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• W2067544586 abstract "Hck is a protein kinase of the Src family specifically expressed in phagocytes as two isoforms, p59Hck and p61Hck, localized at the plasma membrane and lysosomes, respectively. Their individual involvement in functions ascribed to Hck, phagocytosis, cell migration, and lysosome mobilization, is still unclarified. To investigate the specific role of p59Hck, a constitutively active variant in fusion with green fluorescent protein (p59Hckca) was expressed in HeLa cells. p59Hckca was found at focal adhesion sites and triggered reorganization of the actin cytoskeleton, leading to plasma membrane protrusions where it co-localized with F-actin. Similarly, microinjection of p59Hckca cDNA in J774.A1 macrophages induced membrane protrusions. Whereas kinase activity and membrane association of p59Hck were dispensable for location at focal adhesions, p59Hck-induced membrane protrusions were dependent on kinase activity, plasma membrane association, and Src homology 2 but not Src homology 3 domain and were inhibited by dominant-negative forms of Cdc42 or Rac but not by blocking Rho activity. A dominant negative form of p59Hck inhibited the Cdc42- and Rac-dependent FcγRIIa-mediated phagocytosis. Expression of the Cdc42/Rac-interacting domain of p21-activated kinase in macrophages abolished the p59Hckca-induced morphological changes. Therefore, p59Hck-triggered remodeling of the actin cytoskeleton depends upon the activity of Cdc42 and Rac to promote formation of membrane protrusions necessary for phagocytosis and cell migration. Hck is a protein kinase of the Src family specifically expressed in phagocytes as two isoforms, p59Hck and p61Hck, localized at the plasma membrane and lysosomes, respectively. Their individual involvement in functions ascribed to Hck, phagocytosis, cell migration, and lysosome mobilization, is still unclarified. To investigate the specific role of p59Hck, a constitutively active variant in fusion with green fluorescent protein (p59Hckca) was expressed in HeLa cells. p59Hckca was found at focal adhesion sites and triggered reorganization of the actin cytoskeleton, leading to plasma membrane protrusions where it co-localized with F-actin. Similarly, microinjection of p59Hckca cDNA in J774.A1 macrophages induced membrane protrusions. Whereas kinase activity and membrane association of p59Hck were dispensable for location at focal adhesions, p59Hck-induced membrane protrusions were dependent on kinase activity, plasma membrane association, and Src homology 2 but not Src homology 3 domain and were inhibited by dominant-negative forms of Cdc42 or Rac but not by blocking Rho activity. A dominant negative form of p59Hck inhibited the Cdc42- and Rac-dependent FcγRIIa-mediated phagocytosis. Expression of the Cdc42/Rac-interacting domain of p21-activated kinase in macrophages abolished the p59Hckca-induced morphological changes. Therefore, p59Hck-triggered remodeling of the actin cytoskeleton depends upon the activity of Cdc42 and Rac to promote formation of membrane protrusions necessary for phagocytosis and cell migration. Hck is a protein-tyrosine kinase (PTK) 1The abbreviations used are: PTKprotein-tyrosine kinaseSH1-2, and -3, Src homology 1, 2, and 3, respectivelyPAKp21 (Cdc42/Rac)-activated kinasePBSphosphate-buffered salineBSAbovine serum albuminTRITCtetramethylrhodamine isothiocyanateGFPgreen fluorescent proteinCRIBCdc42/Rac-interacting binding of the Src family mainly expressed in phagocytes (1Quintrell N. Lebo R. Varmus H. Bishop J.M. Pettenati M.J., Le Beau M.M. Diaz M.O. Rowley J.D. Mol. Cell. Biol. 1987; 7: 2267-2275Crossref PubMed Scopus (203) Google Scholar). Src PTKs are key elements of diverse signaling cascades, which act via at least two different ways: a tyrosine kinase activity and/or an adaptor function (for a review, see Ref. 2Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Crossref PubMed Scopus (2175) Google Scholar). These two roles are achieved by three highly conserved domains shared by all members of the Src family. The Src homology domain 1 (SH1) lying at the C terminus of the protein supports the catalytic activity of the enzyme, and the SH2 and SH3 domains allow interactions with cellular proteins through recognition of phosphotyrosine and proline-rich motif, respectively (3Yu H. Rosen M.K. Shin T.B. Seidel-Dugan C. Brugge J.S. Schreiber S.L. Science. 1992; 258: 1665-1668Crossref PubMed Scopus (285) Google Scholar, 4Moran M.F. Koch C.A. Anderson D. Ellis C. England L. Martin G.S. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8622-8626Crossref PubMed Scopus (331) Google Scholar). Src PTKs are regulated by the phosphorylation state of a conserved carboxyl-terminal tyrosine. When phosphorylated, this tyrosine is recognized by the SH2 domain, leading to intramolecular interactions that stabilize the kinase under its inactive form. Its dephosphorylation disrupts these intramolecular interactions and subsequently unmasks the SH3, SH2, and catalytic domains. Therefore, this protein “opening” activates both the adaptor and the kinase functions of Src PTKs (5Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1047) Google Scholar). protein-tyrosine kinase -2, and -3, Src homology 1, 2, and 3, respectively p21 (Cdc42/Rac)-activated kinase phosphate-buffered saline bovine serum albumin tetramethylrhodamine isothiocyanate green fluorescent protein Cdc42/Rac-interacting binding Hck is the unique example among the Src PTKs to be expressed as two isoforms generated in equal amounts by alternative translation. p61Hck translation is initiated at a CTG codon 21 codons upstream from the p59Hck ATG codon (6Lock P. Ralph S. Stanley E. Boulet I. Ramsay R. Dunn A.R. Mol. Cell. Biol. 1991; 11: 4363-4370Crossref PubMed Scopus (106) Google Scholar). Like other members of the Src family, both Hck isoforms have a unique amino-terminal domain comprising about 80 amino acids with acylation motifs (7Resh M.D. Cell. 1994; 76: 411-413Abstract Full Text PDF PubMed Scopus (593) Google Scholar). p61Hck has 21 additional N-terminal amino acids containing the Met1-Gly2-X 3-X 4-X 5-Ser6/Thr6N-terminal sequence that supports covalent myristoylation of glycine 2, whereas the Met1-Gly2-Cys3-X 4-X 5-Ser6/Thr6N terminus of p59Hck guides permanent myristoylation of glycine 2 and reversible palmitoylation of cysteine 3 (8Robbins S.M. Quintrell N.A. Bishop J.M. Mol. Cell. Biol. 1995; 15: 3507-3515Crossref PubMed Scopus (230) Google Scholar). These different acylations govern association of both isoforms with distinct cellular membranes; while the double acylated form of p59Hck is anchored at the plasma membrane, the monoacylated forms (p61Hck and the nonpalmitoylated form of p59Hck) are associated with lysosomal membranes, both isoforms being present at the Golgi apparatus (9Carreno S. Gouze M.E. Schaak S. Emorine L.J. Maridonneau-Parini I. J. Biol. Chem. 2000; 275: 36223-36229Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). These distinct subcellular localizations are probably a key element of the differential functions of Hck isoforms by offering them access to different substrates. Since Hck has been involved in the lysosome mobilization process (10Welch H. Maridonneau-Parini I. J. Biol. Chem. 1997; 272: 102-109Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 11N′diaye E.N. Darzacq X. Astarie-Dequeker C. Daffe M. Calafat J. Maridonneau-Parini I. J. Immunol. 1998; 161: 4983-4991PubMed Google Scholar) and in the signaling of membrane receptors such as phagocytic ones (12Fitzer-Attas C.J. Lowry M. Crowley M.T. Finn A.J. Meng F. Defranco A.L. Lowell C.A. J. Exp. Med. 2000; 191: 669-682Crossref PubMed Scopus (199) Google Scholar, 13Ghazizadeh S. Bolen J.B. Fleit H.B. J. Biol. Chem. 1994; 269: 8878-8884Abstract Full Text PDF PubMed Google Scholar, 14Wang A.V. Scholl P.R. Geha R.S. J. Exp. Med. 1994; 180: 1165-1170Crossref PubMed Scopus (130) Google Scholar), we have proposed that the monoacylated lysosomal p61Hck and p59Hck could control lysosome exocytosis, whereas the plasma membrane-associated isoform p59Hck could transduce signals from membrane receptors (9Carreno S. Gouze M.E. Schaak S. Emorine L.J. Maridonneau-Parini I. J. Biol. Chem. 2000; 275: 36223-36229Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). However, no attempt has been made to identify the respective functions of each isoform. In this work, we focused on p59Hck and investigated the function devoted to this plasma membrane-associated isoform. Since we have previously shown that ectopic expression of Hck isoforms in HeLa cells leads to the same subcellular distribution as the endogenous kinase in human neutrophils and monocytes-macrophages (9Carreno S. Gouze M.E. Schaak S. Emorine L.J. Maridonneau-Parini I. J. Biol. Chem. 2000; 275: 36223-36229Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), we first decided to study p59Hck function in these human epithelial cells. We took advantage of the nonexpression of Hck in these cells to examine whether expression of this phagocyte-specific kinase would trigger a phagocyte-specific phenotype. Ectopic expression of a constitutively active form of p59Hck in fusion with GFP in these cells led to reorganization of the actin cytoskeleton, which triggered the formation of plasma membrane protrusions. When the Hck-GFP construct was microinjected in J774.A1 macrophages, a similar phenotype was observed. Using targeted mutagenesis and deletion constructs, we showed that these cytoskeletal changes were strictly dependent on the association of p59Hck with the plasma membrane and involved both its tyrosine kinase activity and its SH2 adaptor domain. Furthermore, we showed that a dominant negative form of p59Hck co-transfected in HeLa cells with the FcγRIIa was able to inhibit phagocytosis mediated by this receptor. Since Cdc42 and Rac, two small GTP-binding proteins of the Rho subfamily that control the actin cytoskeleton, have been involved in FcγR-mediated phagocytosis (15Caron E. Hall A. Science. 1998; 282: 1717-1721Crossref PubMed Scopus (809) Google Scholar, 16Massol P. Montcourrier P. Guillemot J.C. Chavrier P. EMBO J. 1998; 17: 6219-6229Crossref PubMed Scopus (203) Google Scholar), we investigated their role in the p59Hck-mediated membrane protrusion. Using dominant negative Cdc42 or Rac or the Cdc42/Rac-interacting domain of p21 (Cdc42/Rac)-activated kinase (PAK), we showed that p59Hck acted upstream of the Cdc42/Rac pathway to promote these cytoskeletal changes both in HeLa cells and in macrophages. We thus propose that p59Hck is part of a signaling pathway between plasma membrane receptors and Cdc42/Rac that promotes actin cytoskeleton rearrangements necessary for phagocytosis or cell migration. The wild-type Hck cDNA was a gift from N. Quintrell (1Quintrell N. Lebo R. Varmus H. Bishop J.M. Pettenati M.J., Le Beau M.M. Diaz M.O. Rowley J.D. Mol. Cell. Biol. 1987; 7: 2267-2275Crossref PubMed Scopus (203) Google Scholar). Construction of p59Hck in fusion with GFP has been described (9Carreno S. Gouze M.E. Schaak S. Emorine L.J. Maridonneau-Parini I. J. Biol. Chem. 2000; 275: 36223-36229Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). p59Hckca was obtained by point mutation of the carboxyl-terminal tyrosine 505 (TAC) into phenylalanine (TTC) and of the stop codon TGA into CGA by PCR using primers that generate XbaI and NheI sites at the 5′- and 3′-ends, respectively. The PCR products were ligated into the NheI site of pEGFP-N3 (CLONTECH, Palo Alto, CA), preserving the 28-amino acid polylinker of the vector between p59Hckca and GFP. The C3S point mutation was introduced into the p59Hckca-GFP vector by inverse PCR mutating the cysteine (TGC) into serine (AGC) and introducing a NcoI site. The p59Hckdn-GFP was obtained by inverse PCR on the p59Hckca-GFP vector mutating the lysine 381 (AAG) responsible of ATP binding into glutamic acid (GAG) and introducing an XcmI site. p61Hckca and p61Hckdn fused with GFP were obtained by the same strategy using the p61Hck-GFP vector as template (9Carreno S. Gouze M.E. Schaak S. Emorine L.J. Maridonneau-Parini I. J. Biol. Chem. 2000; 275: 36223-36229Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). p59(ΔSH2-SH3)Hckca, p59(ΔSH2)Hckca, and p59(ΔSH3)Hckca were obtained by inverse PCR on the p59Hckca-GFP vector using primers allowing the deletion of amino acids 57–220, 123–220, and 57–117, respectively. Conformity of each mutations was verified by sequencing (Genome Express, Grenoble, France). In addition, all constructs were tested for expression by Western blotting (see below). For phagocytic assay we used the plasmid pKC3 encoding the human FcγRIIa provided by C. Sautes-Fridman (17Cassard L. Dragon-Durey M.A. Ralli A. Tartour E. Salamero J. Fridman W.H. Sautes-Fridman C. Immunol. Lett. 2000; 75: 1-8Crossref PubMed Scopus (9) Google Scholar). cDNAs coding for Myc-tagged Cdc42N17 and RacN17, subcloned into pRK5 vector, were provided by A. Hall (15Caron E. Hall A. Science. 1998; 282: 1717-1721Crossref PubMed Scopus (809) Google Scholar). The Cdc42/Rac binding domain of PAK (PAKCRIB (18Sander E.E. Ten Klooster J.P. Van Delft S. Van Der Kammen R.A. Collard J.G. J. Cell Biol. 1999; 147: 1009-1022Crossref PubMed Scopus (738) Google Scholar)) was subcloned into the eukaryotic expression vector pRK5myc (19Lamarche N. Tapon N. Stowers L. Burbelo P.D. Aspenstrom P. Bridges T. Chant J. Hall A. Cell. 1996; 87: 519-529Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). HeLa cells were cultured at 37 °C, 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 1% l-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin. HeLa cells were seeded in 24-well plates (2 × 104 cells/well on glass coverslips) for immunofluorescence experiments or in 9-cm Petri dishes (106 cells) for immunoblotting experiments. The following day, phosphate precipitate was added to the cells (1 μg of DNA per 40 μl of DNA/calcium per well containing 360 μl of fresh complete medium). For double transfection experiments, DNA/calcium phosphate precipitates were made with 500 ng of each cDNA. Cells were washed free of DNA/calcium phosphate precipitates after 16–18 h and incubated in fresh medium for an additional period of 60 h before analyses. The murine macrophage cell line J774.A1 was maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat inactivated fetal calf serum and penicillin/streptomycin (100 units/ml and 100 μg/ml). Transfected cells grown on glass coverslips were washed twice with PBS and fixed in 3.7% paraformaldehyde for 30 min at room temperature, and unreacted aldehyde groups were neutralized in 50 mm NH4Cl for 1 min. After washing and permeabilization (0.3% Triton X-100 in PBS, 5 min), cells were blocked for 10 min in PBS containing 1% bovine serum albumin (PBS-BSA). Coverslips were then overlaid as indicated with 20 μl of one of the following primary antibodies diluted into PBS-BSA: monoclonal anti-vinculin antibody (1:50; Sigma), monoclonal anti-FcγRIIa IV.3 antibody (300 ng/ml; kindly provided by C. Sautes-Fridman (20Vely F. Gruel N. Moncuit J. Cochet O. Rouard H. Dare S. Galon J. Sautes C. Fridman W.H. Teillaud J.L. Hybridoma. 1997; 16: 519-528Crossref PubMed Scopus (36) Google Scholar)), monoclonal anti-Golgi CTR33 (kindly provided by M. Bornens (9Carreno S. Gouze M.E. Schaak S. Emorine L.J. Maridonneau-Parini I. J. Biol. Chem. 2000; 275: 36223-36229Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar)), and monoclonal anti-Myc 9E10 (1:100; Sigma). After a 30-min incubation period, the coverslips were washed three times in PBS and incubated for 30 min at room temperature with a 1:100 dilution of affinity-purified TRITC-conjugated secondary antibodies (Sigma) directed against mouse IgG. The coverslips were washed three times in PBS. Labeling of F-actin was performed on cells fixed as described above, after permeabilization (0.3% Triton X-100, 5 min), and coverslips were overlaid with 20 μl of permeabilization buffer supplemented with 0.3 units of rhodamine-phalloidin (Molecular Probes, Leiden, The Netherlands). In some experiments, cells were incubated for 24 h with 20 μg/ml recombinant C3 exoenzyme from Clostridium botulinum kindly provided by P. Boquet and prepared as previously described (21Doussau F. Gasman S. Humeau Y. Vitiello F. Popoff M. Boquet P. Bader M.F. Poulain B. J. Biol. Chem. 2000; 275: 7764-7770Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). All coverslips were mounted in Mowiol and viewed using a Leica DM-RB fluorescence microscope or a Leica TCS-SP2 confocal scanning microscope. Epifluorescence images were captured, and negatives were digitalized with a Nikon LS2000. All images were prepared for publication using Adobe Photoshop software. J774 macrophages were used for microinjection as previously described (15Caron E. Hall A. Science. 1998; 282: 1717-1721Crossref PubMed Scopus (809) Google Scholar). Briefly, macrophages were seeded on glass coverslips at a density of 1 × 105 cells/ml. Immediately prior to injection, cells were transferred to 10 mm Hepes-buffered, serum-free Dulbecco's modified Eagle's medium. cDNA constructs prepared for microinjection by standard CsCl gradient methods were injected (0.1 mg/ml) into the nucleus of 50–100 cells in a temperature (37 °C)- and CO2 (10%)-controlled chamber using phase-contrast microscopy. Cells were returned to the incubator for ∼2.5 h for optimal expression. Cells were fixed in 4% (w/v) paraformaldehyde for 20 min at room temperature prior to permeabilization with 0.1% Triton X-100/PBS for 5 min and quenching in NH4Cl/PBS (2.7 mg/ml) for 10 min. For immunostaining, cells were blocked with 0.5% BSA for 30 min and then incubated with antibodies diluted in PBS for 30 min. Where appropriate, all antibody mixes contained excess human IgG (Sigma) to prevent nonspecific binding to the Fc receptors. Myc-tagged constructs were visualized using mouse monoclonal anti-Myc (9E10) followed by Cy5-conjugated anti-mouse IgG. F-actin was stained using rhodamine-conjugated phalloidin (Sigma). Coverslips were mounted in mowiol mountant (Calbiochem) containing p-phenylenediamine as an antibleaching agent. Cells were examined with a Zeiss Axiophot microscope using a Zeiss 63 × 1.4 oil immersion objective. Fluorescence images were captured using a Hamamatsu C5985–10 video camera and Openlab software and processed using Adobe Photoshop. 60 h after transfection, HeLa cells (3 × 106 cells) were washed in PBS, lysed in 1 ml of Laemmli buffer, and boiled for 5 min. Neutrophils from healthy donors were isolated (22Maridonneau-Parini I. Tringale S.M. Tauber A.I. J. Immunol. 1986; 137: 2925-2929PubMed Google Scholar) by Dextran T500 sedimentation (AmershamBiosciences) and Ficoll centrifugation (Eurobio) and were resuspended in boiling Laemmli buffer. Proteins were electrophoresed through 8% SDS-PAGE, transferred to nitrocellulose membrane, which was blocked in Tris-buffered saline buffer containing 5% nonfat milk, and then incubated with anti-Hck antibodies (1:2000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The primary antibody was revealed with horseradish peroxidase-conjugated anti-mouse secondary antibodies (1:10,000; Bio-Rad, Hercules, CA) followed by ECL (AmershamBiosciences). Zymosan particles (Sigma) were swollen in PBS for 30 min. They were then sedimented by centrifugation, washed, and incubated in 0.2m Na2CO3/NaHCO3, pH 9.2, with 250 μg/ml rhodamine B isothiocyanate (Sigma) for 1 h at room temperature, and the reaction was stopped by 50 mmNH4Cl. Labeled zymosan was washed several times in PBS, resuspended, and opsonized in pooled human sera for 30 min at 37 °C. Opsonized and rhodamine-labeled zymosan was washed twice and resuspended in PBS. HeLa cells were used 60 h after transfection. Cells were starved in serum for 3 h, and then opsonized zymosan was added at a concentration of 100 particles/cell and incubated at 37 °C for 3 h. Cells were then extensively washed to remove adherent zymosan, fixed in paraformaldehyde. External particles was labeled using fluorescein-conjugated anti-human IgG (1:100; Diagnostics Pasteur, Paris, France) and then appeared as doubly green and red fluorescence. FcγRIIa was detected using a monoclonal anti-FcγRIIa antibody revealed by TRITC-conjugated secondary antibodies as described above. Cells positive for both GFP fluorescence and FcγRIIa labeling were then counted by fluorescence microscopy for the presence of at least one particle inside the cell. To study the cellular effects triggered by p59Hck, we made use of a constitutive active form of p59Hck (p59Hckca), obtained by point-mutating p59Hck cDNA to replace the C-terminal regulatory tyrosine (responsible for the intramolecular regulation) by a phenylalanine (23Chiaradonna F. Fontana L. Iavarone C. Carriero M.V. Scholz G. Barone M.V. Stoppelli M.P. EMBO J. 1999; 18: 3013-3023Crossref PubMed Scopus (58) Google Scholar). Subcellular localization and estimation of the expression level of p59Hck were allowed by a C-terminal fusion with GFP, which has been previously shown not to interfere with the kinase activity of Hck or with its localization (9Carreno S. Gouze M.E. Schaak S. Emorine L.J. Maridonneau-Parini I. J. Biol. Chem. 2000; 275: 36223-36229Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Direct confocal fluorescence analysis of HeLa cells transiently expressing p59Hck or p59Hckca showed the association of both proteins with the plasma membrane (Fig. 1) (9Carreno S. Gouze M.E. Schaak S. Emorine L.J. Maridonneau-Parini I. J. Biol. Chem. 2000; 275: 36223-36229Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The shape of cells expressing p59Hckca was dramatically modified by the formation of plasma membrane protrusions (Fig. 1). This phenotype was observed in 57.2 ± 5.3% of the cells expressing p59Hckca (488 total cells counted out of three representative experiments). It is notable that this phenotype was obtained even at low levels of p59Hckca expression as assessed by the heterogeneous intensity of fluorescence in cells forming protrusions (see the cell shown by an arrow in Fig. 1, C and D). Because membrane extensions are associated with reorganization of the actin cytoskeleton, the effects of activated p59Hck on F-actin organization were examined by a rhodamine-phalloidin labeling. Cells expressing p59Hck showed a similar pattern of F-actin as nontransfected cells, whereas cells expressing p59Hckca displayed a clear reorganization of their actin filaments with disappearance of stress fibers and actin polymerization at the periphery in GFP-enriched membrane protrusions corresponding to sites of p59Hckca localization (Fig. 2, D–I). It can also be noted that, as previously described, the Golgi apparatus was stained by p59Hck (Figs. 1 A and 2 A) (9Carreno S. Gouze M.E. Schaak S. Emorine L.J. Maridonneau-Parini I. J. Biol. Chem. 2000; 275: 36223-36229Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). p59Hckca was also associated with the Golgi, which was dispersed as vesicles into the cytoplasm (Fig. 2, J–L). A similar Golgi phentotype is observed in cells treated with microtubule depolymerizing agents (24Thyberg J. Moskalewski S. Exp. Cell Res. 1999; 246: 263-279Crossref PubMed Scopus (297) Google Scholar). However, the microtubule network was not affected in cells expressing the constitutively active p59Hckca (data not shown). The mechanisms involved in disruption of the Golgi apparatus by the action of p59Hckcaare presently under study in the laboratory. Because Hck is a phagocyte-specific Src-like kinase, we also studied the effect of its overexpression in the murine macrophage cell line, J774.A1. When J774 cells are grown on glass coverslips, their morphology is generally reminiscent of that of motile cells (i.e. they adopt an elongated, polarized shape that shows an F-actin-rich leading edge). cDNA constructs were microinjected in the nucleus of cells. As shown in Fig. 3, microinjection per se did not affect cell morphology. In some cases, GFP-expressing cells displayed numerous, long filopodia, contrasting with the few short filopodia associated with control cells (12.8 ± 1.8% of 226 cells counted of three experiments). By contrast, overexpression of GFP-tagged p59Hck (data not shown) or p59Hckca induced the formation of membrane protrusions and localized actin-rich ruffles in which Hck-GFP accumulated (Fig. 3) in 53.1 ± 1.9% (252 cells counted of three experiments) or 57.9 ± 3.7% (90 cells counted of three experiments) of the cells, respectively. Since the association of p59Hck with the plasma membrane is dependent on palmitoylation (9Carreno S. Gouze M.E. Schaak S. Emorine L.J. Maridonneau-Parini I. J. Biol. Chem. 2000; 275: 36223-36229Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), we constructed a palmitoylation mutant of p59Hckca to determine whether formation of membrane protrusions is due to its presence at the plasma membrane. The palmitoylated cysteine residue at position 3 of p59Hckca was substituted by a serine (p59C3SHckca). This mutant was not associated with the plasma membrane but was redistributed into cytoplasmic vesicles previously characterized as lysosomes (9Carreno S. Gouze M.E. Schaak S. Emorine L.J. Maridonneau-Parini I. J. Biol. Chem. 2000; 275: 36223-36229Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Whereas about 60% of the cells expressing p59Hckca had membrane protrusions, none of the cells expressing p59C3SHckcapresented these structures (Fig. 4,A–C). Src kinases comprise a tyrosine kinase activity and SH2 and SH3 adaptor functions. To distinguish which of these two properties was involved in the formation of plasma membrane protrusions, the lysine residue responsible for ATP binding was substituted by a glutamic acid in p59Hckca. This mutation has previously been shown to lead to a kinase-less, adaptor-plus form of Src kinases with dominant negative properties for kinase activity (p59Hckdn) (25Fan G. Shumay E. Malbon C.C. Wang H. J. Biol. Chem. 2001; 276: 13240-13247Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar,26Lionberger J.M. Wilson M.B. Smithgall T.E. J. Biol. Chem. 2000; 275: 18581-18585Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). HeLa cells expressing p59Hckdn never showed formation of membrane protrusions or disorganization of stress fibers (Fig. 4,D–F). We noticed that p59C3SHckcaor p59Hckdn mutants were redistributed to focal adhesion structures, where they colocalized with vinculin, a usual marker of these sites (27Geiger B. Volk T. Volberg T. J. Cell Biol. 1985; 101: 1523-1531Crossref PubMed Scopus (113) Google Scholar) (Fig. 4, G–L). Similarly, p59Hckca co-localized with vinculin, mostly at the edge of membrane extensions (Fig. 4, M–O). Therefore, neither kinase activity nor plasma membrane attachment is required for association to focal adhesions. The presence of p59Hckca at focal adhesion sites was not isoform-specific, since we observed that p61Hckca, the lysosome-associated isoform, was also targeted to focal adhesions as determined by its colocalization with vinculin (Fig. 5). p61Hckdnwas also found in focal adhesion plaques, indicating that the adaptor function of the protein is sufficient for this targeting. The SH2 domain is responsible for phosphotyrosine recognition and the SH3 domain for binding to proline-rich domains. To determine whether adaptor domains were implicated in formation of membrane protrusions, several deletions were made in p59Hckca cDNA (Fig. 6 A). The deletion mutants expressed in HeLa cells migrated in SDS gels at the expected molecular weights (p59(ΔSH2-SH3)Hckca, p59(ΔSH2)Hckca, and p59(ΔSH3)Hckca) (Fig. 6 B). Each of these mutants remained localized at the plasma membrane, but those deleted for both SH2 and SH3 domains failed to promote membrane protrusions (Fig. 6 C) and modifications of the actin cytoskeleton (data not shown). Similarly, the p59(ΔSH2)Hckca was unable to promote membrane protrusions, whereas cells expressing p59(ΔSH3)Hckcashowed these cytoskeletal modifications at a similar rate as cells transfected by p59Hckca (Fig. 6 C), indicating that interaction of the kinase with its partners involved in formation of membrane protrusions requires the SH2 but not the SH3 domain. Pseudopodia are membrane structures involved in phagocytosis of IgG-coated particles by FcγRs (28Kaplan G. Scand. J. Immunol. 1977; 6: 797-807Crossref PubMed Scopus (169) Google Scholar). Src PTKs are activated upon FcγR aggregation (29Daeron M. Annu. Rev. Immunol. 1997; 15: 203-234Crossref PubMed Scopus (1050) Google Scholar), and Hck has been described to be physically associated with the FcγRIIa receptor (13Ghazizadeh S. Bolen J.B. Fleit H.B. J. Biol. Chem. 1994; 269: 8878-8884Abstract Full Text PDF PubMed Google Scholar). Since we have shown above that constitutive activation of p59Hck is sufficient to trigger actin rearrangements leading to formation of membrane protrusions, this led us to hypothesize that p59Hck might link the Fcγ receptors to the actin cytoskeleton. Expression of the FcγRIIa receptor confers phagocytic capacities to non-phagocytic cells in contact with IgG-coated particles (30Downey G.P. Botelho R.J. Butler J.R. Moltyaner Y. Chien P. Schreiber A.D. Grinstein S. J. Biol. Chem. 1999; 274: 28436-28444Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Because of the well known functional redundancy of Src PTKs, it is likely that ubiquitous members of the Src PTK family replace the phagocyte-specific kinases that are lacking in these cells. Indeed, in macrophages from Hck, Lyn, and Fgr triple knockout mice, phagocytosis of IgG-coated particles is still dri" @default.
• W2067544586 created "2016-06-24" @default.
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• W2067544586 creator A5024675355 @default.
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• W2067544586 date "2002-06-01" @default.
• W2067544586 modified "2023-10-18" @default.
• W2067544586 title "p59Hck Isoform Induces F-actin Reorganization to Form Protrusions of the Plasma Membrane in a Cdc42- and Rac-dependent Manner" @default.
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