-promoted Rac activation was suppressed by additional expression of a repressive mutant of Iba1. In conjunction with the parallel results shown with mutant PLC-gamma and WT Iba1, the functions of Iba1 and PLC-gamma in Rac activation are considered to be closely related. It is likely that Iba1 may modulate the PLC-gamma -dependent signaling pathway. During membrane ruffling, complicated machineries are constructed by Iba1, PLC-gamma , and other signaling molecules, including Rac, which co-operate in interactions between each them. However, unfortunately, we have no clear evidence to demonstrate direct binding among Iba1, PLC-gamma , and Rac. We are now investigating conditions that would support their association.

Activated PLC-gamma translocates to the inside surface of the cell membrane and catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to form diacylglycerol and inositol 1,4,5-trisphosphate, which are capable of activating protein kinase C (PKC) and mobilizing intracellular calcium ([Ca2+]i), respectively. In our preliminary experiments, membrane ruffling of Iba1-expressing Swiss 3T3 cells was not suppressed by pre-treatment with phorbol ester to down-regulate PKC (data not shown). On the other hand, M-CSF-induced membrane ruffling of MG5 cells was accompanied by [Ca2+]i spikes and was completely inhibited by chelation of cytoplasmic free calcium with O,O'-Bis(2-aminophenyl)ethylene glycol-N,N,N',N'-tetraacetic acid, tetraacetoxyethyl ester (12). Iba1 actually possesses calcium-binding activity; furthermore, an Iba1 mutant without calcium-binding activity suppressed M-CSF-induced membrane ruffling (12). Several pieces of evidence indicate the significance of calcium signaling in actin remodeling (3, 39). These observations point to the importance of [Ca2+]i in Iba1-related membrane ruffling and suggest that [Ca2+]i mobilization is a strong candidate for linking PLC-gamma and Iba1 molecules.

Iba1 also translocates to the cell membrane during membrane ruffling (12). In our preliminary studies, Iba1 was demonstrated to bind to phosphatidylserine in the presence of calcium (data not shown), suggesting that Iba1 translocates to the phosphatidylserine-rich inner surface of the cell membrane in a calcium-dependent manner. Iba1 was further shown to bind to phosphatidylinositol 4,5-bisphosphate, the significant substrate of PLC-gamma . Iba1 may, directly or indirectly, support translocation of PLC-gamma to the cell membrane and its substrate, and may induce Rac activation by potentiating the activity of PLC-gamma . Indeed, overexpression of PLC-gamma in CHO cells enhanced activation of Rac (Fig. 6C). Although it seems rather inconsistent with data presented here, mutant Iba1 suppressed membrane ruffling induced by activated RacV12 in our previous paper (12). Preliminarily, mutant Iba1 exhibited loss of ability to translocate, localized constitutively to the cell membrane, and disrupted surrounding actin architecture (data not shown). RacV12 showed a tendency to be excluded from the site where mutant Iba1 accumulated (data not shown). In our hypothesis, Iba1 may also function in translocation of Rac, and mutant Iba1 may suppress membrane ruffling by inhibiting appropriate translocation of RacV12.

Macrophages are extremely motile. To express this phenotype, macrophages have to contain highly integrated mechanisms that regulate dynamic reorganization of the actin cytoskeleton. In addition to the well-known PI3K-organized Rac regulation, Iba1- and PLC-gamma -based mechanisms are likely to direct Rac activity in macrophages. Herein, we have shown direct evidence that PLC-gamma activates Rac and causes membrane ruffling in the presence of Iba1. This finding indicates that macrophages/microglia have at least dual control pathways to regulate Rac activity and exert extreme motile activity by these systems. In fact, some studies suggest cross-talk between PI3K and PLC-gamma pathways (31, 40, 41). Iba1 is therefore considered to be a crucial molecule for the function of activated macrophages/microglia. Further studies are definitely required to determine the precise molecular mechanisms underlying Iba1- and PLC-gamma -dependent activation of Rac and membrane ruffling.

    ACKNOWLEDGEMENTS

We thank Dr. Carl-Henrik Heldin (Ludwig Institute for Cancer Research) and Dr. Koutaro Yokote (Chiba University) for providing us with PAE cells, Dr. Andrius Kazlauskas (Harvard Medical School) for pLXSN plasmids carrying the human WT or mutant beta -PDGFRs, Dr. Pann-Ghill Suh (Pohang University of Science and Technology) for pFLAG-CMV2 plasmids carrying the WT or mutant PLC-gamma 1, and Dr. Tadaomi Takenawa and Dr. Kiyoko Fukami (Institute of Medical Science, University of Tokyo) for expression plasmids of GST-PLCgamma 1-2SH2 and GST-PI3-K SH2(N). We also thank Dr. Koutaro Yokote and Dr. Kiyoko Fukami for helpful discussion.

    FOOTNOTES

* This work was supported by the Organization for Pharmaceutical Safety and Research, by a grant from the Japanese Ministry of Health, Labour and Welfare, and by a grant-in-aid for scientific research on priority areas from the Japanese Ministry of Education, Science, Sports, Culture and Technology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Tel.: 81-42-346-1721; Fax: 81-42-346-1751; E-mail:kohsaka@ncnp.go.jp.

Published, JBC Papers in Press, March 26, 2002, DOI 10.1074/jbc.M109218200

    ABBREVIATIONS

The abbreviations used are: PDGFR, platelet-derived growth factor receptor; M-CSF, macrophage colony-stimulating factor; PAE, porcine aorta endothelial; CHO, Chinese hamster ovary; PI3K, phosphatidylinositol-3 kinase; PLC, phospholipase C; FCS, fetal bovine serum; WT, wild type; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; HRP, horseradish peroxidase; LPA, lysophosphatidic acid; PBS, phosphate-buffered saline; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; PKC, protein kinase C; [Ca2+]i, intracellular calcium; PAK, p21-activated kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Stossel, T. P. (1993) Science 260, 1086-1094[Abstract/Free Full Text]
2. Mitchison, T. J., and Cramer, L. P. (1996) Cell 84, 371-379[CrossRef][Medline] [Order article via Infotrieve]
3. Hartwig, J. H., and Yin, H. L. (1988) Cell Motil. Cytoskeleton 10, 117-125[CrossRef][Medline] [Order article via Infotrieve]
4. Downey, G. P. (1994) Curr. Opin. Immunol. 6, 113-124[CrossRef][Medline] [Order article via Infotrieve]
5. Nakajima, K., and Kohsaka, S. (1993) Neurosci. Res. 7, 187-203
6. Kreutzberg, G. W. (1996) Trends Neurosci. 19, 312-318[CrossRef][Medline] [Order article via Infotrieve]
7. Hall, A. (1998) Science 279, 509-514[Abstract/Free Full Text]
8. Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D., and Hall, A. (1992) Cell 70, 401-410[CrossRef][Medline] [Order article via Infotrieve]
9. Imai, Y., Ibata, I., Ito, D., Ohsawa, K., and Kohsaka, S. (1996) Biochem. Biophys. Res. Commun. 224, 855-862[CrossRef][Medline] [Order article via Infotrieve]
10. Ito, D., Imai, Y., Ohsawa, K., Nakajima, K., Fukuuchi, Y., and Kohsaka, S. (1998) Brain Res. Mol. Brain Res. 57, 1-9[Medline] [Order article via Infotrieve]
11. Ohsawa, K., Imai, Y., Nakajima, K., and Kohsaka, S. (1997) Glia 21, 285-298[CrossRef][Medline] [Order article via Infotrieve]
12. Ohsawa, K., Imai, Y., Kanazawa, H., Sasaki, Y., and Kohsaka, S. (2000) J. Cell Sci. 113, 3073-3084[Abstract]
13. Allen, W. E., Jones, G. E., Pollard, J. W., and Ridley, A. J. (1997) J. Cell Sci. 110, 707-720[Abstract]
14. Westermark, B., Siegbahn, A., Heldin, C.-H., and Claesson-Welsh, L. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 128-132[Abstract/Free Full Text]
15. Valius, M., and Kazlauskas, A. (1993) Cell 73, 321-334[CrossRef][Medline] [Order article via Infotrieve]
16. Kim, H. K., Kim, J. W., Zilberstein, A., Margolis, B., Kim, J. G., Schlessinger, J., and Rhee, S. G. (1991) Cell 65, 435-441[CrossRef][Medline] [Order article via Infotrieve]
17. Homma, Y., and Takenawa, T. (1992) J. Biol. Chem. 267, 21844-21849[Abstract/Free Full Text]
18. Yokote, K., Mori, S., Siegbahn, A., Rönnstrand, L., Wernstedt, C., Heldin, C.-H., and Claesson-Welsh, L. (1996) J. Biol. Chem. 271, 5101-5111[Abstract/Free Full Text]
19. Manser, E., Loo, T.-H., Koh, C.-G., Zhao, Z.-S., Chen, X.-Q., Tan, L., Tan, I., Leung, T., and Lim, L. (1998) Mol. Cell 1, 183-192[CrossRef][Medline] [Order article via Infotrieve]
20. Ridley, A. J., and Hall, A. (1992) Cell 70, 389-399[CrossRef][Medline] [Order article via Infotrieve]
21. Kozma, R., Ahmed, S., Best, A., and Lim, L. (1995) Mol. Cell. Biol. 15, 1942-1952[Abstract]
22. Nobes, C. D., and Hall, A. (1995) Cell 81, 53-62[CrossRef][Medline] [Order article via Infotrieve]
23. Sánchez-Madrid, F., and del Pozo, M. A. (1999) EMBO J. 18, 501-511[CrossRef][Medline] [Order article via Infotrieve]
24. Nobes, C. D., Hawkins, P., Stephens, L., and Hall, A. (1995) J. Cell Sci. 108, 225-233[Abstract]
25. Wennström, S., Siegbahn, A., Yokote, K., Arvidsson, A.-K., Heldin, C.-H., Mori, S., and Claesson-Welsh, L. (1994) Oncogene 9, 651-660[Medline] [Order article via Infotrieve]
26. Ma, Y.-H., Reusch, H. P., Wilson, E., Escobedo, J. A., Fantl, W. J., Williams, L. T., and Ives, H. E. (1994) J. Biol. Chem. 269, 30734-30739[Abstract/Free Full Text]
27. Mustelin, T., Coggeshall, K. M., Isakov, N., and Altman, A. (1990) Science 247, 1584-1587[Abstract/Free Full Text]
28. Kotani, K., Yonezawa, K., Hara, K., Ueda, H., Kitamura, Y., Sakaue, H., Ando, A., Chavanieu, A., Calas, B., Grigorescu, F., Nishiyama, M., Waterfield, M. D., and Kasuga, M. (1994) EMBO J. 13, 2313-2321[Medline] [Order article via Infotrieve]
29. Arrieumerlou, C., Donnadieu, E., Brennan, P., Keryer, G., Bismuth, G., Cantrell, D., and Trautmann, A. (1998) Eur. J. Immunol. 28, 1877-1885[CrossRef][Medline] [Order article via Infotrieve]
30. Hooshmand-Rad, R., Claesson-Welsh, L., Wennström, S., Yokote, K., Siegbahn, A., and Heldin, C.-H. (1997) Exp. Cell Res. 234, 434-441[CrossRef][Medline] [Order article via Infotrieve]
31. Rönnstrand, L., Siegbahn, A., Rorsman, C., Johnell, M., Hansen, K., and Heldin, C.-H. (1999) J. Biol. Chem. 274, 22089-22094[Abstract/Free Full Text]
32. Hawkins, P. T., Eguinoa, A., Qiu, R.-G., Stokoe, D., Cooke, F. T., Walters, R., Wennström, S., Claesson-Welsh, L., Evans, T., Symons, M., and Stephens, L. (1995) Curr. Biol. 5, 393-403[CrossRef][Medline] [Order article via Infotrieve]
33. Araki, N., Johnson, M. T., and Swanson, J. A. (1996) J. Cell Biol. 135, 1249-1260[Abstract/Free Full Text]
34. Benard, V., Bohl, B. P., and Bokoch, G. M. (1999) J. Biol. Chem. 274, 13198-13204[Abstract/Free Full Text]
35. Akasaki, T., Koga, H., and Sumimoto, H. (1999) J. Biol. Chem. 274, 18055-18059[Abstract/Free Full Text]
36. Geijsen, N., van Delft, S., Raaijmakers, J. A. M., Lammers, J.-W., Collard, J. G., Koenderman, L., and Coffer, P. J. (1999) Blood 94, 1121-1130[Abstract/Free Full Text]
37. Kundra, V., Escobedo, J. A., Kazlauskas, A., Kim, H. K., Rhee, S. G., Williams, L. T., and Zetter, B. R. (1994) Nature 367, 474-476[CrossRef][Medline] [Order article via Infotrieve]
38. Hashimoto, A., Okada, H., Jiang, A., Kurosaki, M., Greenberg, S., Clark, E. A., and Kurosaki, T. (1998) J. Exp. Med. 188, 1287-1295[Abstract/Free Full Text]
39. Janmey, P. A. (1994) Annu. Rev. Physiol. 56, 169-191[Medline] [Order article via Infotrieve]
40. Bae, Y. S., Cantley, L. G., Chen, C. S., Kim, S. R., Kwon, K. S., and Rhee, S. G. (1998) J. Biol. Chem. 273, 4465-4469[Abstract/Free Full Text]
41. Falasca, M., Logan, S. K., Lehto, V. P., Baccante, G., Lemmon, M. A., and Schlessinger, J. (1998) EMBO J. 17, 414-422[CrossRef][Medline] [Order article via Infotrieve]

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 StrokeHome page
K. Hayakawa, K. Mishima, M. Nozako, M. Hazekawa, S. Mishima, M. Fujioka, K. Orito, N. Egashira, K. Iwasaki, and M. Fujiwara
Delayed Treatment With Minocycline Ameliorates Neurologic Impairment Through Activated Microglia Expressing a High-Mobility Group Box1-Inhibiting Mechanism
Stroke, March 1, 2008; 39(3): 951 - 958.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
L. Esposito, J. Raber, L. Kekonius, F. Yan, G.-Q. Yu, N. Bien-Ly, J. Puolivali, K. Scearce-Levie, E. Masliah, and L. Mucke
Reduction in mitochondrial superoxide dismutase modulates Alzheimer's disease-like pathology and accelerates the onset of behavioral changes in human amyloid precursor protein transgenic mice.
J. Neurosci., May 10, 2006; 26(19): 5167 - 5179.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
B. Janji, A. Giganti, V. De Corte, M. Catillon, E. Bruyneel, D. Lentz, J. Plastino, J. Gettemans, and E. Friederich
Phosphorylation on Ser5 increases the F-actin-binding activity of L-plastin and promotes its targeting to sites of actin assembly in cells.
J. Cell Sci., May 1, 2006; 119(Pt 9): 1947 - 1960.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. Villeneuve, P. Tremblay, and L. Vallieres
Tumor Necrosis Factor Reduces Brain Tumor Growth by Enhancing Macrophage Recruitment and Microcyst Formation
Cancer Res., May 1, 2005; 65(9): 3928 - 3936.
[Abstract] [Full Text] [PDF]

Home page