A Lipid Raft Environment Enhances Lyn Kinase Activity by Protecting the Active Site Tyrosine from Dephosphorylation

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 (16Sheets E.D. Holowka D. Baird B. J. Cell Biol. 1999; 145: 877-887Crossref PubMed Scopus (288) Google and by with a detergent for a of either Triton X-100 or used detergents Triton and in (11Field K.A. Holowka D. Baird B. J. Biol. Chem. 1997; 272: 4276-4280Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). were for on and by × for at 4 °C to a In of the Triton X-100-lysed cells were on a sucrose the other were for and the The of Lyn solubilized by by of and The of Lyn solubilized in the Triton X-100 by Lyn from with Triton X-100 cells were at × cells/ml in Triton X-100 in for on The were to sucrose by a with an sucrose and of this to the of an × of sucrose and of sucrose were the were described for at × K.A. Holowka D. Baird B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9201-9205Crossref PubMed Scopus (272) Google Scholar). were to lipid raft and raft were with for of Lyn. Lyn and in from of detergent by with for on and for with of A at 4 were with detergent and with kinase and Lyn were to in kinase by of kinase either or and of an were at either °C for or °C for with for were by the of K.A. Holowka D. Baird B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9201-9205Crossref PubMed Scopus (272) Google Scholar) by were by on and proteins were to an membrane with a to in a of and and were by with for and with on the and a both in a of bovine serum albumin in were described K.A. Holowka D. Baird B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9201-9205Crossref PubMed Scopus (272) Google Scholar). of Lyn and were from with an and of on a were to a in Lyn phosphorylation is the of phosphorylation on Lyn an in kinase in the absence of Lyn specific activity is the of phosphorylation on Lyn following an in kinase in the presence of the phosphorylation of a parallel were by the of tyrosine phosphorylation on Lyn from the by the of Lyn from the in the from following specific activity the of phosphorylated following an in kinase in the presence of and in the kinase by the of Lyn in the of of to and × of RBL-2H3 cells in the detergent Lyn were to in the in kinase to and Lyn from the by with the and to to the at in the The following of to and this to in the more and were solubilized in of and of the on a and to an membrane by were for 4 with bovine serum albumin in and with and by described Lyn from Triton X-100 the specific activity of Lyn isolated from RBL-2H3 cells with Triton X-100 with that for Lyn isolated from cells with RBL-2H3 cells were in the presence of phosphatase and Lyn from the with showed that of the cellular Lyn recovered from the only of Lyn recovered from the Triton X-100 The of solubilized in is for Triton X-100 M. D. and B. of FcϵRI were solubilized by these two detergent by in M. D. and B. with we both tyrosine phosphorylation of Lyn and in kinase of Lyn to in a from laboratory K.A. Holowka D. Baird B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9201-9205Crossref PubMed Scopus (272) Google Scholar). were and the of Lyn in by tyrosine phosphorylation of Lyn from both Triton and Lyn is small with tyrosine phosphorylation of Lyn following the with Lyn from has higher of The specific kinase activity for Lyn is higher in Lyn recovered from with Triton X-100 Thus, Lyn isolated following Triton X-100 represents a subset of Lyn with relatively low specific activity that is than of the cellular Lyn To the of the two on Lyn specific activity, we To whether Lyn kinase activity, we Triton Lyn with to the in kinase We in kinase activity of Lyn with and the differential of detergents on Lyn kinase M. D. and B. of the large in Lyn from and Triton X-100 we that were of the of Lyn the of used for the in kinase The Triton X-100 of insolubility of a percentage of cellular Lyn following RBL-2H3 in Triton X-100 to the of solubilized Lyn in the Triton X-100 by sucrose gradient the of this Lyn, with the of Lyn from cells in the of Triton X-100 but a sucrose gradient The in that the Triton X-100 is of lipid raft-associated of the Lyn from the fractionates in the lipid raft of the of the cellular Lyn is in the raft these were for in Triton that Lyn solubility is not by the of detergent these M. D. and B. D. Sheets E.D. Baird B. J. Cell Sci. PubMed Google the and of Lyn are more in the nonraft in results, that Triton X-100 a that is of lipid raft-associated Lyn. Lyn of that the in specific activity between Lyn from Triton X-100 and be due to of raft-associated Lyn. is with studies by S. S. G. B. Biol. Cell. 1999; PubMed Scopus Google Scholar) and J. Eur. J. Immunol. PubMed Scopus Google Scholar) that the Src family and are by their lipid environment. To whether the lipid raft environment Lyn activity, we isolated Lyn from either lipid raft or nonraft from sucrose gradient of RBL cells by Triton A high of cells used in these to of Lyn from gradient for and in kinase The of detergent to cells is to that used in studies to 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). these IgE-FcϵRI with the raft plasma membrane M. D. and B. The sucrose gradient of Lyn is in and is to that in and in these (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 recovered from the between the and sucrose and nonraft Lyn recovered from the in the sucrose were and Lyn and in from these are in with in we find that Lyn isolated from lipid rafts has a higher specific activity than Lyn isolated from nonraft the of phosphorylation are higher for Lyn from the lipid raft environment with solubilized Lyn Thus, lipid raft-associated Lyn has substantially more kinase activity than Lyn from a more membrane environment in this Furthermore, this higher specific activity correlates with higher phosphorylation of Lyn. To the of on in Lyn we in kinase an We that is phosphorylated by Lyn by a of phosphorylation in the Lyn M. D. and B. by analysis of the with and of Lyn by the for the A is in and the specific for Lyn from lipid raft and nonraft with the in and raft-associated Lyn has a higher specific activity than Lyn. of Lyn in studies, the Lyn kinase activity associated with FcϵRI to increase following antigen but the specific activity of Lyn an to be T. Mao S.Y. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11251-11255Crossref PubMed Scopus (160) Google Scholar, C. Metzger H. Biochemistry. 2001; 40: PubMed Scopus Google Scholar). To whether the specific activity of cellular Lyn is by antigen RBL-2H3 cells were with an of antigen serum for Lyn from and in kinase activity with an The are in with from Metzger and colleagues T. Mao S.Y. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11251-11255Crossref PubMed Scopus (160) Google Scholar, C. Metzger H. Biochemistry. 2001; 40: PubMed Scopus Google is in Lyn specific kinase activity following for at Lyn specific activity to by of this be to the in FcϵRI tyrosine phosphorylation at of antigen D. Sheets E.D. Baird B. J. Cell Sci. PubMed Google Scholar) that FcϵRI cross-linking does not detectable of Lyn kinase Lyn kinase activity in cells is for FcϵRI phosphorylation that from cross-linking by We that the increase in FcϵRI phosphorylation commonly cross-linking from in FcϵRI proximity to active in Lyn kinase specific of Lyn specific for Lyn in for Lyn in in a the of on Lyn from RBL in and 4 that the higher specific activity of Lyn isolated from a lipid raft environment correlates with higher tyrosine higher specific activity is following of Lyn from the ordered of the raft by Thus, it is that a of Lyn, is for the higher activity Lyn, other Src family kinases, has two of tyrosine phosphorylation that in the active site at residue A and a at the at residue J. Cell Biol. 1997; PubMed Scopus Google Scholar). of has to increase the specific activity of Lyn by N. E. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google phosphorylation of the Lyn activity, to other Src family T. N. M. T. S. M. C. Y. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). To the abundance of tyrosine phosphorylation at these both and the in kinase we of Lyn is to the active site and site with of and these Lyn and to an in kinase with or in by with in Lyn phosphorylation is detectable in an with the for the is in the that is the of the to in A. H. T. M. H. Cell. Biol. 1994; 14: PubMed Scopus Google Scholar). the in kinase with Lyn is in the to in two studies N. E. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, S. H. M. H. H. T. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). of Lyn by it to whether the is the but the that phosphorylation at the active site loop not to tyrosine phosphorylation of Lyn in RBL Furthermore, the of phosphorylation following the in kinase in this and in and show that only a small of Lyn is phosphorylated in the that Lyn activity is primarily by phosphorylation at and that phosphorylation plays a role in the of Lyn kinase activity in RBL-2H3 for of raft-associated Lyn has a higher of phosphorylation than nonraft Lyn and Lyn to be phosphorylated primarily at We that lipid raft-associated Lyn has a higher of tyrosine phosphorylation than nonraft Lyn in RBL and this to its higher specific We were unable to isolate Lyn from sucrose gradient to of phosphorylated Lyn from lipid However, a of hypothesis is that the kinase activity of raft-associated Lyn be reduced by the of tyrosine To this we the specific of lipid raft-associated and nonraft Lyn from cells either in the presence in or in the absence of the tyrosine phosphatase and in lipid raft-associated Lyn isolated in the absence of phosphatase has a specific activity with Lyn isolated in the presence of phosphatase In raft-associated Lyn isolated in the absence of phosphatase has a kinase activity to that of Lyn isolated from nonraft environments. Thus, dephosphorylation of Lyn in the absence of tyrosine phosphatase substantially kinase activity of Lyn from lipid rafts, that this Lyn its higher specific activity from phosphorylation that is in the presence of phosphatase inhibitors. with the results, these that raft-associated Lyn is more active it has a higher of phosphorylation at the specific activity of Lyn is increased isolated from cells in the absence of phosphatase that the specific activity of Lyn the lipid raft environment be negatively by phosphorylation at studies (11Field K.A. Holowka D. Baird B. J. Biol. Chem. 1997; 272: 4276-4280Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, E.D. Holowka D. Baird B. J. Cell Biol. 1999; 145: 877-887Crossref PubMed Scopus (288) Google Scholar) evidence that FcϵRI phosphorylation by Lyn tyrosine kinase on lipid rafts, but it from studies how the raft environment this process. depletion causes both Lyn and cross-linked FcϵRI to their association with lipid rafts, of FcϵRI phosphorylation by this be due to the loss of an ordered lipid environment for either Lyn or FcϵRI or to the role of the lipid raft environment on Lyn kinase that the lipid raft environment regulates the specific activity of Lyn Lyn from a lipid raft environment has a substantially higher in kinase activity than raft-associated Lyn and Furthermore, this activity with or does not increase following antigen of Lyn that the active site is a site for tyrosine phosphorylation in phosphorylation at is primarily on Lyn isolated from lipid rafts, and dephosphorylation by of tyrosine phosphatase raft-associated Lyn kinase activity to to that of Lyn isolated from more regions of the We that of active Lyn with FcϵRI in lipid rafts antigen cross-linking in increased receptor phosphorylation and thereby initiation of initial used detergents to Lyn for and in kinase and we that Triton X-100 in of than of the cellular Lyn in the not and the of Lyn kinase were with the of Triton and Furthermore, the specific activity of Lyn solubilized by Triton X-100 to be substantially than that from and sucrose gradient analysis showed that the Triton X-100 of lipid raft-associated Lyn and lipid rafts K.A. Chen M.G. D.A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) not sucrose gradient fractionation is not for cells in these initial to the and of Lyn sucrose gradient of Triton X-100-lysed In the absence of a to a of the Lyn is recovered in the lipid raft The is either in in the of the gradient or at the of the where and other are results, with sucrose gradient analysis of the that Lyn associated with lipid rafts in the that with Triton X-100 in a of association of lipid rafts with the at the of in the sucrose this interaction is Lyn and raft-associated to at low Previous studies showed that lipid rafts on cells are for of the to the plasma membrane D. Sheets E.D. Baird B. J. Cell Sci. PubMed Google Scholar, T. Eur. J. Immunol. 1999; PubMed Scopus Google and other studies that Triton rafts a by the cellular following S. Biol. Cell. 1995; PubMed Scopus Google Scholar, M. S. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). that Lyn kinase activity from Triton X-100 represents a subset of its cellular activity that is of lipid raft-associated Lyn. to cellular Lyn, we find that antigen does not a detectable increase in Lyn tyrosine kinase activity of but rather in a in this activity at T. H. M. T. C. Y. Cell. Biol. PubMed Scopus Google Scholar) that a increase in Lyn kinase activity, which phosphorylation of and other occurs at in RBL-2H3 they solubilized cells in a of Triton X-100 and it is whether they a subset in lipid raft-associated Lyn. by H. N. M. T. J. Immunol. 2002; PubMed Scopus Google Scholar) via FcϵRI, is to lipid rafts via its to and this in a in FcϵRI tyrosine The in specific activity of Lyn we following antigen from the phosphorylation of raft-associated Lyn by and this to the in FcϵRI phosphorylation that We showed that of by causes FcϵRI phosphorylation to more in to antigen D. Sheets E.D. Baird B. J. Cell Sci. PubMed Google Scholar). Thus, it be to whether regulates Lyn kinase activity and whether is in this process. that tyrosine phosphorylation of the active site is a of Lyn kinase activity, both and antigen are with a role for phosphorylation a mechanism to Lyn kinase activity at of A in RBL-2H3 mast cells that of can increase of Lyn by phosphorylation T. N. M. T. S. M. C. Y. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). of Lyn its in these cells phosphorylation of FcϵRI and a increase by that phosphorylation of Lyn to its but is not the of antigen-stimulated FcϵRI the of this it is clear that phosphorylation plays a role in its activity in cells Annu. Rev. Cell Biol. 1997; 13: PubMed Scopus Google but this kinase only a increase in activity due to phosphorylation of its active site loop C. D. P. Eur. J. PubMed Scopus Google Scholar). In the increase in Lyn kinase activity that from phosphorylation this phosphorylation site more important in the overall of Lyn kinase activity N. E. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). Based on results, we propose a model for signal initiation in which Lyn in an ordered lipid raft environment is from dephosphorylation at its active In this Lyn with tyrosine which is from lipid rafts J. Cell Biol. 1996; PubMed Scopus Google Scholar). is not on RBL-2H3 cells M. Siraganian R.P. J. Immunol. 1995; 155: Google but it represents a large family of or more of which a role on Lyn kinase In model, cross-linking of IgE-FcϵRI by antigen lipid raft active Lyn more active Lyn and cross-linked FcϵRI. this active Lyn ITAM sequences on FcϵRI β and γ subunits to the signaling The of this lipid raft association of FcϵRI by sucrose gradient analysis (11Field K.A. Holowka D. Baird B. J. Biol. Chem. 1997; 272: 4276-4280Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, D. Sheets E.D. Baird B. J. Cell Sci. PubMed Google Scholar). in these lipid rafts be from which are and from Lyn or FcϵRI. Metzger and colleagues M. Metzger H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S.Y. Metzger H. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus (36) Google Scholar) that FcϵRI and other of Lyn or kinase are not from dephosphorylation is by Based on these and results, they that lipid rafts to from the of tyrosine However, in of cross-linked receptors Holowka D. Baird B. J. Cell Biol. PubMed Scopus Google and this of lipid In Kovarova M. P. R. L. Rivera J. P. Cell. Biol. 2001; PubMed Scopus Google Scholar) that Lyn with a acyl to with lipid rafts, but antigen-stimulated phosphorylation of FcϵRI with this Lyn with a saturated acyl be to with an ordered lipid but its interaction be and it be to the of Triton X-100 R. Biochemistry. 2001; 40: PubMed Scopus Google Scholar). Furthermore, the in these a domain to its it is whether this can be negatively by which to the of Lyn in the membrane environment is not clear whether the for FcϵRI dephosphorylation that occurs cross-linking are in the active site of these differential interactions with lipid this model and of these with FcϵRI and Lyn in which antigen-stimulated FcϵRI phosphorylation can be model that FcϵRI phosphorylation on of cross-linked receptor and Lyn from a phosphatase that is from lipid The role of lipid rafts to interactions between active Lyn and cross-linked FcϵRI is to the initial events of signal in RBL-2H3 mast We for on tyrosine