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Negative regulation of Lyn protein-tyrosine kinase by c-Cbl ubiquitin-protein ligase in FceRI-mediated mast cell activation

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Negative regulation of Lyn protein-tyrosine kinase by c-Cbl ubiquitin-protein ligase in FceRI-mediated mast cell activation
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   ©Blackwell Publishing Limited  Genes to Cells(2003)  8  ,825–836  825  DOI: 10.1046/j.1365-2443.2003.00679.x  BlackwellPublishingLtd.Oxford,UKGTCGenestoCells1356-9597©BlackwellPublishingLtd?20038?OriginalArticleUbiquitinationofLyninmastcellsSKyoetal.  Negative regulation of Lyn protein-tyrosine kinase by c-Cbl ubiquitin-protein ligase in Fc εεεε  RI-mediated mast cell activation  Shinkou Kyo, Kiyonao Sada, Xiujuan Qu, Koichiro Maeno, S. M. Shahjahan Miah, Keiko Kawauchi-Kamata and Hirohei Yamamura*   Division of Proteomics, Department of Genome Sciences, Kobe University Graduate School of Medicine, Kobe, Japan Abstract  Background   :Recent studies have demonstrated thatc-Cbl functions as a ubiquitin-protein ligase towardimmune receptors and non-receptor protein-tyrosinekinase Syk by facilitating their ubiquitination andsubsequent targeting to proteasomes. However, it wasnot clear whether Src family kinase Lyn is regulatedby the Cbl family of ubiquitin-protein ligases.  Results  :Aggregation of the high affinity IgE receptor(Fc εεεε  RI) induces the rapid ubiquitination of Lyn inrat basophilic leukaemia RBL-2H3 cells. Treatmentof cells with a proteasome inhibitor enhances theubiquitination of Lyn. Stimulation of Fc εεεε  RI resultsin the association of Lyn with c-Cbl and Cbl-b, bothof which then become tyrosine phosphorylated.Co-transfection study shows that both c-Cbl andCbl-b could induce the ubiquitination of activatedLyn in COS cells. Furthermore, over-expression of membrane-anchored form of c-Cbl inhibits theFc εεεε  RI-mediated degranulation and cytokine geneproduction in RBL-2H3 cells by the down-regulationof the kinase activity of Lyn through the enhancedubiquitination.  Conclusions  :These results demonstrate that Lyn isdown-regulated by c-Cbl-mediated ubiquitination andsubsequent degradation in proteasome after Fc εεεε  RIstimulation in mast cells. Targeting of c-Cbl in the lipidraft results in the inhibition of Fc εεεε  RI-mediated mastcell activation.  Introduction   Activation of mast cells plays a critical role in the allergicreactions. Aggregation of the high-affinity IgE receptors(Fc   ε   RI) on mast cells initiates a number of biochemicalevents leading to the release of inflammatory mediatorsby degranulation and producing the multiple cytokinesand leukotrienes that cause allergic symptoms. Fc   ε   RIsignals are induced by the activation of the non-receptor type of protein-tyrosine kinase (PTK) Lyn which phos-phorylates tyrosine residues in the immunoreceptor tyrosine-based activation motif (ITAM) in the β   and γ    subunits of Fc   ε   RI (Nishizumi & Yamamoto 1997). Lynconstitutively localizes in the lipid raft (GEMs, glycolipid-enriched microdomains) due to its lipid modification(Field et al    . 1995). Phosphorylation of the ITAM of Fc   ε   RI then recruits and activates Syk, which is essentialfor propagating the downstream signals (Costello et al    .1996; Zhang et al    . 1996).Ubiquitin is a highly conserved 76-amino acid eukaryoticprotein that becomes conjugated to proteins through theconcerted action of three enzymes: a ubiquitin-activatingenzyme (E1), a ubiquitin-conjugating enzyme (E2) and aubiquitin-protein ligase (E3). Post-translational modifi-cation of proteins with ubiquitin is associated with thedegradation of protein by targeting to proteasomes(Weissman 2001). Recent studies have demonstratedthat the Cbl family of RING type E3 ubiquitin-proteinligases can recognize tyrosine phosphorylated both receptor and non-receptor type PTKs through the Src-homology2 (SH2) domain and induce their ubiquitination(Levkowitz et al    . 1998; Miyake et al    . 1998; Rao et al    . 2001).In mast cells, it was shown that the over-expression of c-Cbl results in an inhibition of the release of inflam-matory mediators (Ota & Samelson 1997). c-Cbl catalyses  Communicated by   : Tadashi Yamamoto*  Correspondence   : E-mail: yamamura@kobe-u.ac.jp   S Kyo et al.  Genes to Cells(2003)  8  ,825–836  ©Blackwell Publishing Limited  826   Fc   ε   RI-mediated ubiquitination of Fc   ε   RI   β   , γ    chains andSyk (Paolini & Kinet 1993; Paolini et al    . 2002; Youssef    et al    . 2002). Autophosphorylation of Tyr317 in the linker region of Syk becomes a putative docking site to recruitc-Cbl to the receptor complex (Deckert et al    . 1998; Yankee et al    . 1999; Sada et al    . 2000; Paolini et al    . 2002).In the present study, we have shown that Fc   ε   RI aggre-gation causes the ubiquitination of Lyn in RBL-2H3 cells.The Cbl-family of E3 ubiquitin-protein ligases associateswith the activated Lyn and mediates its ubiquitinationafter the receptor aggregation. Over-expression of themembrane-anchored form of c-Cbl results in a down-regulation of the kinase activity of Lyn. Furthermore, wefound that another Src family PTK Fyn, which is impor-tant for phosphatidylinositol-3 kinase (PI3-kinase) acti-vating complementary pathway in mast cells is alsoubiquitinated on Fc   ε   RI-stimulation (Parravicini et al    .2002). Expression of the membrane-anchored form of c-Cbl inhibits Fc   ε   RI-mediated mast cell activation,suggesting that the negative regulation of Lyn byubiquitin-protein ligases has a therapeutic potential for developing the novel anti-allergic drugs.  Results  Aggregation of Fc εεεε  RI induces the ubiquitination of Lyn in RBL-2H3 mast cells   Previously, the yeast two-hybrid screening demonstratedthat RING type E3 ubiquitin-protein ligase c-Cbl directlyassociates with Lyn (Tezuka et al    . 1996). Lyn associates withFc   ε   RI   β   in the unstimulated cells and triggers the acti-vation of mast cells upon antigen stimulation (Jouvin et al    .1994). Therefore, we tested whether or not stimulation of Fc   ε   RI induces the ubiquitination of Lyn. SensitizedRBL-2H3 monolayers were stimulated with the antigen,and cell lysates were immunoprecipitated with anti-Lynantibody (Fig. 1A, left panel). There were smeared slow-migrated proteins which reacted with anti-ubiquitin mAbin the immunoprecipitates of Lyn after the antigen-stimulation. A similar pattern was observed when thestripped membrane was re-probed with anti-Lyn anti-body (long exposure) (Fig. 1A, right panel). However,the amount of precipitated Lyn was not apparentlydecreased, suggesting that the limited amount of Lynprotein was ubiquitinated by Fc   ε   RI-stimulation (Fig. 1A,right panel, short exposure). To characterize the ubiqui-tination of Lyn in mast cells in detail, we transfected theexpression construct of Flag-tagged ubiquitin (pSVL-Flag-Ub) into RBL-2H3 cells and generated the stableclones. Ubiquitination of proteins was detectable byusing the immunoblotting with anti-Flag mAb (Soubeyran   et al    . 2002). Cloned lines were stimulated with the anti-gen for the indicated times and the ubiquitination of Lynwas analysed by the immunoblotting with anti-FlagmAb (Fig. 1B, left panel). Ubiquitination of Lyn rapidlyreached the maximum at 15 s and subsequently disap-peared at 300 s after the aggregation of Fc   ε   RI. A similar pattern was observed by re-probing the stripped mem-brane with anti-Lyn antibody (long exposure) (Fig. 1B,right panel). The smeared proteins were also observed inthe immunoblotting of anti-Flag immunoprecipitateswith anti-Lyn antibody at the same time course (Fig. 1C).Furthermore, the pre-treatment of cells with the protea-some inhibitor MG132 enhanced the ubiquitination of Lyn, compared with the control cells (Fig. 1D). Therefore,these results demonstrate that the aggregation of Fc   ε   RIinduces the ubiquitination and proteasome-mediateddegradation of Lyn in RBL-2H3 cells.Src-family kinase Fyn is critical for Fc   ε   RI-mediatedphosphorylation of Gab2 and activation of PI3-kinase inmast cells (Parravicini et al    . 2002). Therefore, we utilizedthe stable cell lines expressing Flag-Ub to examinewhether aggregation of Fc   ε   RI also induces the ubiqui-tination of Fyn or not. Immunoprecipitation and immu-noblotting analysis revealed that Fyn is ubiquitinated inRBL-2H3 cells upon Fc   ε   RI aggregation (Fig. 1E). Itsuggests that Fyn-Gab2-PI3-kinase pathway is negativelyregulated by the ubiquitination in mast cells.  Antigen-stimulation induces the temporal association of Lyn with Cbl family ubiquitin-protein ligases in RBL-2H3 cells   Recent findings have demonstrated that Cbl family pro-teins have E3 ubiquitin-protein ligase activity (Joazeiro   et al    . 1999). We have identified that RBL-2H3 cellsexpress a second member of the Cbl family protein Cbl-b, besides c-Cbl (Fig. 2). Antigen-stimulation inducedtyrosine phosphorylation of both c-Cbl and Cbl-b at thesimilar time course (Fig. 2A and B). In addition to a major band around 120 kDa which corresponds to either Cblor Cbl-b, there were other double bands which appearedwith a different time course (Fig. 2A and B, top panels).Stripping and re-probing the same membranes withanti-Lyn antibody revealed that both c-Cbl and Cbl-btemporarily associates with Lyn after the receptor aggre-gation in RBL-2H3 cells (Fig. 2A and B, lower panels).Presumably this association was mediated by the proline-rich regions of Cbl family proteins because GST-Lyn-SH3,but not SH2 domain, was capable of binding with bothc-Cbl and Cbl-b (Fig. 2C). Unlike the immunoprecipi-tation experiments, GST-Lyn-SH3 domain could bindto c-Cbl and Cbl-b in unstimulated cells. The discrepancy   Ubiquitination of Lyn in mast cells  ©Blackwell Publishing Limited  Genes to Cells(2003)  8  ,825–836  827 Figure 1 Aggregation of Fc ε RI induces the ubiquitination of Lyn and Fyn in RBL-2H3 cells. (A) RBL-2H3 cells were culturedovernight with anti-DNP monoclonal IgE for sensitization, and then stimulated for 1 min without (–) or with (+) 1 µ g/mL of antigen-DNP-BSA. Cells were solubilized in the denature lysis buffer to dissociate protein complexes. Cell lysates were pre-cleared with proteinA-agarose beads alone and then immunoprecipitated with anti-Lyn antibody pre-coupled to protein A-agarose beads.Immunoprecipitated Lyn was eluted by boiling at 100 ° C for 5 min in 2 ×  sample buffer, separated by SDS–PAGE and analysed byimmunoblotting with anti-ubiquitin mAb and anti-Lyn antibody (long and short exposures). (B) RBL-2H3 cells expressing Flag-Ub andparental control cells (ctrl.) were stimulated without (–Ag) or with (Ag) 1 µ g/mL of antigen for the indicated times. Cells were solubilizedin the denature lysis buffer and anti-Lyn immunoprecipitates were analysed by anti-Flag mAb and anti-Lyn antibody (long and shortexposures). (C) RBL-2H3 cells expressing Flag-Ub were stimulated without (–Ag) or with (Ag) antigen for the indicated times. Cellswere solubilized in the denature lysis buffer and anti-Flag immunoprecipitates were analysed by anti-Lyn antibody. (D) RBL-2H3 cellsexpressing Flag-Ub were pre-incubated with solvent alone (0.1% DMSO in the normal medium) or 50 µ m  MG132 for 4 h, thenstimulated with the antigen for 60 s. Cells were solubilized in the denature lysis buffer and anti-Lyn immunoprecipitates were analysedby immunoblotting with anti-Flag mAb. (E) Aggregation of Fc ε RI induces the ubiquitination of Fyn in RBL-2H3 cells. RBL-2H3 cellsexpressing Flag-Ub and control cells were incubated without (–Ag) or with (Ag) 1 µ g/mL of antigen for the indicated times. Cells weresolubilized in the denature lysis buffer and anti-Fyn immunoprecipitates were analysed by anti-Flag mAb and anti-Fyn antibody (longand short exposures). (B–E) Similar results were obtained when the other cloned lines expressing Flag-Ub were tested. Ubiquitinationof Lyn and Fyn (Lyn-Ub and Fyn-Ub) was indicated.   S Kyo et al.  Genes to Cells(2003)  8  ,825–836  ©Blackwell Publishing Limited  828   of Lyn-c-Cbl/Cbl-b interactions in unstimulated cellsbetween the immunoprecipitation and pull-down experi-ments may be due to the accessibility of the Lyn-SH3domain because Lyn is thought to form a closed-inactiveconformation in unstimulated cells. In fact, c-Cbl islocalized mainly in cytoplasmic fraction in unstimulatedcells and is translocated to the plasma membrane after theFc   ε   RI stimulation (Lafont & Simons 2001). Therefore,these results demonstrated that antigen stimulationinduces the temporal association of Lyn with the Cblfamily of ubiquitin-protein ligases in mast cells.  c-Cbl down-regulates Lyn by the ubiquitination in mast cells   To elucidate the function of Cbl family proteins on Lyn,first we tested their catalytic activities by using COS-7cells (Fig. 3A). As shown, co-expression of c-Cbl or Cbl-b facilitated the ubiquitination of the active form of Lyn(Y508F) rather than wild-type. This ubiquitination of Lyn was not attenuated by the mutation of SH2 domainin c-Cbl (G306E) or Cbl-b (G298E) (data not shown).Expression of the kinase-inactive form of Lyn (K275R)resulted in the decreased c-Cbl/Cbl-b-mediated ubiqui-tination (Fig. 3A). The mutant form of Lyn Y508F/K275R was not ubiquitinated as well as the constitu-tively active form of Lyn Y508F, suggesting that both thekinase activity of Lyn and the unlatching by the lack of C-terminal regulatory tyrosine residue are necessary for c-Cbl-mediated ubiquitination of Lyn (Harrison 2003)(Fig. 3A). The kinase activity of Lyn has a role in releas-ing its SH3 domain to form the active conformation of Lyn to interact with c-Cbl. A similar pattern was observedwhen the stripped membrane was re-probed with anti-Lynantibody (data not shown). The expression amount of Lyn Y508F was less than that of wild-type and thekinase-inactive form of Lyn, consistent with a previousreport which demonstrated that a kinase-active form of c-Src was less stable than wild-type (Harris et al    . 1999).Although there is a structural similarity between c-Cbl and Cbl-b, c-Cbl appears to be more important for T-cell receptor signalling by degradation of activatedmolecules in GEMs (Liu & Gu 2002). In mast cells,Fc   ε   RI stimulation results in the accumulation of activated Figure 2 Aggregation of Fc ε RI inducesthe association of Lyn with Cbl familyubiquitin-protein ligases. (A and B) For theindicated times, either unstimulated or antigen-stimulated RBL-2H3 cells weresolubilized in Triton lysis buffer and celllysates were immunoprecipitated withspecific anti-c-Cbl (A) or anti-Cbl-b (B)antibody. Immunoprecipitates were analysedby immunoblotting with the indicatedantibodies. (C)   Pull-down experiments.Either unstimulated or antigen-stimulatedRBL-2H3 cells were solubilized in thebinding buffer. Pre-cleared cell lysates wereincubated with GST, GST-Lyn-SH2, or GST-Lyn-SH3 fusion protein as indicated.Co-precipitated proteins and cell lysateswere separated by SDS–PAGE and analysedby immunoblotting with anti-c-Cbl or anti-Cbl-b antibody.   Ubiquitination of Lyn in mast cells  ©Blackwell Publishing Limited  Genes to Cells(2003)  8  ,825–836  829   signalling molecules into GEMs (Field et al    . 1997;Wilson et al    . 2001). In particular, Fc   ε   RI and c-Cbl asso-ciate with GEMs after activation of IgE signalling(Lafont & Simons 2001). Based on this idea, we con-structed a novel mutant of c-Cbl (   GEM    -Cbl), which isconstitutively anchored to the plasma membrane. Addi-tion of the 16 amino acids from the N-terminal regionof c-Src with single amino acid alteration (Ser3 to Cys)can direct the cytoplasmic molecule to GEMs (Honda   et al    . 2000; Sada et al    . 2001). The cloned RBL-2H3 celllines over-expressing GEM    -Cbl were generated by cDNAtransfection and positive clones were utilized for further experiments. The subcellular fractionation study showedthat the expression of GEM    -Cbl was restricted in the mem-brane fraction, prior to the antigen stimulation (Fig. 3B).By using the stable cell lines, we tested the followingexperiments. Compared with the parental RBL-2H3 cells,the over-expression of GEM    -Cbl resulted in the increasedFc   ε   RI-mediated ubiquitination of Lyn, suggesting that   GEM    -Cbl acts as a dominant-active form of c-Cbl (Fig.3C). Then, we analysed the antigen-induced tyrosinephosphorylation of Fc   ε RI to examine the effect of theexpression of GEM  -Cbl on the kinase activity of Lyn. Asshown, inducible tyrosine phosphorylation of Fc ε RI β and γ chains was drastically decreased in the cells over-expressing GEM  -Cbl, although the precipitated amountof the receptor protein was comparable (Fig. 3D). Then,the kinase activity of Lyn was measured by the in vitro  proteinkinase assay using the exogenous substrate enolase (Fig. 3E).Over-expression of GEM  -Cbl resulted in the reductionof the in vitro  protein kinase activity of Lyn after the anti-gen stimulation (Fig. 3E). Densitometric analysis showedthat phosphorylation of enolase by Lyn was decreased inthe cells over-expressing GEM  -Cbl (Fig. 3E). Furthermore,the pre-treatment of RBL-2H3 cells with the proteasomeinhibitor MG132 increased in the kinase activity of Lyn (Fig. 3F). These results demonstrate that targeting of c-Cbl to the plasma membrane down-regulates the kinaseactivity of Lyn by the ubiquitination and subsequentproteasome-mediated degradation. It suggests that theantigen stimulation causes the ubiquitination and degra-dation of activated Lyn in the Fc ε RI signalling pathway. Over-expression of c-Cbl in the plasma membrane inhibits the Fc εεεε RI-mediated degranulation and cytokine gene production in RBL-2H3 mast cells Finally, we examined the biological relevance of thetargeting of c-Cbl to the plasma membrane. At first weexamined the antigen-induced tyrosine phosphorylation of Syk, which is an essential molecule for the degranulation,cytokine synthesis and arachidonic acid metabolism(Costello et al  . 1996; Zhang et al  . 1996). As shown, theincrease in Syk tyrosine phosphorylation was dra-matically suppressed in the cells over-expressing GEM  -Cbl compared with that in the control cells (Fig. 4A).Then we tested the antigen-induced degranulationas assessed by the measurement of the release of β -hexosaminidase (Fig. 4B). Control cells and cells over-expressing GEM  -Cbl were stimulated with differentconcentrations of antigen. Antigen-induced degranulationwas inhibited by the over-expression of GEM  -Cbl. Tyrosinephosphorylation of Fc ε RI γ   and Syk was dramaticallyinhibited by the over-expression of GEM  -Cbl, while thedegranulation response was partially inhibited (Figs 3Dand 4A and B). This discrepancy might be explained thatSyk could multiply the Fc ε RI signals by phosphorylat-ing a number of substrates leading to the degranulation.In addition, we attempted to analyse the functionalinvolvement of GEM  -Cbl on Fc ε RI-mediated mast cellactivation by utilizing the cells transiently over-expressing GEM  -Cbl or GEM  -70Z/Cbl (Fig. 4C). 70Z/Cblloses the ability to promote the ubiquitination by thelack of 17 amino acids (Fig. 4C) (Andoniou et al  . 1994).Expression of GEM  -Cbl suppressed the antigen-induceddegranulation, while GEM  -70Z/Cbl did not suppressthe antigen-induced degranulation. This result confirmsthat the ubiquitin ligase activity is necessary for GEM  -Cbl-mediated negative regulation of degranulation inmast cells. Finally, we compared the antigen-inducedcytokine gene production between the parental RBL-2H3 cells and cells over-expressing GEM  -Cbl (Fig. 4D).Over-expression of GEM  -Cbl suppressed the increase inthe transcription of interleukin-3 (IL-3), IL-6, and tumour necrosis factor- α  (TNF- α ). Therefore, these observationsdemonstrate that over-expression of GEM  -Cbl inhibitsthe antigen-induced functional mast cell activation. Discussion Fc εεεε RI stimulation induces the ubiquitination of Lyn The ubiquitin proteolytic pathway plays a critical role inthe degradation of regulatory proteins which are impor-tant in a variety of basic cellular processes (Schwartz &Ciechanover 1999). Recently, it was shown that aggre-gation of Fc ε RI results in the immediate ubiquitinationof Fc ε RI β , γ   chains and Syk in mast cells and ubiquitin-protein ligase c-Cbl is responsible for the ubiquitinationof these proteins (Paolini et al  . 2002). In the presentstudy, we have demonstrated that aggregation of Fc ε RIrapidly induces the ubiquitination of two Src familyPTKs, Lyn and Fyn, which are essential for Fc ε RI-mediated downstream signalling in RBL-2H3 cells
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