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• W2014148669 abstract "The plasma membrane contains ordered lipid domains, commonly called lipid rafts, enriched in cholesterol, sphingolipids, and certain signaling proteins. Lipid rafts play a structural role in signal initiation by the high affinity receptor for IgE. Cross-linking of IgE-receptor complexes by antigen causes their coalescence with lipid rafts, where they are phosphorylated by the Src family tyrosine kinase, Lyn. To understand how lipid rafts participate in functional coupling between Lyn and FcϵRI, we investigated whether the lipid raft environment influences the specific activity of Lyn. We used differential detergent solubility and sucrose gradient fractionation to isolate Lyn from raft and nonraft regions of the plasma membrane in the presence or absence of tyrosine phosphatase inhibitors. We show that Lyn recovered from lipid rafts has a substantially higher specific activity than Lyn from nonraft environments. Furthermore, this higher specific activity correlates with increased tyrosine phosphorylation at the active site loop of the kinase domain. Based on these results, we propose that lipid rafts exclude a phosphatase that negatively regulates Lyn kinase activity by constitutive dephosphorylation of the kinase domain tyrosine residue of Lyn. In this model, cross-linking of FcϵRI promotes its proximity to active Lyn in a lipid raft environment. The plasma membrane contains ordered lipid domains, commonly called lipid rafts, enriched in cholesterol, sphingolipids, and certain signaling proteins. Lipid rafts play a structural role in signal initiation by the high affinity receptor for IgE. Cross-linking of IgE-receptor complexes by antigen causes their coalescence with lipid rafts, where they are phosphorylated by the Src family tyrosine kinase, Lyn. To understand how lipid rafts participate in functional coupling between Lyn and FcϵRI, we investigated whether the lipid raft environment influences the specific activity of Lyn. We used differential detergent solubility and sucrose gradient fractionation to isolate Lyn from raft and nonraft regions of the plasma membrane in the presence or absence of tyrosine phosphatase inhibitors. We show that Lyn recovered from lipid rafts has a substantially higher specific activity than Lyn from nonraft environments. Furthermore, this higher specific activity correlates with increased tyrosine phosphorylation at the active site loop of the kinase domain. Based on these results, we propose that lipid rafts exclude a phosphatase that negatively regulates Lyn kinase activity by constitutive dephosphorylation of the kinase domain tyrosine residue of Lyn. In this model, cross-linking of FcϵRI promotes its proximity to active Lyn in a lipid raft environment. Cross-linking of IgE-FcϵRI complexes on mast cells by multivalent antigens initiates a series of signaling events that culminate in the exocytosis of mediators of the allergic response. The earliest detectable biochemical event following receptor aggregation is phosphorylation of the ITAM sequences on FcϵRI β and γ subunits by Src family tyrosine kinases, primarily Lyn (1Kinet J.P. Annu. Rev. Immunol. 1999; 17: 931-972Crossref PubMed Scopus (860) Google Scholar, 2Parravicini V. Gadina M. Kovarova M. Odom S. Gonzalez-Espinosa C. Furumoto Y. Saitoh S. Samelson L.E. O'Shea J.J. Rivera J. Nat. Immunol. 2002; 3: 741-748Crossref PubMed Scopus (402) Google Scholar). The mechanism by which cross-linking promotes this initial phosphorylation remains unclear. One current hypothesis focuses on the interaction of Lyn kinase with FcϵRI. Metzger and colleagues (3Pribluda V.S. Pribluda C. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11246-11250Crossref PubMed Scopus (175) Google Scholar, 4Yamashita T. Mao S.Y. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11251-11255Crossref PubMed Scopus (160) Google Scholar) described evidence that a small percentage of Lyn is constitutively associated with FcϵRI on RBL-2H3 mast cells but is unable to phosphorylate these receptors in the absence of aggregation. In the transphosphorylation model proposed in these studies, Lyn can only phosphorylate an adjacent FcϵRI following aggregation of two or more receptors. Subsequent studies from the Metzger laboratory showed that the β subunit of FcϵRI is capable of weak interactions with Lyn in the absence of phosphorylation (5Vonakis B.M. Chen H. Haleem-Smith H. Metzger H. J. Biol. Chem. 1997; 272: 24072-24080Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 6Vonakis B.M. Haleem-Smith H. Benjamin P. Metzger H. J. Biol. Chem. 2001; 276: 1041-1050Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). However, other studies showed that signaling occurs via human FcϵRI in the absence of the β subunit (7Alber G. Miller L. Jelsema C.L. Varin-Blank N. Metzger H. J. Biol. Chem. 1991; 266: 22613-22620Abstract Full Text PDF PubMed Google Scholar, 8Jurgens M. Wollenberg A. Hanau D. de la Salle H. Bieber T. J. Immunol. 1995; 155: 5184-5189PubMed Google Scholar) and that β plays an amplifying role in FcϵRI signaling (9Dombrowicz D. Lin S. Flamand V. Brini A.T. Koller B.H. Kinet J.P. Immunity. 1998; 8: 517-529Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 10Lin S. Cicala C. Scharenberg A.M. Kinet J.P. Cell. 1996; 85: 985-995Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). An alternative model focuses on the ordered lipid environment of lipid rafts that are proposed to facilitate the productive interaction between aggregated FcϵRI and Lyn (11Field K.A. Holowka D. Baird B. J. Biol. Chem. 1997; 272: 4276-4280Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 12Holowka D. Baird B. Semin. Immunol. 2001; 13: 99-105Crossref PubMed Scopus (88) Google Scholar). Lipid rafts are enriched in cholesterol, sphingolipids, and glycerophospholipids with saturated acyl chains and can be isolated due to their insolubility in nonionic detergents at 4 °C (13Fridriksson E.K. Shipkova P.A. Sheets E.D. Holowka D. Baird B. McLafferty F.W. Biochemistry. 1999; 38: 8056-8063Crossref PubMed Scopus (246) Google Scholar, 14Brown D.A. London E. J. Membr. Biol. 1998; 164: 103-114Crossref PubMed Scopus (841) Google Scholar). A large percentage of cellular Lyn fractionates with lipid rafts following sucrose gradient analysis of Triton X-100-lysed RBL mast cells (11Field K.A. Holowka D. Baird B. J. Biol. Chem. 1997; 272: 4276-4280Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 15Field K.A. Holowka D. Baird B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9201-9205Crossref PubMed Scopus (272) Google Scholar). Cholesterol depletion by methyl-β-cyclodextrin reversibly inhibits antigen-stimulated tyrosine phosphorylation of FcϵRI and in parallel causes reversible loss of both Lyn and cross-linked FcϵRI from lipid rafts (16Sheets E.D. Holowka D. Baird B. J. Cell Biol. 1999; 145: 877-887Crossref PubMed Scopus (288) Google Scholar). Thus, either the association of Lyn or FcϵRI or both with rafts is important for initiating this process. Previous studies that focused on the relatively low abundance of proteins in isolated lipid rafts suggested that localization of signaling proteins in rafts serves to concentrate them and thereby promote signaling (17Xavier R. Brennan T. Li Q. McCormack C. Seed B. Immunity. 1998; 8: 723-732Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar, 18Liu P. Anderson R.G. J. Biol. Chem. 1995; 270: 27179-27185Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar). However, it is now clear that these ordered regions of the plasma membrane constitute a large percentage of its lipid and surface area (19Gidwani A. Holowka D. Baird B. Biochemistry. 2001; 40: 12422-12429Crossref PubMed Scopus (138) Google Scholar, 20Maxfield F. Curr. Opin. Cell Biol. 2002; 14: 483-487Crossref PubMed Scopus (246) Google Scholar). To gain more insight about the mechanism by which lipid rafts facilitate functional coupling between cross-linked FcϵRI and Lyn, we investigated the role of membrane environment on the kinase activity of Lyn. We find that Lyn solubilized from RBL mast cells by Triton X-100 represents a subset of Lyn with reduced kinase activity, and this subset largely fractionates with nonraft proteins in sucrose gradients. Lyn isolated with lipid rafts has a substantially higher kinase activity than Lyn from nonraft fractions, and this increase in activity correlates with greater tyrosine phosphorylation of raft-associated Lyn in its kinase domain. Cross-linking of FcϵRI does not increase the overall kinase activity of Lyn in these cells but rather promotes functional coupling by causing co-compartmentalization of these receptors with the active form of Lyn in the raft environment. Differential Detergent Solubility—RBL-2H3 cells (21Barsumian E.L. Isersky C. Petrino M.G. Siraganian R.P. Eur. J. Immunol. 1981; 11: 317-323Crossref PubMed Scopus (485) Google Scholar) were suspended at 8–10 × 106 cells/ml in bovine serum albumin containing buffered saline solution (BSS (16Sheets E.D. Holowka D. Baird B. J. Cell Biol. 1999; 145: 877-887Crossref PubMed Scopus (288) Google Scholar)) and lysed by mixing 1:1 (v/v) with a detergent solution for a final concentration of either 0.5% Triton X-100 or RIPA 1The abbreviations used are: RIPA, radioimmune precipitation assay; PNS, postnuclear supernatant; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. detergents (0.5% Triton X-100, 0.5% deoxycholate, and 0.05% SDS) in lysis buffer (11Field K.A. Holowka D. Baird B. J. Biol. Chem. 1997; 272: 4276-4280Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Cells were lysed for 10 min on ice and then clarified by microcentrifugation (14,800 × g) for 20 min at 4 °C to yield a postnuclear supernatant (PNS). In some experiments, half of the Triton X-100-lysed cells were loaded directly on a sucrose gradient, the other half were microcentrifuged for 20 min, and the PNS was loaded. The amount of Lyn solubilized by RIPA was determined by quantitative anti-Lyn Western blot data (see below) through comparisons of RIPA-lysed samples before and after clarification. The relative amount of Lyn solubilized in the Triton X-100 lysate was determined by comparing Lyn immunoprecipitation yields from RIPA PNS with Triton X-100 PNS. Sucrose Gradient Fractionation—RBL-2H3 cells were lysed at 3 × 107 cells/ml in 0.25% Triton X-100 in lysis buffer for 10 min on ice. The lysates were then brought to 40% sucrose by a 1:1 (v/v) dilution with an 80% sucrose stock solution, and 1 ml of this solution was applied to the bottom of an 11 × 60-mm centrifuge tube (Beckman Instruments, Inc., Palo Alto, CA). 2 ml of 30% sucrose and then 1 ml of 5% sucrose were layered above the lysate. Samples were ultracentrifuged as previously described for 12–18 h at 250,000 × g (15Field K.A. Holowka D. Baird B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9201-9205Crossref PubMed Scopus (272) Google Scholar). After ultracentrifugation, samples were fractionated to obtain lipid raft and nonlipid raft fractions. These fractions were diluted 2-fold with lysis buffer containing RIPA for subsequent immunoprecipitation of Lyn. Lyn Immunoprecipitation and in Vitro Kinase Assay—Lyn was immunoprecipitated from 0.5–1 ml of detergent extracts by incubation with 2 μg anti-Lyn mouse monoclonal antibody H6 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1–2 h on ice and then rotated for 45 min with 35 μl of ImmunoPure Immobilized Protein A (Pierce) at 4 °C. Immunoprecipitates were washed twice with lysis buffer without detergent and then once with kinase assay buffer (20 mm Tris, pH 7.6, 10 mm MgCl2, and 1 mm Na3VO4). After washing, Lyn immunoprecipitates were subjected to in vitro kinase assays by adding 200 μl of kinase assay buffer containing either 1 mm ATP, no ATP, or 1 mm ATP and 100 μg of dephosphorylated α-casein (Sigma) as an exogenous substrate. Samples were then incubated at either 37 °C for samples without α-casein, or 30 °C for samples with α-casein, for 15 min. Reactions were quenched by the addition of 50 μlof5× nonreducing SDS sample buffer (15Field K.A. Holowka D. Baird B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9201-9205Crossref PubMed Scopus (272) Google Scholar) followed immediately by boiling. Immunoblotting—Samples were separated by electrophoresis on 10% acrylamide SDS gels under nonreducing conditions, and then electrophoresed proteins were transferred to an Immobilon P membrane (Millipore Corp., Bedford, MA) with a Panther semidry electroblotter (Owl Separation Systems, Inc., Portsmouth, NH). Anti-phosphotyrosine blotting was performed using 0.1 μg/ml 4G10 mouse monoclonal antibody conjugated to horseradish peroxidase (Upstream Biotechnology, Inc., Lake Placid, NY) diluted in a 1:1 (v/v) mixture of Tris-buffered saline solution (TBST) (50 mm Tris, pH 7.6, 150 mm NaCl2, and 0.1% (v/v) Tween 20) and StabilZyme SELECT (SurModics, Eden Prairie, MN). Blots were then stripped by incubation with 0.2 m NaOH for 5–30 min and quenched with TBST. Anti-Lyn blotting was performed on the stripped blots using 0.2 μg/ml anti-Lyn rabbit polyclonal antibody 44 (Santa Cruz Biotechnology) and a horseradish peroxidase-labeled antirabbit Ig secondary antibody (Amersham Biosciences), both in a solution of 0.8% bovine serum albumin in TBST. All immunoblots were developed as previously described (15Field K.A. Holowka D. Baird B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9201-9205Crossref PubMed Scopus (272) Google Scholar). Quantitation of Lyn Basal Phosphorylation and Specific Activity— Western blots were scanned from film with an Epson Expression 1600 digital scanner (Epson, Long Beach, CA), and density was determined using UnScanit software (Silk Scientific, Orem, UT); intensities of multiple samples on a single blot were normalized to a single sample in each blot. Lyn basal phosphorylation is defined as the amount of phosphorylation on immunopurified Lyn after an in vitro kinase incubation in the absence of ATP. Lyn specific activity is defined as the amount of phosphorylation on immunopurified Lyn following an in vitro kinase assay incubation in the presence of ATP minus the basal phosphorylation of a parallel sample. Values were calculated by dividing the normalized intensity of tyrosine phosphorylation on Lyn from the 4G10 blot by the normalized intensity of Lyn from the reprobed anti-Lyn blot in the same lane from nonsaturated film following Western blotting. Alternatively, specific activity was determined as the intensity of phosphorylated α-casein following an in vitro kinase assay incubation in the presence of ATP and α-casein in the kinase assay buffer, divided by the amount of Lyn in the same lane. Phosphopeptide Mapping of Lyn—Lyn was immunopurified using 35 μl of anti-Lyn conjugated to agarose beads (Santa Cruz Biotechnology) and 1–2 × 108 cell equivalents of RBL-2H3 cells lysed in the RIPA detergent buffer as above. Some Lyn samples were subjected to autophosphorylation in the in vitro kinase assay prior to elution and mapping. Lyn was eluted from the anti-Lyn beads by incubation with 0.1 m glycine HCl, pH 2.5. Next, the sample was dried using vacuum centrifugation (Thermo Savant, Holbrook, NY) and exchanged to 70% formic acid containing 100 mg/ml cyanogen bromide (CNBr) (ICN Biomedicals, Inc., Aurora, OH) to digest the protein overnight at room temperature in the dark. The following day, 500 μl of H2O was added to digests, and this mixture was then evaporated to dryness in the vacuum centrifuge. This wash step was repeated three more times, and dried samples were solubilized in 30–50 μl of 1× sample buffer (125 mm Tris, pH 6.8, 4% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.2 mg/ml bromphenol blue). Half of the sample was run on a 16.5% acrylamide Tricine gel (Bio-Rad) and then transferred to an Immobilon PSQ membrane (Millipore Corp.) by semidry transfer. Membranes were subsequently blocked for 4 h with 4% bovine serum albumin in TBST and then probed with anti-phosphotyrosine 4G10, washed, and detected by chemiluminescence as described above. Lyn Isolated from Triton X-100 Postnuclear Supernatants Has Reduced Specific Activity—Initial experiments compared the specific activity of Lyn isolated from RBL-2H3 cells lysed with 0.5% Triton X-100 with that for Lyn isolated from cells lysed with RIPA detergents. Unstimulated RBL-2H3 cells were lysed in the presence of phosphatase inhibitors, and Lyn was immunoprecipitated from the PNS. Western blotting with anti-Lyn showed that 95 ± 1% of the total cellular Lyn was recovered from the RIPA PNS, whereas only 40 ± 9% of Lyn was recovered from the Triton X-100 PNS (Fig. 1A). The amount of protein solubilized in each PNS is approximately equal for Triton X-100 versus RIPA lysis. 2R. M. Young, D. Holowka, and B. Baird, unpublished results. Additionally, equivalent amounts of uncross-linked FcϵRI were solubilized by these two detergent preparations as determined by fluorescent IgE quantitation in PNSs. 2R. M. Young, D. Holowka, and B. Baird, unpublished results. By Western blotting with anti-phosphotyrosine monoclonal antibody 4G10, we determined both basal tyrosine phosphorylation of Lyn and in vitro kinase autophosphorylation of Lyn under conditions similar to those in a previous study from our laboratory (15Field K.A. Holowka D. Baird B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9201-9205Crossref PubMed Scopus (272) Google Scholar). Blots were then stripped, and the amount of Lyn present in each sample was determined by anti-Lyn Western blotting (Fig. 1B). Basal tyrosine phosphorylation of Lyn from both Triton X-100- and RIPA-solubilized Lyn (Fig. 1C, white bars) is small compared with tyrosine phosphorylation of Lyn following the incubation with ATP, although Lyn from RIPA PNS consistently has slightly higher levels of basal phosphorylation. The specific kinase activity for Lyn autophosphorylation (Fig. 1C, shaded bars) is 4.5-fold higher in Lyn recovered from RIPA PNS compared with Triton X-100 PNS. Thus, Lyn isolated following Triton X-100 solubilization represents a subset of total Lyn with relatively low specific activity that is less than 10% of the total cellular Lyn activity. To investigate further the effects of the two different lysis conditions on Lyn specific activity, we performed additional control experiments. To test whether RIPA enhances Lyn kinase activity, we washed Triton X-100-solubilized, immunoprecipitated Lyn with RIPA prior to the in vitro kinase reaction. We found no difference in kinase activity of Lyn with and without the RIPA wash, indicating no differential effects of detergents on Lyn kinase activity. 2R. M. Young, D. Holowka, and B. Baird, unpublished results. Also, because of the large differences in Lyn recovery from RIPA and Triton X-100 PNS, we verified that results obtained were independent of the concentration of Lyn under the range of concentrations used for the in vitro kinase assay (Fig. 1C, inset). The Triton X-100 Postnuclear Supernatant Is Depleted of Raft-associated Lyn—The insolubility of a substantial percentage of cellular Lyn following RBL-2H3 lysis in 0.5% Triton X-100 led us to examine the distribution of solubilized Lyn in the Triton X-100 PNS by sucrose gradient analysis. Fig. 2 compares the distribution of this Lyn, obtained as PNS (Fig. 1A), with the distribution of Lyn from cells lysed in the same concentration of Triton X-100 but directly loaded onto a sucrose gradient without precentrifugation. The representative blots shown in Fig. 2 illustrate that the Triton X-100 PNS is significantly depleted of lipid raft-associated Lyn; 4% of the Lyn from the PNS fractionates in the lipid raft region of the gradient, whereas 45% of the total cellular Lyn is found in the raft fraction under these conditions. Similar results were observed for lysis in 1% Triton X-100, indicating that Lyn solubility is not limited by the amount of detergent under these conditions. 2R. M. Young, D. Holowka, and B. Baird, unpublished results. As observed previously (25Holowka D. Sheets E.D. Baird B. J. Cell Sci. 2000; 113: 1009-1019Crossref PubMed Google Scholar), the alternatively spliced 53- and 56-kDa forms of Lyn are more highly resolved in the nonraft fractions in Fig. 2. These results, taken together, indicate that Triton X-100 lysis yields a PNS that is selectively depleted of lipid raft-associated Lyn. Raft-associated Lyn Has Enhanced Kinase Activity—The results of Fig. 2 suggest that the difference in specific activity between Lyn from Triton X-100 and RIPA PNSs could be due to different amounts of raft-associated Lyn. This interpretation is consistent with studies by Ilangumaran et al. (22Ilangumaran S. Arni S. van Echten-Deckert G. Borisch B. Hoessli D.C. Mol. Biol. Cell. 1999; 10: 891-905Crossref PubMed Scopus (117) Google Scholar) and Kabouridis et al. (23Kabouridis P.S. Janzen J. Magee A.L. Ley S.C. Eur. J. Immunol. 2000; 30: 954-963Crossref PubMed Scopus (292) Google Scholar) indicating that the Src family kinases Lck and Fyn are differentially regulated by their lipid environment. To investigate directly whether the lipid raft environment affects Lyn activity, we isolated Lyn from either lipid raft or nonraft fractions obtained from sucrose gradient separation of RBL cells lysed by Triton X-100. A high concentration of cells was used in these experiments to permit sufficient recovery of Lyn from gradient fractions for immunoprecipitation and in vitro kinase analysis. The ratio of detergent to cells chosen is similar to that used in previous studies to preserve the interactions between cross-linked IgE-FcϵRI complexes and lipid rafts (11Field K.A. Holowka D. Baird B. J. Biol. Chem. 1997; 272: 4276-4280Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 15Field K.A. Holowka D. Baird B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9201-9205Crossref PubMed Scopus (272) Google Scholar). Under these conditions, uncross-linked IgE-FcϵRI was fully solubilized, fractionating with the nonlipid raft components, indicating complete plasma membrane lysis. 2R. M. Young, D. Holowka, and B. Baird, unpublished results. The sucrose gradient distribution of Lyn is shown in Fig. 3A and is similar to that in Fig. 2 and in previous experiments carried out under these conditions (11Field K.A. Holowka D. Baird B. J. Biol. Chem. 1997; 272: 4276-4280Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Lipid raft-associated Lyn was recovered from the interface between the 5 and 30% sucrose layers (fraction 2; Fig. 2), and nonraft Lyn was recovered from the soluble lysate in the 40% sucrose fraction (fraction 5; Fig. 2). Both fractions were then diluted into RIPA buffer, and Lyn was immunoprecipitated and analyzed as in Fig. 1. Representative Western blots from these experiments are shown in Fig. 3B. Consistent with results in Fig. 1C, we find that Lyn isolated from lipid rafts has a 3.6-fold higher specific activity than Lyn isolated from nonraft fractions (Fig. 3C, shaded bars). Also, the levels of basal phosphorylation are 6-fold higher for Lyn from the detergent-resistant lipid raft environment compared with solubilized Lyn (Fig. 3C, open bars). Thus, lipid raft-associated Lyn has substantially more kinase activity than Lyn from a more disordered membrane environment in this autophosphorylation assay. Furthermore, this higher specific activity correlates with higher basal phosphorylation of Lyn. To test the validity of conclusions based on in vitro Lyn autophosphosphorylation, we performed in vitro kinase assays using dephosphorylated α-casein as an exogenous substrate. We first determined that dephosphorylated α-casein is specifically phosphorylated by Lyn as indicated by a lack of phosphorylation in mock immunoprecipitations done without the Lyn antibody. 2R. M. Young, D. Holowka, and B. Baird, unpublished results. Activity was detected by quantitative Western blot analysis of the α-casein band with anti-phosphotyrosine and calculated per amount of Lyn detected by reprobing the blot as for the autophosphorylation assay. A representative Western blot is shown in Fig. 4A, and Fig. 4B summarizes the relative specific activities for Lyn from lipid raft and nonraft fractions. Consistent with the autophosphorylation results obtained in Figs. 1 and 3, raft-associated Lyn has a 4.7-fold higher specific activity toward α-casein than nonraft-associated Lyn. Specific Activity of Lyn Does Not Increase in Stimulated Cells—In previous studies, the total Lyn kinase activity associated with FcϵRI was shown to increase following antigen stimulation, but the specific activity of receptor-associated Lyn toward an exogenous substrate was found to be unchanged (4Yamashita T. Mao S.Y. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11251-11255Crossref PubMed Scopus (160) Google Scholar, 24Torigoe C. Metzger H. Biochemistry. 2001; 40: 4016-4025Crossref PubMed Scopus (14) Google Scholar). To determine whether the specific activity of total cellular Lyn is altered by antigen stimulation, IgE-sensitized RBL-2H3 cells were stimulated with an optimal dose of antigen (0.9 μg/ml denitrophenylated-bovine serum albumin) for various times. Lyn was then immunoprecipitated from RIPA-lysed cells, and in vitro kinase activity was determined with α-casein as an exogenous substrate. The results are summarized in Table I. Consistent with previous results from Metzger and colleagues (4Yamashita T. Mao S.Y. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11251-11255Crossref PubMed Scopus (160) Google Scholar, 24Torigoe C. Metzger H. Biochemistry. 2001; 40: 4016-4025Crossref PubMed Scopus (14) Google Scholar), there is little change in Lyn specific kinase activity following stimulation for 2 min at 37 °C. Interestingly, Lyn specific activity was found to decrease by 60% after 5 min of stimulation (Table I); this may be related to the decline in FcϵRI tyrosine phosphorylation observed at later times of antigen stimulation (25Holowka D. Sheets E.D. Baird B. J. Cell Sci. 2000; 113: 1009-1019Crossref PubMed Google Scholar) (see “Discussion”). These results indicate that FcϵRI cross-linking does not cause detectable activation of Lyn kinase per se. Rather, Lyn kinase activity in resting cells is sufficient for stimulated FcϵRI phosphorylation that results from cross-linking by antigen. We conclude that the increase in FcϵRI phosphorylation commonly observed after cross-linking results from changes in FcϵRI proximity to active Lyn.Table IAntigen-stimulated changes in Lyn kinase specific activityTime of stimulationRelative Lyn specific activityaDetermined for RIPA-solubilized Lyn using α-casein as in Fig. 4.min01.0020.94 ± 0.27bS.D. (n = 3).50.37 ± 0.09a Determined for RIPA-solubilized Lyn using α-casein as in Fig. 4.b S.D. (n = 3). Open table in a new tab Tyrosine 397 Phosphorylation Is the Predominant Detectable Site of Tyrosine Phosphorylation on Lyn from Unstimulated RBL Cells—Results summarized in Figs. 1, 3, and 4 indicate that the higher specific activity of Lyn isolated from a lipid raft environment correlates with higher basal tyrosine phosphorylation. This higher specific activity is preserved following solubilization of Lyn away from the ordered lipids of the raft membranes by RIPA. Thus, it is likely that a covalent modification of Lyn, such as phosphorylation, is responsible for the higher activity state. Lyn, like other Src family kinases, has two major sites of tyrosine phosphorylation that regulate activity: one in the active site at residue 397 (Lyn A notation) and a second at the C terminus at residue 508 (26Hibbs M.L. Dunn A.R. Int. J. Biochem. Cell Biol. 1997; 29: 397-400Crossref PubMed Scopus (41) Google Scholar). Phosphorylation of Tyr397 has been reported to increase the specific activity of Lyn by 17-fold (27Sotirellis N. Johnson T.M. Hibbs M.L. Stanley I.J. Stanley E. Dunn A.R. Cheng H.C. J. Biol. Chem. 1995; 270: 29773-29780Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), whereas phosphorylation of the C-terminal regulatory site, Tyr508, reduces Lyn activity, similar to other Src family members (28Honda Z. Suzuki T. Hirose N. Aihara M. Shimizu T. Nada S. Okada M. Ra C. Morita Y. Ito K. J. Biol. Chem. 1997; 272: 25753-25760Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). To investigate the relative abundance of tyrosine phosphorylation at these sites both before and after the in vitro kinase assay, we performed CNBr peptide mapping. CNBr cleavage of Lyn is predicted to yield separate fragments containing the active site and C-terminal site tyrosines with sizes of 8.2 and 4.1 kDa, respectively (Fig. 5). For these experiments, RIPA-solubilized Lyn was immunoprecipitated and subjected to an in vitro kinase incubation, with or without ATP, as in previous experiments, followed by treatment with CNBr. As shown in Fig. 5, basal Lyn phosphorylation is readily detectable in an ∼8-kDa fragment, consistent with the size expected for the minimal peptide containing Tyr397. Much less is detected in the ∼4-kDa peptide that is the size of the C-terminal fragment, similar to previous results in B-cells (29Hata A. Sabe H. Kurosaki T. Takata M. Hanafusa H. Mol. Cell. Biol. 1994; 14: 7306-7313Crossref PubMed Scopus (79) Google Scholar). After the in vitro kinase incubation with ATP, Lyn autophosphorylation is again preferentially found in the ∼8-kDa fragment, similar to trends observed in two previous studies (27Sotirellis N. Johnson T.M. Hibbs M.L. Stanley I.J. Stanley E. Dunn A.R. Cheng H.C. J. Biol. Chem. 1995; 270: 29773-29780Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 44Yanagi S. Sugawara H. Kurosaki M. Sabe H. Yamamura H. Kurosaki T. J. Biol. Chem. 1996; 271: 30487-30492Abstract Full Text Full Text PDF PubMed Scopus (" @default.
• W2014148669 created "2016-06-24" @default.
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• W2014148669 creator A5048526342 @default.
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• W2014148669 date "2003-06-01" @default.
• W2014148669 modified "2023-10-09" @default.
• W2014148669 title "A Lipid Raft Environment Enhances Lyn Kinase Activity by Protecting the Active Site Tyrosine from Dephosphorylation" @default.
• W2014148669 cites W1526116399 @default.
• W2014148669 cites W1950163772 @default.
• W2014148669 cites W1968664666 @default.
• W2014148669 cites W1969408386 @default.
• W2014148669 cites W1972145067 @default.
• W2014148669 cites W1990044954 @default.
• W2014148669 cites W1992692912 @default.
• W2014148669 cites W1993180174 @default.
• W2014148669 cites W1998865212 @default.
• W2014148669 cites W2000589601 @default.
• W2014148669 cites W2001759749 @default.
• W2014148669 cites W2016178838 @default.
• W2014148669 cites W2023965621 @default.
• W2014148669 cites W2024203551 @default.
• W2014148669 cites W2024912701 @default.
• W2014148669 cites W2032711419 @default.
• W2014148669 cites W2041255103 @default.
• W2014148669 cites W2046588698 @default.
• W2014148669 cites W2047533539 @default.
• W2014148669 cites W2048345403 @default.
• W2014148669 cites W2053204073 @default.
• W2014148669 cites W2059534402 @default.
• W2014148669 cites W2060922997 @default.
• W2014148669 cites W2064567527 @default.
• W2014148669 cites W2069488204 @default.
• W2014148669 cites W2072274588 @default.
• W2014148669 cites W2076833228 @default.
• W2014148669 cites W2080644669 @default.
• W2014148669 cites W2082393878 @default.
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