The Mechanism of CSF-1-induced Wiskott-Aldrich Syndrome Protein Activation in Vivo
2009; Elsevier BV; Volume: 284; Issue: 35 Linguagem: Inglês
10.1074/jbc.m109.036384
ISSN1083-351X
AutoresMichael Cammer, Jean-Claude Gevrey, Mike Lorenz, Athanassios Dovas, John S. Condeelis, Dianne Cox,
Tópico(s)3D Printing in Biomedical Research
ResumoA role for Wiskott-Aldrich syndrome protein (WASP) in chemotaxis to various agents has been demonstrated in monocyte-derived cell types. Although WASP has been shown to be activated by multiple mechanisms in vitro, it is unclear how WASP is regulated in vivo. A WASP biosensor (WASPbs), which uses intramolecular fluorescence resonance energy transfer to report WASP activation in vivo, was constructed, and following transfection of macrophages, activation of WASPbs upon treatment with colony-stimulating factor-1 (CSF-1) was detected globally as early as 30 s and remained localized to protrusive regions at later time points. Similar results were obtained when endogenous WASP activation was determined using conformation-sensitive antibodies. In vivo CSF-1-induced WASP activation was fully Cdc42-dependent. Activation of WASP in response to treatment with CSF-1 was also shown to be phosphatidylinositol 3-kinase-dependent. However, treatment with the Src family kinase inhibitors PP2 or SU6656 or disruption of the major tyrosine phosphorylation site of WASPbs (Y291F mutation) did not reduce the level of CSF-1-induced WASP activation. Our results indicate that WASP activation downstream of CSF-1R is phosphatidylinositol 3-kinase- and Cdc42-dependent consistent with an involvement of these molecules in macrophage migration. However, although tyrosine phosphorylation of WASP has been proposed to stimulate WASP activity, we found no evidence to indicate that this occurs in vivo. A role for Wiskott-Aldrich syndrome protein (WASP) in chemotaxis to various agents has been demonstrated in monocyte-derived cell types. Although WASP has been shown to be activated by multiple mechanisms in vitro, it is unclear how WASP is regulated in vivo. A WASP biosensor (WASPbs), which uses intramolecular fluorescence resonance energy transfer to report WASP activation in vivo, was constructed, and following transfection of macrophages, activation of WASPbs upon treatment with colony-stimulating factor-1 (CSF-1) was detected globally as early as 30 s and remained localized to protrusive regions at later time points. Similar results were obtained when endogenous WASP activation was determined using conformation-sensitive antibodies. In vivo CSF-1-induced WASP activation was fully Cdc42-dependent. Activation of WASP in response to treatment with CSF-1 was also shown to be phosphatidylinositol 3-kinase-dependent. However, treatment with the Src family kinase inhibitors PP2 or SU6656 or disruption of the major tyrosine phosphorylation site of WASPbs (Y291F mutation) did not reduce the level of CSF-1-induced WASP activation. Our results indicate that WASP activation downstream of CSF-1R is phosphatidylinositol 3-kinase- and Cdc42-dependent consistent with an involvement of these molecules in macrophage migration. However, although tyrosine phosphorylation of WASP has been proposed to stimulate WASP activity, we found no evidence to indicate that this occurs in vivo. Macrophages, terminally differentiated cells of the mononuclear phagocytic lineage, are found throughout the body and play important roles in normal tissue development and immune defense. However, in certain circumstances, excessive recruitment of macrophages has been shown to participate in the progression of several diseases, inflammatory (rheumatoid arthritis) or metabolic (atherosclerosis), as well as in tumor progression (1.Campbell I.K. Rich M.J. Bischof R.J. Hamilton J.A. J. Leukoc. Biol. 2000; 68: 144-150PubMed Google Scholar, 2.Smith J.D. Trogan E. Ginsberg M. Grigaux C. Tian J. Miyata M. Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 8264-8268Crossref PubMed Scopus (575) Google Scholar, 3.Lin E.Y. Nguyen A.V. Russell R.G. Pollard J.W. J. Exp. Med. 2001; 193: 727-740Crossref PubMed Scopus (1307) Google Scholar). Importantly expression of colony-stimulating factor-1 (CSF-1), 4The abbreviations used are: CSF-1colony-stimulating factor-1CFPcyan fluorescent proteinFRETfluorescence resonance energy transferPI3Kphosphatidylinositol 3-kinaseWASPWiskott-Aldrich syndrome proteinWASPbsWASP biosensorCSAWASP/N-WASP conformation-sensitive antibodyYFPyellow fluorescent proteinWAVEWiskott-Aldrich syndrome verprolin-homologousVCAverprolin homology, cofilin-like, and acidic regionN-WASPneuronal WASPSH3Src homology 3. the most pleiotropic macrophage growth factor, has been correlated with the progression of these disease states (for a review, see Ref. 4.Pixley F.J. Stanley E.R. Trends Cell Biol. 2004; 14: 628-638Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar). Inhibition of undesirable macrophage recruitment to specific sites in response to CSF-1 is therefore an attractive goal for therapies (5.Aharinejad S. Sioud M. Lucas T. Abraham D. Methods Mol. Biol. 2007; 361: 227-238PubMed Google Scholar). colony-stimulating factor-1 cyan fluorescent protein fluorescence resonance energy transfer phosphatidylinositol 3-kinase Wiskott-Aldrich syndrome protein WASP biosensor WASP/N-WASP conformation-sensitive antibody yellow fluorescent protein Wiskott-Aldrich syndrome verprolin-homologous verprolin homology, cofilin-like, and acidic region neuronal WASP Src homology 3. In addition to stimulating survival, proliferation, and differentiation of monocytes and macrophages, CSF-1 is also a potent chemotactic factor inducing the migration of these cell types (for a review, see Ref. 4.Pixley F.J. Stanley E.R. Trends Cell Biol. 2004; 14: 628-638Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar). CSF-1 stimulation leads to the rapid production of F-actin-rich protrusions and the spreading and migration of macrophages (4.Pixley F.J. Stanley E.R. Trends Cell Biol. 2004; 14: 628-638Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar). All CSF-1 effects are mediated through its tyrosine kinase receptor (CSF-1R), which upon activation leads to phosphorylation of tyrosine residues in a number of signaling molecules. Downstream molecules essential for macrophage migration in response to CSF-1 include phosphatidylinositol 3-kinase (PI3K) isoforms β and δ (6.Vanhaesebroeck B. Jones G.E. Allen W.E. Zicha D. Hooshmand-Rad R. Sawyer C. Wells C. Waterfield M.D. Ridley A.J. Nat. Cell Biol. 1999; 1: 69-71Crossref PubMed Scopus (201) Google Scholar, 7.Jones G.E. Prigmore E. Calvez R. Hogan C. Dunn G.A. Hirsch E. Wymann M.P. Ridley A.J. Exp. Cell Res. 2003; 290: 120-131Crossref PubMed Scopus (95) Google Scholar). PI3K may potentially regulate migration through the activation of guanine nucleotide exchange factor activity to Rac1 and Cdc42, which are required for CSF-1-elicited protrusions (8.Cox D. Chang P. Zhang Q. Reddy P.G. Bokoch G.M. Greenberg S. J. Exp. Med. 1997; 186: 1487-1494Crossref PubMed Scopus (371) Google Scholar, 9.Allen W.E. Jones G.E. Pollard J.W. Ridley A.J. J. Cell Sci. 1997; 110: 707-720Crossref PubMed Google Scholar) and chemotaxis (10.Allen W.E. Zicha D. Ridley A.J. Jones G.E. J. Cell Biol. 1998; 141: 1147-1157Crossref PubMed Scopus (445) Google Scholar). The major means by which Rac and Cdc42 regulate the Arp2/3 complex is through the Wiskott-Aldrich syndrome protein/Wiskott-Aldrich syndrome verprolin-homologous (WASP/WAVE) family of proteins (11.Takenawa T. Miki H. J. Cell Sci. 2001; 114: 1801-1809Crossref PubMed Google Scholar). A Rac1-IRSp53-Abi1-WAVE2 complex has been shown to mediate CSF-1-induced macrophage motility (12.Kheir W.A. Gevrey J.C. Yamaguchi H. Isaac B. Cox D. J. Cell Sci. 2005; 118: 5369-5379Crossref PubMed Scopus (63) Google Scholar, 13.Abou-Kheir W. Isaac B. Yamaguchi H. Cox D. J. Cell Sci. 2008; 121: 379-390Crossref PubMed Scopus (66) Google Scholar), and a unique role for WASP in macrophage chemotaxis to CSF-1, formylmethionylleucylphenylalanine, MCP-1, and MIP-1α has been demonstrated (14.Zicha D. Allen W.E. Brickell P.M. Kinnon C. Dunn G.A. Jones G.E. Thrasher A.J. Br. J. Haematol. 1998; 101: 659-665Crossref PubMed Scopus (202) Google Scholar, 15.Badolato R. Sozzani S. Malacarne F. Bresciani S. Fiorini M. Borsatti A. Albertini A. Mantovani A. Ugazio A.G. Notarangelo L.D. J. Immunol. 1998; 161: 1026-1033PubMed Google Scholar). WASP is a hematopoietic cell-specific regulator of Arp2/3-dependent actin remodeling. The catalytically active domain of WASP lies in its C terminus, which is conserved among all WASP/WAVE proteins and contains a VCA (verprolin homology, cofilin-like, and acidic region) domain capable of activating the Arp2/3 complex. The other domains found in WASP can regulate, directly or indirectly, the activity of its VCA domain (for a review, see Ref. 16.Takenawa T. Suetsugu S. Nat. Rev. Mol. Cell Biol. 2007; 8: 37-48Crossref PubMed Scopus (712) Google Scholar). Both WASP and N-WASP bind activated Cdc42 through their GTPase-binding domain, which is believed to cause a structural transition that results in dissociation of the intramolecular contacts leaving the VCA domain accessible for Arp2/3 binding (17.Kim A.S. Kakalis L.T. Abdul-Manan N. Liu G.A. Rosen M.K. Nature. 2000; 404: 151-158Crossref PubMed Scopus (625) Google Scholar, 18.Rohatgi R. Ma L. Miki H. Lopez M. Kirchhausen T. Takenawa T. Kirschner M.W. Cell. 1999; 97: 221-231Abstract Full Text Full Text PDF PubMed Scopus (1082) Google Scholar). In addition, biochemical studies have revealed that several signaling molecules, including WASP-interacting SH3 protein, WASP-interacting protein, Grb2, phosphoinositides, and Src family kinases, activate N-WASP (for reviews, see Refs. 16.Takenawa T. Suetsugu S. Nat. Rev. Mol. Cell Biol. 2007; 8: 37-48Crossref PubMed Scopus (712) Google Scholar and 19.Miki H. Takenawa T. J. Biochem. 2003; 134: 309-313Crossref PubMed Scopus (140) Google Scholar). Phosphorylation of WASP has also been proposed to activate Arp2/3-mediated actin polymerization in vitro (20.Cory G.O. Cramer R. Blanchoin L. Ridley A.J. Mol. Cell. 2003; 11: 1229-1239Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 21.Cory G.O. Garg R. Cramer R. Ridley A.J. J. Biol. Chem. 2002; 277: 45115-45121Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 22.Torres E. Rosen M.K. Mol. Cell. 2003; 11: 1215-1227Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Recently different probes have been developed that detect a conformational change in N-WASP and therefore reflect its activation (23.Ward M.E. Wu J.Y. Rao Y. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 970-974Crossref PubMed Scopus (26) Google Scholar, 24.Lorenz M. Yamaguchi H. Wang Y. Singer R.H. Condeelis J. Curr. Biol. 2004; 14: 697-703Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 25.Sukumvanich P. DesMarais V. Sarmiento C.V. Wang Y. Ichetovkin I. Mouneimne G. Almo S. Condeelis J. Cell. Motil. Cytoskeleton. 2004; 59: 141-152Crossref PubMed Scopus (23) Google Scholar). Using either a fluorescence resonance energy transfer (FRET)-based biosensor that detects a conformational change in N-WASP (23.Ward M.E. Wu J.Y. Rao Y. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 970-974Crossref PubMed Scopus (26) Google Scholar, 24.Lorenz M. Yamaguchi H. Wang Y. Singer R.H. Condeelis J. Curr. Biol. 2004; 14: 697-703Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) or antibodies that can only bind to the open conformation of N-WASP (25.Sukumvanich P. DesMarais V. Sarmiento C.V. Wang Y. Ichetovkin I. Mouneimne G. Almo S. Condeelis J. Cell. Motil. Cytoskeleton. 2004; 59: 141-152Crossref PubMed Scopus (23) Google Scholar), N-WASP has been shown to be activated in response to epidermal growth factor in HEK293 cells and in MTLn3 carcinoma cells. This activity has been temporally localized to subcellular compartments important for carcinoma cell chemotaxis and invasion (24.Lorenz M. Yamaguchi H. Wang Y. Singer R.H. Condeelis J. Curr. Biol. 2004; 14: 697-703Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). We have adapted these approaches to explore the signal transduction pathways responsible for the activation of WASP in vivo. The WASP biosensor (WASPbs) construct was generated according to a previously described procedure (24.Lorenz M. Yamaguchi H. Wang Y. Singer R.H. Condeelis J. Curr. Biol. 2004; 14: 697-703Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Briefly human WASP was used as a template for PCR amplification and insertion into the ECFP-EYFP-biosensor vector (24.Lorenz M. Yamaguchi H. Wang Y. Singer R.H. Condeelis J. Curr. Biol. 2004; 14: 697-703Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Point mutations were introduced using the QuikChange kit from Stratagene (La Jolla, CA) according to the manufacturer's instructions. COS cells and HEK293 cells (ATCC, Manassas, VA) were cultured according to their specifications. RAW/LR5 cells were derived from the murine monocyte/macrophage RAW 264.7 cell line (8.Cox D. Chang P. Zhang Q. Reddy P.G. Bokoch G.M. Greenberg S. J. Exp. Med. 1997; 186: 1487-1494Crossref PubMed Scopus (371) Google Scholar) and were grown in RPMI 1640 medium (Mediatech, Inc.) containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin (all from Sigma). All cells were maintained at 37 °C in a 5% CO2 incubator. Transient transfections were performed using the SuperFect reagent from Qiagen according to the manufacturer's instructions. Reduction of Cdc42 expression was achieved through the retroviral infection of RAW/LR5 cells with short hairpin RNAs directed against the Cdc42 mRNA using pSUPER.reto.puro plasmids (Oligoengine, Seattle, WA). 5A. Dovas, I.-C. Gevrey, A. Grossi, W. G. Abou-Kheir, and D. Cox, manuscript submitted. For all experiments involving a CSF-1 stimulation cells were treated according to Cox et al. (8.Cox D. Chang P. Zhang Q. Reddy P.G. Bokoch G.M. Greenberg S. J. Exp. Med. 1997; 186: 1487-1494Crossref PubMed Scopus (371) Google Scholar). 20 ng/ml murine recombinant CSF-1 (R&D Systems, Minneapolis, MN) was added or not to the cells for the indicated times at 37 °C. Following fixation in 3.7% formaldehyde and permeabilization in 0.2% Triton X-100 cells were stained using either WASP/N-WASP conformation-sensitive antibody (CSA) (25.Sukumvanich P. DesMarais V. Sarmiento C.V. Wang Y. Ichetovkin I. Mouneimne G. Almo S. Condeelis J. Cell. Motil. Cytoskeleton. 2004; 59: 141-152Crossref PubMed Scopus (23) Google Scholar) or anti-Myc antibodies (Roche Applied Science) followed by incubation with labeled secondary antibodies and Alexa Fluor 568 phalloidin (Molecular Probes, Invitrogen). Mean fluorescence intensity of entire cells was measured at 20× and plotted versus time after CSF-1 addition. Spectral analysis of WASPbs expressing HEK293 cells (see Fig. 1B) was done using a Leica TCS SP2 AOBS confocal microscope with a 63×, numerical aperture 1.4 objective (Leica Microsystems, Exton, PA). Cells were illuminated with a 405 nm laser, and fluorescence was detected from 445 (440–450) to 595 nm (590–600) with a 10-nm-wide slit stepping up the spectrum. The intensity of the whole image was plotted versus the wavelength to get the fluorescence spectra and normalized to the signal at 495 nm. FRET image sequences of WASPbs-expressing cells were obtained essentially as described in Lorenz et al. (24.Lorenz M. Yamaguchi H. Wang Y. Singer R.H. Condeelis J. Curr. Biol. 2004; 14: 697-703Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Acquisition was performed with IP Lab v3.51 (Scanalytics Inc.), and FRET analyses were performed with IP Lab v3.51 and with ImageJ (W. S. Rasband, ImageJ, National Institutes of Health, Bethesda, MD, 1997–2006). For ratiometric FRET analysis, after background subtraction the total cellular donor fluorescence intensity was divided by the total FRET fluorescence intensity. Only cells expressing low levels of the WASPbs, as measured by the acceptor fluorescence intensity, were analyzed because overexpression of WASP induced artifacts similar to those reported for cells overexpressing N-WASP (24.Lorenz M. Yamaguchi H. Wang Y. Singer R.H. Condeelis J. Curr. Biol. 2004; 14: 697-703Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Absolute FRET values were variable between experiments due to varying illumination intensity and exposure conditions. Therefore we only compared paired conditions within the same experiment to avoid instrument or other variability that was not related to specific regulation of WASP activity. To compensate for this, for each experiment, each condition was compared with the unstimulated condition of the same experiment and expressed as a percentage. The percentage of control values from at least three independent experiments were subsequently averaged together. Results were then reported as donor/FRET values for individual experiments or as percent change compared with the basal (or unstimulated) controls when multiple experiments were compared. RAW/LR5 cells (see Fig. 5A) were pretreated with 10 μm PP2 (Calbiochem, EMD Bioscience) or with DMSO (vehicle) for 1 h at 37 °C before being subjected to CSF-1 stimulation as described above. COS cells transfected with the indicated WASPbs constructs with or without Myc-Cdc42Q61L (see Fig. 6B) or RAW/LR5 cells transfected with Myc-WASP (see Fig. 5A) were incubated in the presence or absence of 12 μm pervanadate for 30 min at 37 °C according to Cory et al. (21.Cory G.O. Garg R. Cramer R. Ridley A.J. J. Biol. Chem. 2002; 277: 45115-45121Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Cells were lysed in ice-cold lysis buffer (1% Triton X-100, 25 mm Tris, 137 mm NaCl, 2 mm EDTA, 1 mm orthovanadate, 1 mm benzamidine, 10 μg/ml aprotinin, and 10 μg/ml leupeptin, pH 7.4). Immunoprecipitations were carried out by incubating the cleared cell lysates at 4 °C with the appropriate antibody prebound to protein A/G-agarose beads (Santa Cruz Biotechnology). Samples were resolved by SDS-PAGE, transferred onto polyvinylidene difluoride membranes (Immobilon-P, Millipore) followed by incubation with the indicated primary antibodies and secondary antibodies conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA). Signals were detected using the Super Signal West Pico chemiluminescent substrate from Pierce, and images were acquired and analyzed using a Kodak Image Station 440. Antibodies used for these applications were as follows: anti-phosphotyrosine (Tyr(P)-99, Santa Cruz Biotechnology), anti-phospho-Hck (Tyr-411, Santa Cruz Biotechnology), anti-green fluorescent protein or anti-Myc (Roche Applied Science), anti-β-actin (AC-15, Sigma), and anti-WASP (B19, Santa Cruz Biotechnology). All data are represented as the mean, and error bars represent the S.E. Significance was analyzed using paired Student's t test, and differences between two means with a p value <0.05 were considered significant. To specifically address how WASP activity is regulated in vivo we have constructed a FRET-based WASPbs. Based on the known autoinhibitory conformation of both WASP and N-WASP, the WASPbs was designed to exhibit intramolecular FRET between cyan fluorescent protein (CFP; donor) and yellow fluorescent protein (YFP; acceptor) fused to the N or C termini of full-length WASP, respectively (see Fig. 1A); it is similar to the previously described N-WASP biosensor (24.Lorenz M. Yamaguchi H. Wang Y. Singer R.H. Condeelis J. Curr. Biol. 2004; 14: 697-703Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Activation of WASP induces a conformational change that would result in increasing the CFP-YFP distance leading to a decreased FRET signal (Fig. 1A). Validating the functionality of the biosensor, HEK293 cells expressing the WASPbs alone (data not shown) or in the presence of dominant-negative Cdc42 (Cdc42N17) exhibited two emission peaks at 475 (CFP) and 525 nm (YFP) after excitation of CFP (Fig. 1B). This observation is consistent with WASP being present in a closed conformation and permitting FRET. Moreover in the presence of constitutively active Cdc42 (Cdc42Q61L) a reduction in the YFP peak at 525 nm was observed suggesting that a structural change had occurred upon Cdc42Q61L binding (Fig. 1B). To confirm that a decrease in FRET was indicative of a conformational change in the WASPbs, experiments were performed using the WASP inhibitor wiskostatin that binds to and stabilizes the closed, autoinhibited conformation of WASP (26.Peterson J.R. Bickford L.C. Morgan D. Kim A.S. Ouerfelli O. Kirschner M.W. Rosen M.K. Nat. Struct. Mol. Biol. 2004; 11: 747-755Crossref PubMed Scopus (156) Google Scholar). Following fixation of COS-7 cells cotransfected with WASPbs and Cdc42Q61L, WASP activity was determined as the loss of FRET and quantified as a ratio of the donor emission divided by the FRET emission (see "Experimental Procedures" for more details). Pretreatment of the cells with 5 μm wiskostatin inhibited the activation of the WASPbs induced by Cdc42Q61L (Fig. 1C) indicating that changes in FRET observed in the presence of Cdc42Q61L reflect changes in WASPbs conformation. Murine monocyte/macrophage cells, RAW/LR5 (8.Cox D. Chang P. Zhang Q. Reddy P.G. Bokoch G.M. Greenberg S. J. Exp. Med. 1997; 186: 1487-1494Crossref PubMed Scopus (371) Google Scholar), were transfected with the WASPbs and then stimulated with CSF-1 for various times before fixation. As shown in Fig. 2A, WASP activation was detected globally as early as 30 s following CSF-1 addition and appeared to be localized to protrusive regions at later times. Quantification of whole cell donor/FRET signals indicated a significant peak in WASP activity at 30 s after CSF-1 addition (Fig. 2, B and C). These results were confirmed using fluorescent lifetime imaging microscopy as an alternative method (not shown). The biosensor had a FRET efficiency in the inactive conformation of ∼19%, similar to the FRET efficiency observed for the highly structural homologue N-WASPbs (24.Lorenz M. Yamaguchi H. Wang Y. Singer R.H. Condeelis J. Curr. Biol. 2004; 14: 697-703Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), and 5% after activation, corresponding to an estimated dye-to-dye distance of 6.4 and 8.2 nm, respectively (data not shown). To ensure that the WASPbs was reflecting the activation of endogenous WASP in RAW/LR5 cells, a CSA was used as an alternative approach to determine the activation state of endogenous WASP. This antibody was generated against a region only accessible when the protein is in an opened, active conformation (25.Sukumvanich P. DesMarais V. Sarmiento C.V. Wang Y. Ichetovkin I. Mouneimne G. Almo S. Condeelis J. Cell. Motil. Cytoskeleton. 2004; 59: 141-152Crossref PubMed Scopus (23) Google Scholar). Consistent with a highly conserved GTPase-binding domain between N-WASP and WASP, CSA was able to detect WASP by Western blot in RAW/LR5 (data not shown). However, because it has been reported that a small amount of N-WASP is also expressed in macrophages (27.Stamm L.M. Pak M.A. Morisaki J.H. Snapper S.B. Rottner K. Lommel S. Brown E.J. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 14837-14842Crossref PubMed Scopus (52) Google Scholar) the affinity of the CSA for WASP and N-WASP was determined by examining lysates of COS cells transfected with either the WASPbs used in this study or the previously reported N-WASP biosensor (24.Lorenz M. Yamaguchi H. Wang Y. Singer R.H. Condeelis J. Curr. Biol. 2004; 14: 697-703Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The affinity of CSA for either WASP or N-WASP was determined as a ratio of CSA staining intensity of WASP/N-WASP following normalization for the expression of the constructs using anti-green fluorescent protein (supplemental Fig. 1). The 5-fold higher affinity of the CSA for WASP and the fact that WASP is expressed at 15 times the level of N-WASP in RAW/LR5 cells 6B. M. Isaac, D. Ishihara, L. Nusblat, J. Condeelis, and D. Cox, manuscript in preparation. suggest that this reagent can be used to determine the activation state of endogenous WASP. When used in immunofluorescence experiments on fixed cells, CSA detected an increase of WASP activity 30 s after CSF-1 addition with hot spots of active WASP observed in CSF-1-elicited protrusions (Fig. 3A). Quantification of stained cells indicated a significant increase in fluorescence intensity 30 s following CSF-1 addition that decreased to base-line levels by 5 min (Fig. 3B), thus paralleling the rapid and transient CSF-1-induced activation of WASP as measured with the FRET biosensor. Activated Cdc42 has been shown to bind to and activate WASP and N-WASP in vitro (18.Rohatgi R. Ma L. Miki H. Lopez M. Kirchhausen T. Takenawa T. Kirschner M.W. Cell. 1999; 97: 221-231Abstract Full Text Full Text PDF PubMed Scopus (1082) Google Scholar). Therefore the involvement of Cdc42 in WASP activation in vivo was assessed. Cotransfection of RAW/LR5 cells with WASPbs and Cdc42N17 resulted in a decrease in WASP activity compared with cells expressing the WASPbs alone (Fig. 4A). This is consistent with an interpretation that despite serum starvation there always was some constitutive level of WASP activity in resting cells. Importantly treatment with CSF-1 was unable to rescue the inhibition of WASP activity in this situation indicating that CSF-1 signaling to WASP cannot bypass Cdc42 in these cells (Fig. 4A). Conversely RAW/LR5 cells showed an enhanced WASP activity when cotransfected with WASPbs and constitutively active Cdc42 (Cdc42V12) compared with WASPbs alone-expressing cells. In addition, CSF-1 stimulation did not potentiate the Cdc42V12-dependent increase in WASPbs activity (Fig. 4A), suggesting that the activation of WASPbs was already maximal. Similar results were also obtained with Cdc42Q61L (data not shown). To confirm a specific role for Cdc42 in WASP activation the ability of CSF-1 to induce WASP activation in cells with reduced endogenous levels of Cdc42 was determined. Cdc42 levels were stably reduced by greater than 70% through expression of a short hairpin RNA sequence targeting Cdc42. 5A. Dovas, I.-C. Gevrey, A. Grossi, W. G. Abou-Kheir, and D. Cox, manuscript submitted. There was no significant increase in WASP activation following CSF-1 addition as determined using the CSA in cells with reduced levels of Cdc42 (Fig. 4B). Control infected cells showed a level of WASP activation in response to CSF-1 similar to that of non-infected cells (compare Figs. 4B and 3B). Altogether these data clearly demonstrate that WASP activation in response to CSF-1 is Cdc42-dependent. It has been speculated that following treatment of monocytes/macrophages with CSF-1 PI3K activates guanine nucleotide exchange factors that in turn activate the Rho GTPases Rac and Cdc42 that are required for chemotaxis (28.Ridley A.J. FEBS Lett. 2001; 498: 168-171Crossref PubMed Scopus (98) Google Scholar). Because Cdc42 binding appeared to be essential for WASP activity in RAW/LR5 cells, we then asked whether its putative activator PI3K was indeed controlling WASP activity in our system. To address this question, cells expressing the WASPbs were preincubated in the presence or absence of PI3K inhibitors, wortmannin, or LY294002 before CSF-1 stimulation for 30 s, and the WASP activation was measured. PI3K inhibition completely blocked WASP activation induced by CSF-1 as measured with either the WASPbs (Fig. 4C) or CSA (Fig. 5C). Expression of Cdc42V12 in WASPbs-expressing cells was able to bypass the requirement of PI3K for WASP activation in response to CSF-1 (Fig. 4B) consistent with a situation where PI3K is upstream of Cdc42, which itself is required for the activation of WASP. Recent reports have suggested that there are also Cdc42-independent mechanisms of WASP activation. Tyrosine phosphorylation of N-WASP or WASP or introduction of a phosphomimicking mutation has been shown to stimulate their activities in vitro (21.Cory G.O. Garg R. Cramer R. Ridley A.J. J. Biol. Chem. 2002; 277: 45115-45121Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 29.Suetsugu S. Hattori M. Miki H. Tezuka T. Yamamoto T. Mikoshiba K. Takenawa T. Dev. Cell. 2002; 3: 645-658Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). The hematopoietic cell-specific Src family kinase Hck has been proposed to phosphorylate and activate WASP in macrophages (21.Cory G.O. Garg R. Cramer R. Ridley A.J. J. Biol. Chem. 2002; 277: 45115-45121Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Immunoprecipitation experiments were performed to identify activated Hck in the phosphotyrosine-containing fraction of RAW/LR5 cell lysates after addition of CSF-1. Hck was activated after addition of CSF-1 with kinetics similar to that of WASP activation (Fig. 5A). As a control, we verified that the CSF-1-induced increase in Hck tyrosine phosphorylation was abolished when RAW/LR5 cells were pretreated with the Src family kinase inhibitor PP2 (Fig. 5A, middle panel). In addition, tyrosine phosphorylation of Myc-tagged WASP expressed in RAW/LR5 cells was shown to be fully dependent on Src family kinase activity (Fig. 5A, lower panel). Then to determine whether Src family kinases were required for WASP activation in vivo, cells expressing the WASPbs were pretreated with PP2, and the ability of CSF-1 to activate WASP was analyzed. At concentrations of PP2 that effectively inhibited CSF-1-induced Hck activation, PP2 had no effect on either the basal level or the CSF-1-induced activation of WASP as shown in Fig. 5B. In addition, there was no significant difference in the intensity of staining using the CSA between CSF-1-stimulated cells in the presence or absence of PP2 or SU6656, a structurally unrelated pan-Src family kinase inhibitor (Fig. 5C). Altogether these data clearly demonstrate that Src family kinases are not required for WASP activation in response to CSF-1. Because several other tyrosine kinases have been shown to have the a
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