FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2068833136> ?p ?o ?g. }

• W2068833136 endingPage "4616" @default.
• W2068833136 startingPage "4606" @default.
• W2068833136 abstract "Article17 August 1998free access syk protein tyrosine kinase regulates Fc receptor γ-chain-mediated transport to lysosomes Christian Bonnerot Christian Bonnerot INSERM CJF 95-01, Institut Curie, Section Recherche, 12 rue Lhomond, 75005 Paris, France INSERM U429, Hopital Necker Enfants Malades, 149 rue de Sèvres, 75015 Paris, France Search for more papers by this author Volker Briken Volker Briken INSERM CJF 95-01, Institut Curie, Section Recherche, 12 rue Lhomond, 75005 Paris, France INSERM U429, Hopital Necker Enfants Malades, 149 rue de Sèvres, 75015 Paris, France Search for more papers by this author Valérie Brachet Valérie Brachet INSERM CJF 95-01, Institut Curie, Section Recherche, 12 rue Lhomond, 75005 Paris, France Search for more papers by this author Danielle Lankar Danielle Lankar INSERM CJF 95-01, Institut Curie, Section Recherche, 12 rue Lhomond, 75005 Paris, France Search for more papers by this author Sylvanie Cassard Sylvanie Cassard INSERM CJF 95-01, Institut Curie, Section Recherche, 12 rue Lhomond, 75005 Paris, France Search for more papers by this author Bana Jabri Bana Jabri INSERM U429, Hopital Necker Enfants Malades, 149 rue de Sèvres, 75015 Paris, France Search for more papers by this author Sebastian Amigorena Sebastian Amigorena INSERM CJF 95-01, Institut Curie, Section Recherche, 12 rue Lhomond, 75005 Paris, France Search for more papers by this author Christian Bonnerot Christian Bonnerot INSERM CJF 95-01, Institut Curie, Section Recherche, 12 rue Lhomond, 75005 Paris, France INSERM U429, Hopital Necker Enfants Malades, 149 rue de Sèvres, 75015 Paris, France Search for more papers by this author Volker Briken Volker Briken INSERM CJF 95-01, Institut Curie, Section Recherche, 12 rue Lhomond, 75005 Paris, France INSERM U429, Hopital Necker Enfants Malades, 149 rue de Sèvres, 75015 Paris, France Search for more papers by this author Valérie Brachet Valérie Brachet INSERM CJF 95-01, Institut Curie, Section Recherche, 12 rue Lhomond, 75005 Paris, France Search for more papers by this author Danielle Lankar Danielle Lankar INSERM CJF 95-01, Institut Curie, Section Recherche, 12 rue Lhomond, 75005 Paris, France Search for more papers by this author Sylvanie Cassard Sylvanie Cassard INSERM CJF 95-01, Institut Curie, Section Recherche, 12 rue Lhomond, 75005 Paris, France Search for more papers by this author Bana Jabri Bana Jabri INSERM U429, Hopital Necker Enfants Malades, 149 rue de Sèvres, 75015 Paris, France Search for more papers by this author Sebastian Amigorena Sebastian Amigorena INSERM CJF 95-01, Institut Curie, Section Recherche, 12 rue Lhomond, 75005 Paris, France Search for more papers by this author Author Information Christian Bonnerot1,2, Volker Briken1,2, Valérie Brachet1, Danielle Lankar1, Sylvanie Cassard1, Bana Jabri2 and Sebastian Amigorena1 1INSERM CJF 95-01, Institut Curie, Section Recherche, 12 rue Lhomond, 75005 Paris, France 2INSERM U429, Hopital Necker Enfants Malades, 149 rue de Sèvres, 75015 Paris, France ‡C.Bonnerot and V.Briken contributed equally to this work *Corresponding authors. E-mail: [email protected] The EMBO Journal (1998)17:4606-4616https://doi.org/10.1093/emboj/17.16.4606 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info B- and T-cell receptors, as well as most Fc receptors (FcR), are part of a large family of membrane proteins named immunoreceptors and are expressed on all cells of the immune system. Immunoreceptors' biological functions rely on two of their fundamental attributes: signal transduction and internalization. The signals required for these two functions are present in the chains associated with immunoreceptors, within conserved amino acid motifs called immunoreceptor tyrosine-based activation motifs (ITAMs). We have examined the role of the protein tyrosine kinase (PTK) syk, a critical effector of immunoreceptor-mediated cell signalling through ITAMs, in FcR-associated γ-chain internalization and lysosomal targeting. A point mutation in the immunoreceptor-associated γ-chain ITAM affecting syk activation, as well as overexpression of a syk dominant negative mutant, inhibited signal transduction without affecting receptor coated-pit localization or internalization. In contrast, blocking of γ-chain-mediated syk activation impaired FcR transport from endosomes to lysosomes and selectively inhibited the presentation of certain T-cell epitopes. Therefore, activation of the PTK syk is dispensable for receptor internalization, but necessary for cell signalling and for γ-chain-mediated FcR delivery to lysosomes. Introduction Antigen recognition by cells of the immune system is mediated by a large family of membrane receptors, called immunoreceptors (Daeron, 1997). B- and T-lymphocytes express clonally distributed receptors which recognize either soluble antigens, in the case of B-cell receptors (BcRs), or peptides associated with major histocompatibility complex (MHC) molecules, for T-cell receptors (TcRs). Virtually all cells of the immune system, including granulocytes, macrophages and dendritic cells, express receptors for antigen–antibody complexes, which recognize the Fc portion of immunoglobulins (FcRs) (Ravetch, 1994). TcRs, BcRs and FcRs are multimeric complexes composed of a ligand-binding module, which determines the specificity of ligand recognition, and a transducing module composed of two to six associated chains. These associated chains contain conserved amino acid motifs in their cytoplasmic tails, called immunoreceptor tyrosine-based activation motifs (ITAMs, DxxYxxL6xYxxL) (Reth, 1989; Cambier, 1995b). ITAMs couple immunoreceptors to intracellular effectors of signal transduction pathways (Cambier, 1995a). Upon multimerization, the ITAM tyrosine residues are phosphorylated by protein tyrosine kinases (PTK) of the src family (Cambier, 1995a). Phosphorylated ITAMs then recruit PTKs of the syk family (syk itself or ZAP70 in T cells), which activate several signalling effectors including the PLCγ and ras pathways, as well as the phosphatidylinositol 3′ kinase (PI3K) (Agarwal et al., 1993; Benhamou et al., 1993; Kiener et al., 1993; Kanakaraj et al., 1994; Kurosaki et al., 1994; Shen et al., 1994). An important step, after the engagement of immunoreceptors by their ligands, is their internalization and delivery to lysosomes. Internalization and degradation of the TcR (downmodulation) are thought to regulate T-cell activation (Valitutti et al., 1997), processes which possibly also operate for other immunoreceptors (such as the BcR or various FcRs). Internalization is also critical for peptide presentation to T lymphocytes after antigen or antigen–antibody complex recognition by the BcR or different FcRs, respectively (Lanzavecchia, 1990). Immunoreceptor endocytosis generally occurs through coated pits and vesicles. However, the signals required for immunoreceptor internalization are as yet ill defined. In the case of the TcR, the associated CD3 ϵ-chain bears a double leucine internalization signal, which also mediates direct transport from the trans-Golgi network (TGN) to endosomes (Letourneur and Klausner, 1992). Internalization of antigen-bound BcR requires tyrosine-based internalization signals present in the cytoplasmic tail of either the heavy chain of certain membrane immunoglobulin (mIg) isotypes (Weiser et al., 1994; Knight et al., 1997) or the mIg-associated Igα/Igβ heterodimer (Bonnerot et al., 1995). The FcR-associated γ-chain also contains internalization signals. Mutation of either of the two tyrosine residues in the γ-chain ITAM blocked both cell activation (Bonnerot et al., 1992) and efficient receptor internalization (Amigorena et al., 1992), suggesting a possible link between signalling and internalization. Thus, signal transduction and internalization/lysosomal transport are initiated simultaneously after immunoreceptor engagement. The cytosolic effectors of cell signalling have been analysed extensively, but very little is known about the pathways and effectors of immunoreceptor internalization and lysosomal transport. It is known, however, that the FcR-associated γ-chain and the PTK syk are involved in phagocytosis in macrophages (Greenberg et al., 1994, 1996; Crowley et al., 1997). Recently, the PTK lck has been shown to target TcRs for lysosomal degradation (D'oro et al., 1997), suggesting that effectors of cell signalling may also determine the intracellular fate of immunoreceptors. In order to analyse the relationships between signalling and intracellular transport of immune receptors, we performed an extensive mutagenesis of the ITAM of the FcR-associated γ-chain. One of the mutants we isolated, leucine 35 to alanine (L35A), blocked cell activation without affecting internalization. This mutant had lost the ability to activate the PTK syk completely, but localized to coated pits and was internalized with normal kinetics and efficiency. Importantly, the L35A mutation also blocked transport to the lysosomes completely, suggesting that the recruitment and/or activation of syk is required for lysosomal delivery. Furthermore, expression of a dominant negative truncated syk prevented cell activation through the γ-chain, but not receptor internalization. This mutant also blocked transport from endosomes to lysosomes and presentation of certain T-cell epitopes. Therefore, a major effector of signal transduction through immunoreceptors, the PTK syk, is also required for their lysosomal transport. Results Cell activation and internalization through the FcR-associated γ-chain The γ-chain is associated with different immunoreceptors, including FcϵRI, FcγRIII, FcγRI, FcγRIIA and some TcRs (Daeron, 1997; Ravetch and Kinet, 1991). To define precisely the signals required for γ-chain-mediated signalling and internalization, we prepared a large number of γ-chain mutants. A chimeric receptor composed of the two extracellular and the transmembrane domains of FcγRII and the cytosolic tail of the γ-chain was used to individually mutate amino acids 18–37 of the γ-chain ITAM to alanine (excluding amino acids 26–29; Figure 1). The recombinant mutated chimeric receptors were expressed stably by cDNA transfection in the FcγR negative IIA1.6 B lymphoma cell line. Bulk cell populations expressing FcγR chimeras were obtained after selection and used directly for testing the ability of the different receptors to induce cell activation and ligand internalization after the cross-linking of receptors with the rat anti-mouse Fc receptor mAb, 2.4G2 and F(ab′)2 fragments of mouse IgG anti-rat IgG (Bonnerot and Daeron, 1994). Cell activation was assessed by measuring the induction of interleukin (IL)-2 secretion in the cell culture supernatant; receptor internalization was assessed by measuring the proportion of intracellular radioactive ligand (iodinated 2.4G2). Figure 1.Mutagenesis analysis of the γ-chain cytoplasmic domain. IIA1.6 cells expressing FcγR/icγ chimeras harbouring the indicated mutation in the γ-chain cytoplasmic tail were tested for the induction of cell activation (black bars) and for the internalization of mutivalent Fc receptors ligands (grey bars). Cell activation through FcγR/icγ chimeras was triggered by culturing the cells with rat anti-mouse FcR 2.4G2 (10 μg/ml) and mouse anti-rat antiserum (30 μg/ml). In control experiments, the cells were stimulated through endogenous mIg using F(ab′)2 fragments of anti-mouse IgG rabbit antiserum. After 18 h of incubation at 37°C, IL-2 secretion was measured in the supernatant by a CTLL2 proliferation assay. The results are presented as the ratio between induced IL-2 secretion through FcγR/icγ chimera and endogenous mIgG2a by the same transfected cells. To measure receptor internalization, cells were incubated for 30 min at 4°C with iodinated 2.4G2 antibodies (10 μg/ml). The cells were then washed and the internalization was triggered with prewarmed solution of mouse anti-rat IgG (30 μg/ml). After 20 min of incubation at 37°C, the cells were put on ice and the rate of internalized 2.4G2 was determined after acidic elution of antibodies remaining at the cell surface. The results are presented as the rate of the cell-associated radioactivity after acidic or no treatment (bars represent the mean of three independent experiments). Download figure Download PowerPoint As shown in Figure 1, mutations at amino acids sites (Y21, Y32, L24 and K36) inhibited the activity of ITAM in both activation and internalization assays. The first three amino acids are highly conserved in different ITAMs and are known to be required for ITAM function. Lysine 36 is not conserved, but nevertheless was essential for both signalling and receptor-mediated internalization. Two mutants were found to selectively affect cell activation or receptor internalization. Mutation of aspartic acid at position 18 to alanine (D18A) blocked receptor internalization without affecting cell activation. Mutation of leucine at position 35 to alanine (L35A) had the opposite effect: cell activation was blocked, whereas internalization was not affected. Therefore, in the case of the FcR-associated γ-chain, receptor internalization and cell activation were not necessarily dependent on each other. Cell activation may occur in the absence of internalization (D18A mutant) and, conversely, internalization can proceed without effective signalling (L35A mutation). To further characterize the effect of the L35A mutant on receptor-mediated cell activation and ligand internalization, cells expressing the L35A chimeric receptors were cloned and three individual clones expressing similar levels of the mutant receptors were isolated and analysed. Results obtained with one of the clones are shown; similar results were obtained with the two other clones. We first tested whether the L35A mutant activated PTK syk. Wild-type and L35A FcγR/icγ-expressing cells were stimulated for 2 min at 37°C through FcRs [using the anti-FcR monoclonal antibody (mAb), 2.4G2] or through endogenous mIgG2a [using F(ab′)2 fragments of rabbit anti-mouse IgG]. Cell lysates were prepared and phosphotyrosine-containing proteins were immunoprecipitated with agarose-coupled anti-phosphotyrosine PT66 antibody and analysed by immunoblotting with HRP-coupled anti-phosphotyrosine mAb Py20 or rabbit anti-syk antisera. As shown in Figure 2A, B-cell stimulation through endogenous BcR or FcγR/icγ chimera induced the phosphorylation of similar pattern cellular substrates and PTK syk (Figure 2A, lower panel). Engagement of the BcR or FcγR/icγ chimera also increased the kinase activity of syk, as measured by the phosphorylation of a specific substrate, a glutathione S-transferase (GST) fusion protein containing HS1 polypeptide. In cells expressing the L35A-mutated FcγR/icγ chimera or the FcγRIIb2 (which contains no ITAM), FcR engagement did not induce either phosphorylation of cellular proteins and syk (Figure 2B, middle panels) or activation of syk PTK activity (Figure 2B). Endogenous mIg induced PTK activity and syk phophorylation efficiently in both cell lines (Figure 2A and B). Therefore, L35A mutation prevents phosphorylation and activation of syk tyrosine kinase. Figure 2.L35 mutation prevents syk phosphorylation induced by FcγR/icγ chimera. IIA1.6 cells expressing FcγRIIb2 receptors, FcγR/icγ chimera or L35A mutant were stimulated, or not stimulated, through endogenous BcR with F(ab′)2 fragments of rabbit anti-mouse IgG antibodies (15 μg/ml) or through Fc receptors with the rat anti-mouse FcR antibody 2.4G2 (20 μg/ml) then F(ab′)2 fragments of mouse anti-rat IgG (30 μg/ml). The cells were then washed and lysed with 0.5% Triton X-100. Cell lysates were immunoprecipitated with antiphosphotyrosine antibodies (A) or rabbit anti-syk antibodies (B). (A) Phosphoproteins were detected by Western blotting using HRP-coupled antiphosphotyrosine antibody, PY20, (upper panel) or a rabbit antiserum specific for the N-terminal end of the syk tyrosine kinase (lower panel). (B) The syk tyrosine kinase activity was determined by measuring the phosphorylation of a specific substrate, the GST–HS1 fusion protein, by Western blotting using HRP-coupled antiphosphotyrosine antibody, PY20, (upper panel). The amount of syk tyrosine kinase was determined, for each point on the same filter, by using a rabbit antiserum specific for the N-terminal end of the syk tyrosine kinase (lower panel). Download figure Download PowerPoint Activation of syk is dispensable for receptor internalization To examine the role of syk activation in γ-chain-mediated internalization, we next assessed, by electron microscopy, the ability of wild-type and L35A-mutated FcγR/icγ chimera to localize to coated pits. We have reported previously that the internalization of heterotrimeric type III Fc receptor or FcγR/icγ chimera, in contrast to type II Fc receptor, is only triggered by efficient receptor cross-linking (Bonnerot and Daeron, 1994). Therefore, to test whether L35 mutation affects the coated-pit localization of the FcR/icγ chimera, cells were sequentially incubated for 30 min at 4°C with the mAb 2.4G2 and a rabbit polyclonal anti-rat IgG and protein A coupled to 10 nm gold particles. After 2 min of receptor engagement at 37°C, both receptors were found in clathrin-coated pits and vesicules (Figure 3A–D). The frequencies of localization to these structures were not affected by the mutation, since 70.7 and 70% of gold-labelled receptors were found in clathrin-coated structures in cells expressing wild-type or L35A-mutated FcγR/icγ chimera, respectively. Figure 3.Recruitment of FcγR/icγ and L35A mutant in clathrin-coated pits. Cells were incubated sequentially for 30 min at 4°C with the mAb 2.4G2 and a rabbit polyclonal anti-rat IgG and protein A coupled to 10 nm gold particles. The cells were then incubated for 2 min at 37°C. In both FcγR/icγ- (A and B) and L35A- (C and D) expressing cells, the gold particles were present at the cell surface, in coated-pits and in coated-vesicles, bar = 100 nm. Download figure Download PowerPoint Next, we evaluated the kinetics and efficiency of immune complex internalization. Horse radish peroxidase (HRP)-containing immune complexes were bound to cells expressing the chimeric wild-type or mutant receptors at 4°C. The cells were then incubated at 37°C for different times and the amounts of internal HRP were measured. As shown in Figure 4A, no differences in the kinetics or efficiency of HRP immune complex internalization were found between the wild-type type III Fc receptor, FcγR/icγ chimera and the L35A mutant receptors. Together, these results show that the L35A mutation did not affect the early steps of receptor endocytosis and suggest that efficient induction of syk PTK activation is not required for coated-pit-mediated, ligand-induced uptake through the immunoreceptor-associated γ-chain. Figure 4.L35A mutation prevents immune complex degradation through the FcγR/icγ chimera but does not affect ligand internalization. (A) Internalization of the HRP/anti-HRP immune complexes by FcγR/icγ and L35A mutant or heterotrimeric type III FcγR (FcγRIII α,γ) was measured as described in Materials and methods. (B) Degradation of iodinated DNP-BSA/anti-DNP immune complexes was measured as described in Materials and methods. Download figure Download PowerPoint L35A mutation prevents transport from endosomes to lysosomes Next, we tested the effect of L35A mutation on the lysosomal transport of the chimeric receptor, first by assessing its ability to mediate degradation of internalized radiolabelled bovine serum albumin (BSA), and secondly by directly measuring transport to lysosomes by subcellular fractionation. Radiolabelled BSA immune complexes were incubated for various times at 37°C with cells expressing either the wild-type or the L35A mutant receptors. Degradation of the internalized immune complexes was estimated by measuring the TCA-soluble fraction of iodinated-immune complexes. As shown in Figure 4B, the type III FcR or FcγR/icγ chimera allowed immune complex degradation at similar amounts, whereas the L35A mutation inhibited the generation of TCA soluble counts by >90% during the first 2 h of culture, suggesting that lysosomal transport was blocked by the mutation. The delivery of HRP-containing immune complexes by wild-type or L35A mutant chimeras to lysosomes was measured directly by subcellular fractionation. Lysosomal fractions were isolated in continuous self-forming 25% Percoll gradients. Dense Percoll fractions contained most of the β-hexosaminidase activity (a lysosomal resident enzyme), whereas light Percoll fractions contained the majority of the alkaline phosphodiesterase activity (APDE, a marker of the plasma membrane), as well as early and late endosomes detected by the presence of rab5 and rab7 (Figure 5A). Figure 5.L35A mutation prevents immune complexes lysosomal transport through FcγR/icγ chimera. (A) Cells were fractionated on Percoll gradients. Fractions containing lysosomes were identified by measuring β-hexosaminidase activity and fractions containing plasma membranes were identified by measuring APDE activity. The content of each fraction was characterized by Western blotting using anti-rab5, anti-rab7 and anti-lamp1 specific antibodies. Each blot was quantified after scanning the films with a photocamera. (B) HRP/anti-HRP immune complexes were bound for 2 h at 4°C on cells expressing FcγR/icγ or L35A mutant then fractionated directly (time 0) or after 1 h incubation at 37°C (time 60 min). HRP activity was measured in each fraction using an enzymological assay. The results are presented as the percentage of the HRP activity contained in each fraction versus the total of the HRP activity contained in all the fractions. (C) The experiment was performed three times and the mean value of HRP activity was calculated in the four fractions containing β-hexosaminidase (dense fractions in black) or in the four fractions containing APDE (light fractions in grey). Download figure Download PowerPoint HRP immune complexes bound at 0°C to cells expressing the wild-type or L35A mutant chimeric receptors were found exclusively in Percoll light fractions (Figure 5B, upper panel). When the cells were incubated for 1 h at 37°C, a significant proportion of the HRP was found in dense Percoll fractions in the case of cells expressing the wild-type chimeric receptors (40–50%, Figure 5B and C). In contrast, in the case of cells expressing the L35A mutant chimeras, delivery to dense Percoll fractions was inhibited, with only 10–20% of the HRP activity found in dense Percoll fractions after 60 min (Figure 5B and C). These results show that the L35A mutation prevented γ-chain-mediated, ligand-induced lysosomal transport. Syk mutant prevents efficient syk kinase activation Different mechanisms might account for the effect of the L35A mutation on immunoreceptor lysosomal transport. One possibility was that overall cell activation through the ITAMs increased the efficiency of lysosomal transport and degradation of immune complex by indirect means. This possibility was excluded by co-activation experiments where cell activation (and syk activation) were induced through surface immunoglobulins (which are also ITAM-dependent). We found that co-activation through mIg did not modify the efficiency of immune complex degradation or transport to lysosomes in cells expressing the L35A mutant chimeras (not shown). A second possibility was that downstream effectors of cell activation through the ITAMs might be involved directly in endosome to lysosome transport of γ-chain-associated immunoreceptors. Indeed, a direct connection between PTK activation and receptor internalization has been established for PTK receptors (PTKR), such as the epidermal growth factor receptor (EGFR), since EGFR kinase activity was shown to be required for receptor recruitment into clathrin-coated pits (Lamaze and Schmid, 1995). To determine whether syk is involved directly in the lysosomal transport of immunoreceptors, we stably expressed a kinase-deficient syk mutant [corresponding to the two syk SH2 domains tagged with a hemagglutinin (HA) epitope] in FcγR/icγ- or FcγRIIb2-expressing B lymphoma cells. Individual clones were isolated by intracellular staining with anti-HA mAb (not shown) and expression of the dominant negative Syk was measured by Western blot using a rabbit anti-sera specific for a peptide contained in the N-terminal portion of the syk SH2 domains. As shown in Figure 6A, the syk mutant (34 kDa) was expressed in a 3- to 5-fold excess as compared with endogenous syk (72 kDa), in two independent clones expressing FcγR/icγ chimeras and in two clones expressing FcγRIIb2. Figure 6.Syk mutant inhibits the activation of endogenous syk tyrosine kinase. (A) Stable overexpression of a cDNA encoding the two SH2 domains of the rat syk tyrosine kinase in FcγRIIb2 receptors or FcγR/icγ chimera expressing cells was analysed by Western blotting using a rabbit antiserum specific for the N-terminal end of syk. The clones G4 and C2 (FcγRIIb2) or C3 and C7 (FcγR/icγ) are representative of a series of 40 independent clones characterized. The arrow heads indicate the endogenous syk tyrosine kinase (70 kDa) and the dominant negative mutant (34 kDa), respectively. (B and C) FcγR/icγ chimera cells overexpressing the dominant negative syk (G2 and C2 clones) mutant were stimulated or not through endogenous BcR or Fc chimera as described in Figure 4. The cells were then washed and lysed with 0.5% Triton X-100. Cell lysates were immunoprecipitated with antiphosphotyrosine antibodies (B) or rabbit anti-syk antibodies (C). (B) Phosphoproteins (upper panel) or syk tyrosine kinase (lower panel) were detected by Western blotting as in Figure 4. (C) The phosphorylation of syk kinase was detected after a kinase assay and products were analysed by 10% SDS–PAGE. The quantity of immunoprecipitated syk was controlled for each point by Western blotting (not shown). Download figure Download PowerPoint The effect of the syk mutant on the activation of the endogenous syk through FcγR/icγ chimera was analysed first. The cells were stimulated, or not, through chimera or endogenous mIgG2a, and phosphoproteins were immunoprecipitated and analysed by Western blotting using PY20 or anti-syk antibodies. Overexpression of the syk dominant negative mutant prevented the phosphorylation of intracellular proteins (Figure 6B, upper panels), as well as the phosphorylation of endogenous syk kinase (Figure 6B, lower panels), through the FcγR/icγ chimera. Induction of phosphorylation of endogenous proteins and syk by engagement of the mIg was also decreased, but not abolished (Figure 6B). This is possibly because of the high level of expression of sIgG2a in these cells. In addition, using in vitro kinase assays, we found that the syk mutant prevented autophosphorylation of syk after engagement of the FcγR/icγ chimera (Figure 6C). We also found that overexpression of syk SH2 domains led to a substantial inhibition of kinase activity after cell stimulation through the FcγR/icγ chimera and endogenous mIg measured by phosphorylation of the GST–HS1 fusion protein in vitro (not shown). Similar results were obtained with another independent clone (not shown). Together, these results show that expression of a dominant negative truncated syk PTK inhibits FcγR/icγ chimera-mediated activation of endogenous wild-type syk. Involvement of the PTK syk in γ-chain-mediated lysosomal transport Stable expression of the syk SH2 domains, and observed inhibition of the γ-chain-mediated activation of the endogenous syk, provided us with an experimental model to determine the involvement of this PTK in receptor internalization and lysosomal transport. We compared the effect of syk mutant overexpression on internalization and degadation of immune complexes FcγR/icγ-chimera- and FcγRIIb2-expressing cells. Internalization of HRP immune complexes and degradation of radiolabelled BSA immune complexes were determined as before. As shown in Figure 7A, FcγR/icγ- and FcγRIIb2-mediated immune complex internalization with the same kinetics and efficiencies in cells expressing, or not expressing, the syk mutant. In contrast, overexpression of the syk mutant strongly decreased the degradation of iodinated BSA immune complexes internalized through the FcγR/icγ chimera, whereas no effect was observed on FcγRIIb2-mediated degradation (Figure 7B). These results suggest that the syk mutant specifically inhibited transport of ITAM-containing immunoreceptors from endosomal to lysosomal compartments. Figure 7.syk mutant selectively affected lysosomal transport and immune complex degradation through γ-chain cytoplasmic tail. FcγRIIb2 receptors (triangles) or FcγR/icγ chimera (circles) positive cells, overexpressing (black) or not (white) syk dominant negative mutant were tested for the internalization of the HRP/anti-HRP immune complexes (A), and for the degradation of iodinated DNP-BSA/anti-DNP immune complexes (B). (C) Lysosomal transport of HRP/anti-HRP immune complexes through FcγR/icγ was compared in cells overexpressing (C3 clone) or not overexpressing syk mutant. Cell fractionation was performed using Percoll gradients as in Figure 4. The results correspond to the percentage of the HRP activity contained in each fraction/total of the HRP activity contained in all the fractions after 1 h incubation at 37°C of the C3 clone overexpressing the syk dominant negative mutant. The same experiment was performed twice and the mean value" @default.
• W2068833136 created "2016-06-24" @default.
• W2068833136 creator A5002659299 @default.
• W2068833136 creator A5011516091 @default.
• W2068833136 creator A5016417429 @default.
• W2068833136 creator A5043850885 @default.
• W2068833136 creator A5044323595 @default.
• W2068833136 creator A5064488610 @default.
• W2068833136 creator A5077261751 @default.
• W2068833136 date "1998-08-17" @default.
• W2068833136 modified "2023-10-18" @default.
• W2068833136 title "syk protein tyrosine kinase regulates Fc receptor gamma -chain-mediated transport to lysosomes" @default.
• W2068833136 cites W1510771719 @default.
• W2068833136 cites W1519260014 @default.
• W2068833136 cites W1533457677 @default.
• W2068833136 cites W1545383294 @default.
• W2068833136 cites W1569997399 @default.
• W2068833136 cites W1576402354 @default.
• W2068833136 cites W1618519163 @default.
• W2068833136 cites W1627612497 @default.
• W2068833136 cites W1965825453 @default.
• W2068833136 cites W1968576216 @default.
• W2068833136 cites W1977156769 @default.
• W2068833136 cites W1979815209 @default.
• W2068833136 cites W1980219322 @default.
• W2068833136 cites W1981260497 @default.
• W2068833136 cites W1982304503 @default.
• W2068833136 cites W1992605956 @default.
• W2068833136 cites W1995900983 @default.
• W2068833136 cites W1996319732 @default.
• W2068833136 cites W1999281989 @default.
• W2068833136 cites W2002258688 @default.
• W2068833136 cites W2003319634 @default.
• W2068833136 cites W2012177306 @default.
• W2068833136 cites W2015366690 @default.
• W2068833136 cites W2020614472 @default.
• W2068833136 cites W2025661319 @default.
• W2068833136 cites W2031370626 @default.
• W2068833136 cites W2042209841 @default.
• W2068833136 cites W2042471874 @default.
• W2068833136 cites W2045219246 @default.
• W2068833136 cites W2047997809 @default.
• W2068833136 cites W2053500488 @default.
• W2068833136 cites W2053817261 @default.
• W2068833136 cites W2062373388 @default.
• W2068833136 cites W2064551184 @default.
• W2068833136 cites W2072034032 @default.
• W2068833136 cites W2078991312 @default.
• W2068833136 cites W2154901790 @default.
• W2068833136 cites W2171387990 @default.
• W2068833136 cites W271097414 @default.
• W2068833136 cites W4249736906 @default.
• W2068833136 cites W80109766 @default.
• W2068833136 doi "https://doi.org/10.1093/emboj/17.16.4606" @default.
• W2068833136 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1170790" @default.
• W2068833136 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9707420" @default.
• W2068833136 hasPublicationYear "1998" @default.
• W2068833136 type Work @default.
• W2068833136 sameAs 2068833136 @default.
• W2068833136 citedByCount "78" @default.
• W2068833136 countsByYear W20688331362012 @default.
• W2068833136 countsByYear W20688331362013 @default.
• W2068833136 countsByYear W20688331362014 @default.
• W2068833136 countsByYear W20688331362015 @default.
• W2068833136 countsByYear W20688331362016 @default.
• W2068833136 countsByYear W20688331362017 @default.
• W2068833136 countsByYear W20688331362019 @default.
• W2068833136 countsByYear W20688331362020 @default.
• W2068833136 countsByYear W20688331362021 @default.
• W2068833136 countsByYear W20688331362022 @default.
• W2068833136 countsByYear W20688331362023 @default.
• W2068833136 crossrefType "journal-article" @default.
• W2068833136 hasAuthorship W2068833136A5002659299 @default.
• W2068833136 hasAuthorship W2068833136A5011516091 @default.
• W2068833136 hasAuthorship W2068833136A5016417429 @default.
• W2068833136 hasAuthorship W2068833136A5043850885 @default.
• W2068833136 hasAuthorship W2068833136A5044323595 @default.
• W2068833136 hasAuthorship W2068833136A5064488610 @default.
• W2068833136 hasAuthorship W2068833136A5077261751 @default.
• W2068833136 hasBestOaLocation W20688331362 @default.
• W2068833136 hasConcept C101544691 @default.
• W2068833136 hasConcept C170493617 @default.
• W2068833136 hasConcept C184235292 @default.
• W2068833136 hasConcept C2776165026 @default.
• W2068833136 hasConcept C2911166042 @default.
• W2068833136 hasConcept C35174551 @default.
• W2068833136 hasConcept C42362537 @default.
• W2068833136 hasConcept C55493867 @default.
• W2068833136 hasConcept C62478195 @default.
• W2068833136 hasConcept C86803240 @default.
• W2068833136 hasConcept C95444343 @default.
• W2068833136 hasConceptScore W2068833136C101544691 @default.
• W2068833136 hasConceptScore W2068833136C170493617 @default.
• W2068833136 hasConceptScore W2068833136C184235292 @default.
• W2068833136 hasConceptScore W2068833136C2776165026 @default.
• W2068833136 hasConceptScore W2068833136C2911166042 @default.
• W2068833136 hasConceptScore W2068833136C35174551 @default.
• W2068833136 hasConceptScore W2068833136C42362537 @default.

• next