The Unique Domain as the Site on Lyn Kinase for Its Constitutive Association with the High Affinity Receptor for IgE

Aggregation of the high affinity receptor for IgE (FcεRI) leads to the phosphorylation of tyrosines on the β and γ chains of the receptor by the Src family kinase Lyn. We have studied the interaction between Lyn and the FcεRI in vivo using a transfection-based approach. FcεRI were stably transfected into Chinese hamster ovary cells. The small amount of endogenous Src family kinase was sufficient to phosphorylate receptor tyrosines upon extensive aggregation of FcεRI but not after addition of dimers of IgE. Upon stable co-transfection of Lyn kinase into the cells, dimers were now able to stimulate receptor phosphorylation and the response to more extensive aggregation was enhanced. In contrast, co-transfection with catalytically inactive Lyn inhibited the aggregation-induced phosphorylation by the endogenous kinase, and a quantitatively similar inhibition was observed in cells transfected with the SH4-containing unique domain of Lyn. Consistent with the results of others using alternative approaches, our additional studies using a yeast two-hybrid system detected a direct interaction between intact Lyn or its unique domain and the C-terminal cytoplasmic domain of the β chain but not with the receptor's other cytoplasmic domains. Aggregation of the high affinity receptor for IgE (FcεRI) leads to the phosphorylation of tyrosines on the β and γ chains of the receptor by the Src family kinase Lyn. We have studied the interaction between Lyn and the FcεRI in vivo using a transfection-based approach. FcεRI were stably transfected into Chinese hamster ovary cells. The small amount of endogenous Src family kinase was sufficient to phosphorylate receptor tyrosines upon extensive aggregation of FcεRI but not after addition of dimers of IgE. Upon stable co-transfection of Lyn kinase into the cells, dimers were now able to stimulate receptor phosphorylation and the response to more extensive aggregation was enhanced. In contrast, co-transfection with catalytically inactive Lyn inhibited the aggregation-induced phosphorylation by the endogenous kinase, and a quantitatively similar inhibition was observed in cells transfected with the SH4-containing unique domain of Lyn. Consistent with the results of others using alternative approaches, our additional studies using a yeast two-hybrid system detected a direct interaction between intact Lyn or its unique domain and the C-terminal cytoplasmic domain of the β chain but not with the receptor's other cytoplasmic domains. The family of proteins known as the “multichain immune recognition receptors” includes the antigen receptors on B and T-lymphocytes and Fc receptors including the receptor with high affinity for IgE (FcεRI) 1The abbreviations used are: FcεRI, high affinity receptor for IgE; bp, base pair(s); BSA, bovine serum albumin; CHO, Chinese hamster ovary; DNP, dinitrophenyl; ITAM, immuno-recognition tyrosine-based activation motif; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Pipes, 1,4-piperazinediethanesulfonic acid; Tricine,N-tris(hydroxymethyl)methylglycine. (1Keegan A.D. Paul W.E. Immunol. Today. 1992; 13: 63-68Abstract Full Text PDF PubMed Scopus (173) Google Scholar). Highly homologous in structure, all these receptors utilize, at least in part, a common mechanism to initiate cellular responses; multivalent interactions with antigen lead to aggregation of the receptors and is followed by enhanced phosphorylation of tyrosines (in the “ITAM” motifs within the cytoplasmic domains) of the receptor itself by a receptor-associated Src family kinase (2Cambier J.C. J. Immunol. 1995; 155: 3281-3285PubMed Google Scholar). For FcεRI, we recently presented direct evidence for a “transphosphorylation” mechanism that accounts for the earliest events (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). The data showed that a small fraction of receptors are constitutively associated with the Src family kinase Lyn (4Yamashita T. Mao S.-Y. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11251-11255Crossref PubMed Scopus (160) Google Scholar, 5Eiseman E. Bolen J.B. Nature. 1992; 355: 78-80Crossref PubMed Scopus (417) Google Scholar) and that the enhanced phosphorylation that follows aggregation of the receptors is likely to result simply from the apposition of the kinase with its substrate. We have also shown that when the kinase available to the receptor is limited, shuttling of the enzyme between individual aggregates can regulate the intensity of the signal (6Torigoe C. Goldstein B. Wofsy C. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1372-1377Crossref PubMed Scopus (46) Google Scholar). 2C. Wofsy, C. Torigoe, U. M. Kent, H. Metzger, and B. Goldstein, submitted for publication. The experiments described in this paper mainly explored the sites of interaction between Lyn kinase and FcεRI. For the most part, the prior studies of others explored the interaction between Lyn and isolated portions of the receptor (7Jouvin M.-H.E. Adamczewski M. Numerof R. Letourneur O. Valle A. Kinet J.-P. J. Biol. Chem. 1994; 269: 5918-5925Abstract Full Text PDF PubMed Google Scholar, 8Lin S. Cicala C. Scharenberg A.M. Kinet J.P. Cell. 1996; 85: 985-995Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 9Wilson B.S. Kapp N. Lee R.J. Pfeiffer J.R. Martinez A.M. Platt Y. Letourneur F. Oliver J.M. J. Biol. Chem. 1995; 270: 4013-4022Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 10Kihara H. Siraganian R.P. J. Biol. Chem. 1994; 269: 22427-22432Abstract Full Text PDF PubMed Google Scholar). The yeast two-hybrid system (11Fields S. Sternglanz R. Trends Genet. 1994; 10: 286-292Abstract Full Text PDF PubMed Scopus (526) Google Scholar) used in some of our studies is an analogous approach. We also employed transfection techniques, which allowed us to examine the kinase-receptor interactions in a more physiological setting. The latter experiments also allowed us to test the effect of varying the level of Lyn on the responsiveness of the receptors to discrete stimuli, and thereby to test certain quantitative predictions made by the current model. The yeast strains (CG1945 and Y187) and cloning vectors (pAS2-1 and pACT) were obtained fromCLONTECH (Palo Alto, CA); the expression vectors pBlueBac, pCDM8, and pZeo, as well as a baculovirus MAXBAC expression kit from Invitrogen (Carlsbad, CA); polyacrylamide gels used for electrophoresis (PAGE) from NOVEX (San Diego, CA); the antibiotics (G418, zeocin) from Life Technologies, Inc. and Invitrogen, respectively; and plasmid DNA purification kits from Qiagen (Santa Clarita, CA). Monoclonal anti-phosphotyrosine (anti-Tyr(P)) antibodies were obtained conjugated to horseradish peroxidase from Transduction Laboratories (PY-20) or Upstate Biotechnology, Inc. (4G10). Polyclonal antibodies to human Src family kinases Lyn and Fyn were purchased from Upstate Biotechnology, Inc.; antibodies to c-Src and c-Yes were from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal anti-DNP IgE (12Liu F.T. Bohn J.W. Ferry E.L. Yamamoto H. Molinaro C.A. Sherman L.A. Klinman N.R. Katz D.H. J. Immunol. 1980; 124: 2728-2737PubMed Google Scholar) and rat IgE (of unknown specificity) (13Bazin H. Querinjean P. Beckers A. Heremans J.F. Dessy F. Immunology. 1974; 26: 713-723PubMed Google Scholar) were purified as described previously (14Holowka D. Metzger H. Mol. Immunol. 1982; 19: 219-227Crossref PubMed Scopus (79) Google Scholar, 15Kulczycki Jr., A. Metzger H. J. Exp. Med. 1974; 140: 1676-1695Crossref PubMed Scopus (223) Google Scholar) and labeled with carrier-free 125I using chloramine T (16McConahey P.J. Dixon F.J. Int. Arch. Allergy Appl. Immunol. 1966; 29: 185-189Crossref PubMed Scopus (1397) Google Scholar). Goat anti-mouse IgE was purchased from ICN (Costa Mesa, CA); rabbit anti-rat IgE was purified as described (17Taurog J.D. Mendoza G.R. Hook W.A. Siraganian R.P. Metzger H. J. Immunol. 1977; 119: 1757-1761PubMed Google Scholar). Covalently cross-linked IgE oligomers were prepared and analyzed as described (6Torigoe C. Goldstein B. Wofsy C. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1372-1377Crossref PubMed Scopus (46) Google Scholar). Rat basophilic leukemia (RBL-2H3) cells were maintained as described previously (18Barsumian E.L. Isersky C. Petrino M.G. Siraganian R.P. Eur. J. Immunol. 1981; 11: 317-323Crossref PubMed Scopus (485) Google Scholar). Chinese hamster ovary cells (CHO) were grown in stationary flasks at 37 °C in a humidified atmosphere containing 5% CO2 in Iscove's modified Eagle's minimum essential medium, and the antibiotics to expression of the transfected cells were maintained in at °C as described previously M. and E. Scholar). The of expression was by DNA using a kit obtained from CA). was prepared from isolated from cells. with or were to the The were prior to The was prepared in the expression U. PubMed Scopus Google Scholar). were prepared by of human Lyn the and the were The was and were with and The of and were by and DNA of The from the Inc. was used to and the of the isolated an of Lyn with from to Lyn B with between and The of Lyn in the was to a previously H. H. Siraganian R.P. Mol. Immunol. 1994; PubMed Scopus Google the of rat Lyn B an of in the of the kinase at a to that previously shown for human and Lyn Y. S. J. N. Yamamoto T. Mol. Biol. PubMed Scopus Google Scholar, Bolen J.B. Mol. Biol. 11: PubMed Scopus Google Scholar) but is to Lyn A. from the previously for rat Lyn B N. C. 1994; PubMed Scopus Google Scholar). of our Lyn B is to the Lyn for the The in the N. C. 1994; PubMed Scopus Google Scholar) from or with a Lyn and a Lyn B isolated from an our cells were transfected with the by and a of the cells was prepared using by and with Lyn that the proteins the the for Lyn and Lyn B not DNA domain the and C-terminal cytoplasmic of the rat were by from the and into sites of The cytoplasmic domain of rat as a was into of to activation domain the Lyn and Lyn B and were by and into sites of were into yeast cells by the were on containing and and to the or and to The of was in a with as or in a with to the The Lyn B was isolated from and into the of The unique domain was by using and for the unique and The was with and into the The catalytically inactive Lyn was prepared using the in system from as of Lyn B from was into the of followed the using the in the kit and a Lyn to The Lyn B was with and and into transfection Technologies, of the and γ of rat FcεRI with previously into cells, and a a high of receptors was expression of receptors with in we the by with IgE and on a The of cells the of receptors were on at were for expression of by the cells to and with IgE. The cells were with and the IgE in the by high stable and was used for all cells were by in 5% of a in FcεRI. The cells were in the of of rat or which by with the was with The human Lyn B was from by and into the homologous of cells were with DNA and the Lyn to Lyn cells were with baculovirus at a of of after in containing and with that the Lyn B was on as was in the cells not cells to with antigen were with anti-DNP in Pipes, and and at was as a to cells at 37 °C for the cells with IgE oligomers were with the at 37 °C for the the receptors were in (3Pribluda V.S. Pribluda C. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11246-11250Crossref PubMed Scopus (175) Google Scholar). For anti-mouse or anti-rat IgE was to of in containing The were by and with the with of for the were as described previously (3Pribluda V.S. Pribluda C. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11246-11250Crossref PubMed Scopus (175) Google and the proteins by in for receptors were by electrophoresis in on polyacrylamide gels with and the proteins detected with an and an enhanced system Metzger H. J. Immunol. 1992; Google Scholar). of were by CA). were to of receptors were in studies in which cells with inactive of Lyn were with cells that not the cells were with IgE that labeled and of were on gel was with the amount of human Lyn B to for in the were and with an to the β chain of the receptor J. Kinet J.-P. J. C. Metzger H. Mol. Immunol. PubMed Scopus (79) Google and was The from the were for in or of In the of was by of an and the the of containing or were prepared with for on which the were on or Lyn gels and with an and an on gel was with a amount of human Lyn B The for the to Lyn were to the human Lyn B cells were at and with IgE for at 37 was by the cells with a of IgE for at 37 were from IgE by Jr., A. Metzger H. J. Exp. Med. 1974; 140: 1676-1695Crossref PubMed Scopus (223) Google Scholar, E. D. M. Mol. Cell. PubMed Scopus Google Scholar). cells were and the and were prepared from the as described previously V.S. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar). proteins were in for at fraction was with an of for prior to gel in were with to the of was by containing the cytoplasmic of the FcεRI to the domain of the with containing Lyn or of Lyn to the activation domain of The for the and C-terminal cytoplasmic of the β of the rat IgE were into to DNA domain proteins the is to the between the DNA domain and the proteins Sternglanz R. S. Google Scholar). the containing the cytoplasmic domain of the γ was not we for and into the which is similar to but the or were The of the and in yeast Lyn and or were as described the and unique domain of Lyn and Lyn B with not the interaction was the interaction detected between the and proteins used as a of with and in more on and into of the containing and In contrast, with the Lyn and in and on and the interaction was detected between or with of Lyn in this the interaction between Lyn and or we the of these in yeast in a In addition to the Lyn and the containing the unique we a of Lyn on the results of C. J. J.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar) and Mol. Biol. 1992; PubMed Scopus Google Scholar). The in this was the to of Lyn to the activation domain shown in the of the from with the unique domain of Lyn or B was as high as that from with the of Lyn. are from the Consistent with the result from the interaction between Lyn and is the of the as that between and containing Lyn or of the interaction between Lyn and was of transfected cells that stably was with Lyn of these cells, of the cells, a at the of and observed for rat Lyn was with a of antibodies to human or c-Yes not from the were with IgE and after with the receptors were with anti-mouse IgE. Upon with evidence for phosphorylation was observed the cells were with multivalent antigen prior to phosphorylation of tyrosines on the β and γ of the transfected receptors was observed of the receptors in vivo by addition of after the to to the of the phosphorylation of receptor tyrosines within not Lyn stable FcεRI was by of FcεRI by were prepared by Lyn into cells and with cells, transfected with FcεRI and were prepared by the not shown in this are and were as For the of the Lyn in a of was by the of FcεRI For for the in this the was The latter by The of Lyn was to for the of the FcεRI was in amount of endogenous hamster Lyn and transfected inactive Lyn was by of with Lyn. The shown the of to Lyn Lyn Lyn stable FcεRI was by of FcεRI by were prepared by Lyn into cells and with cells, transfected with FcεRI and were prepared by the not The shown in this are and were as For the of the Lyn in a of was by the of FcεRI For for the in this the was The latter by The of Lyn was to for the of the FcεRI was in The amount of endogenous hamster Lyn and transfected inactive Lyn was by of with Lyn. The shown the of to in a cells can by IgE with antigen or by the cells with dimers of IgE In contrast, of the cells with IgE to phosphorylation of the receptors results are with a amount of kinase associated with the receptors in these cells of stable of the cells with rat Lyn were The of of are shown in the of of of the transfected cells that the transfected Lyn was as a as for a Src family kinase PubMed Scopus Google Scholar). The were with IgE dimers or with IgE and to examine the between the cellular of Lyn and the responsiveness of the cells. was to of receptors were shown in was a between the amount of Lyn and the amount of receptor phosphorylation on the β and γ upon aggregation of the receptors with all of the cells transfected Lyn now to dimers of IgE. extensive phosphorylation was observed in cells receptors were with antigen with the by dimers was more to the amount of Lyn as can by the of the response in which the was showed a of phosphorylation of the receptors of a with an of but in phosphorylation of tyrosines on other cellular proteins when with not for in phosphorylation that have to cells were transfected with and isolated and Upon with IgE from to the showed in phosphorylation of the β and γ of the receptor with cells In a similar the to varying of antigen of were with cells. similar of phosphorylation of the receptors was observed were in the or of cellular proteins that the level of endogenous Lyn was also the of FcεRI on the between and the of phosphorylation was to of the of receptors the used in this The in the cells of an endogenous kinase of the us to the on Lyn with the FcεRI by a We transfected the cells with of Lyn that with the receptor but that were catalytically We the responsiveness of to aggregation of FcεRI to or to cells with the catalytically inactive Lyn B kinase was prepared by to shown in in a in the Lyn was the and in a can a kinase the Lyn was stable of the Lyn were isolated and and The catalytically inactive Lyn was or as a a receptor stable showed phosphorylation of receptor tyrosines cells transfected with the a a that phosphorylation of tyrosines on FcεRI to that inhibited and by our results from the we transfected the unique domain of Lyn kinase into cells and The isolated unique domain was also or in a and between the to of the and cells not transfected with Lyn. Upon with multivalent a inhibition of phosphorylation of receptor tyrosines was observed of of the unique domain showed that of the domain to inhibition a inhibition of phosphorylation of the β and γ chains was observed at and at of of phosphorylation of tyrosines in FcεRI by transfected unique domain of Lyn cells and cells were with of IgE as described in the to A. cells were for varying of with IgE. of is the as in were for with varying of IgE. The data shown are the of and the the between have studied the interaction between FcεRI and Lyn kinase by a of Consistent with (7Jouvin M.-H.E. Adamczewski M. Numerof R. Letourneur O. Valle A. Kinet J.-P. J. Biol. Chem. 1994; 269: 5918-5925Abstract Full Text PDF PubMed Google Scholar, 8Lin S. Cicala C. Scharenberg A.M. Kinet J.P. Cell. 1996; 85: 985-995Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 9Wilson B.S. Kapp N. Lee R.J. Pfeiffer J.R. Martinez A.M. Platt Y. Letourneur F. Oliver J.M. J. Biol. Chem. 1995; 270: 4013-4022Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 10Kihara H. Siraganian R.P. J. Biol. Chem. 1994; 269: 22427-22432Abstract Full Text PDF PubMed Google the results from our studies in the yeast two-hybrid system a direct interaction between the kinase and the C-terminal cytoplasmic of the receptor's β interaction was detected between Lyn and or by the of a the interaction is is with the in of Lyn with FcεRI in the of (4Yamashita T. Mao S.-Y. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11251-11255Crossref PubMed Scopus (160) Google Scholar). results of in that the Lyn and Lyn B E. Bolen J.B. Nature. 1992; 355: 78-80Crossref PubMed Scopus (417) Google Scholar). result is also with our that the of Lyn to the receptor after (4Yamashita T. Mao S.-Y. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11251-11255Crossref PubMed Scopus (160) Google Scholar). we that the unique domain with the receptor as as the kinase, but the of the interaction to the of interaction more by this is that in this system the receptor is but this in the of the receptor is phosphorylation in the of aggregation V.S. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar, R. Kinet J.-P. Nature. PubMed Scopus Google Scholar). the interactions we observed the of Lyn with the receptor the interaction of Lyn with the receptor (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). The results with the cells transfected with Lyn evidence that the amount of Lyn available to the receptor the of the system to initiate The results with the cells transfected with catalytically inactive of Lyn evidence that an between receptor-associated and Lyn. The mechanism we that the of small aggregates of receptors to initiate a response to the amount of Lyn receptor (3Pribluda V.S. Pribluda C. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11246-11250Crossref PubMed Scopus (175) Google The of the in cells with dimers of IgE are more to the of Lyn with inactive Lyn inhibited by the receptor with cells The effect was more using of IgE in cells transfected with the unique and inhibition was detected at this is with the that small aggregates more to the of The effect of the catalytically inactive Lyn that the interaction between Lyn and the receptor is not on an intact on the the unique domain was as as the catalytically inactive Lyn in the interaction between the receptor and the endogenous Src family kinase in the cells. was not for the We previously that after an phosphorylation of the receptor by the constitutively Lyn kinase is for the of additional of Lyn to the receptor (3Pribluda V.S. Pribluda C. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11246-11250Crossref PubMed Scopus (175) Google Scholar). direct studies in have shown that the domain of Lyn can with the of the β H. Siraganian R.P. J. Biol. Chem. 1994; 269: 22427-22432Abstract Full Text PDF PubMed Google is to that the the interaction of the of the Lyn kinase with the high expression of the catalytically inactive which its have the level of phosphorylation the unique domain For in addition to of the endogenous kinase, have the of kinase to the the inactive Lyn have the from D. B. M. R.J. N. A. J. J. 1992; 11: PubMed Scopus Google Scholar). these lead to on the level of we the that the quantitatively other more likely is that the of Lyn not a in the level of phosphorylation of the receptor and not the of the to the likely that the interaction the experiments are is the interaction between Lyn and the For in experiments on the antigen receptor of B have an interaction of the unique domain of Lyn and the Src family kinase Fyn with the but not the from J.C. J. 1994; 13: PubMed Scopus Google Scholar). studies on the of Fyn with the of the of the receptor Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) and of with A. Bolen J.B. Cell. Full Text PDF PubMed Scopus Google Scholar) have the analogous in have the homologous within the of the Src family kinases and have presented evidence that this which is for the of the kinases to PubMed Scopus Google Scholar). the in the interaction of Lyn and the β S. Cicala C. Scharenberg A.M. Kinet J.P. Cell. 1996; 85: 985-995Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). have studied the of to and its interaction with of the T receptor using J. Biol. 1996; PubMed Scopus Google Scholar). that within the and were for that in the interaction of with the chain well have by the interaction of with the Lyn these with and is that this a homologous in this interaction with the FcεRI. of Lyn used to the of this but studies have to extensive to more the studies additional studies evidence which in Lyn are the is at the level of and we are our in that also for the that these interactions in the of D. B. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). our are with the of the current that the of small aggregates to initiate a response to the fraction of receptors constitutively associated with enhanced is for the of of kinase to the The that is the interaction that is is also with of the by B.S. Kapp N. Lee R.J. Pfeiffer J.R. Martinez A.M. Platt Y. Letourneur F. Oliver J.M. J. Biol. Chem. 1995; 270: 4013-4022Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). observed that a the domain when transfected into cells to but inhibited and aggregation-induced phosphorylation of the endogenous FcεRI. result likely by the transfected β chain for of constitutively associated is in effect the to the we We for the of and in the of for with expression in the baculovirus and for of the