THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 269, No. 24, Issue of June 17, pp, 16902-16908, 1994 Printed in U.S.A.

Activation of Protein Tyrosine Kinase ~ 7 2 " ' ~ by FceRI Aggregation in Rat Basophilic Leukemia Cells ~ 7 2 " ~ ~ IS A MINOR COMPONENT BUT THE MAJOR PROTEIN TYROSINE KINASE OF pp72*

(Received for publication, December 1, 1993, and in revised form, February 28, 1994)

Kenji MinoguchiS0, Marc Benhamoul, William D. SwaimS, Yuko Kawakamiy, Toshiaki Kawakamin, and Reuben P. SiraganianS From the gaboratory of Immunology, NIDR, National Institutes of Health, Bethesda, Maryland 20892 and the IDivision of Immunobiology, La Jolla Institute for Allergy and Immunology, La Jolla, California 92037

Aggregation of the high affinity IgE receptors (FccRI) on rat basophilic leukemia RBL-2H3 cells resdts in pro- tein tyrosine phosphorylations. Previously we reported that there is prominent tyrosine phosphorylation of - 72-kDa proteins (pp72) and that the tyrosine kinase ~724" is one component of pp72. Here we studied further the relationship of p7ZWk to pp72. The aggregation of FccRI induced the activation of p72Qk which was paral- lel to its tyrosine phosphorylation. By in vitro kinase assay of immune complexes purified with anti-phospho- tyrosine antibodies, p72OYk was the major pp72 tyrosine kinase. However, by immunoblotting with anti-phospho- tyrosine antibodies, ~ 7 2 " ~ was a minor component of pp72. The heterogeneous nature of pp72 was indicated by different studies. Under optimum conditions of one- dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, pp72 consisted of a heterogeneous group of 69-, 71-, and 72-kDa tyrosine-phosphorylated proteins. There were differences in the tyrosine phos- phorylation of these proteins in cells activated in the absence of extracellular calcium or when stimulation was with the calcium ionophore A23187 or with phorbol myristate acetate. One of the proteins migrating at 69 kDa was ~72"'. By two-dimensional gel electrophoresis pp72 was found to consist of multiple tyrosine-phospho- rylated proteins including 71-80-kDa proteins that asso- ciate with ~53/56'~". A 75-kDa tyrosine-phosphorylated protein, different from pp72, was identified as ~ 7 5 ~ ' ' (SPY75). These results demonstrate the heterogeneous nature of the pp72 and that ~ 7 2 " ' ~ is activated after FccRI aggregation.

Aggregation of the high affinity receptor for IgE (FceRI)' on rat basophilic leukemia RBL-2H3 cells initiates biochemical events that result in degranulation and release of inflamma- tory mediators (1-3). Protein tyrosine phosphorylation is an early event in this FceRI-mediated signal transduction path- way (4-6). These tyrosine phosphorylations include the p and y subunits of FceRI, phospholipase Cyl, p53/56lY", PTK72,

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

munology, Bldg. 10, Rm. 1N106, NIDR, NIH, Bethesda, MD 20892. Tel.: To whom correspondence should be addressed: Laboratory of Lm-

301-496-5105; Fax: 301-496-2443.

IgE; PMA, phorbol myristate acetate; RIPA, radioimmune precipitation; The abbreviations used are: FceRI, receptor with high affinity for

Pipes, 1,4-piperazinediethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylammoniol-l- propanesulfonic acid; G,,,, Il3(NeuAc,)-GgOse4Cer.

Nck, p95""", p12EiFAK, mitogen-activated protein kinase, and sev- eral other molecules that have not yet been identified (7-20).

The aggregation of FceRI in RBL-2H3 celIs results within 15-30 s in the tyrosine phosphorylation of - 72-kDa proteins (pp721, the major tyrosine-phosphorylated proteins after FceRI activation (4). The phosphorylation of pp72 is independent of calcium influx or the activation of phospholipase C or protein kinase C (12,21,22). Thus, tyrosine phosphorylation of pp72 is part of a distinct signaling pathway activated by FceRI aggre- gation.

Several 70-72-kDa non-receptor protein tyrosine kinases have been isolated or characterized in different cells (23-29). These include p72@ (261, ZAP-70 (231, ~ 7 2 " ' (27) (also known as ~ 7 2 ~ ' ~ (29) or p72'"' (30)), and p72fY"-related kinase (28). One group is the Syk family of protein tyrosine kinases which in- cludes ZAP-70, a kinase involved in T cell receptor signaling (23-26). p72"yk, expressed in several hematopoietic cells, is ty- rosine phosphorylated and/or activated by stimulations from such cell surface receptors as IgM and IgD in B cells (311, FceRI in RBL-2H3 cells (121, FcyR in monocytes (32), and thrombin receptors in platelets (33). A 72-kDa protein tyrosine kinase, PTK72, has been characterized biochemically, and antibodies raised to this protein demonstrate that it associates with B cell receptors and with the FceRI in RBL-2H3 cells (13, 34, 35). Although PTK72 and p72"Yk have similar susceptibility to pro- teolytic digestion, it has not been established whether they are identical or related molecules.

Recently we isolated the cDNA for rat p7ZSyR and demon- strated its presence in RBL-2H3 cells (12). There is rapid ty- rosine phosphorylation of ~ 7 2 ' ~ ~ after FceRI aggregation but not after calcium influx or protein kinase C activation. There is also association of p72"yk with the y subunit of FceRI after receptor aggregation. However, depletion of p72'Yk by antibod- ies indicates that it is only a minor component of pp72 (12). The present experiments investigated the relationship between pp72 and p72"yk in RBL-2H3 cells and explored further the molecular heterogeneity of pp72. Here we report that multiple proteins accounted for pp72. Furthermore, FceRI aggregation induced p72"yk activation, and although p72'Yk was a minor com- ponent of pp72, it was the major pp72 protein tyrosine kinase activated in the RBL-2H3 cells.

EXPERIMENTAL PROCEDURES Antibodies-Two peptides based on the rat ~72~~~-deduced amino acid

sequence were synthesized with cysteine residues at either the carboxyl or the amino terminus. Polyclonal anti-rat-Sykl antibodies were pre- pared by immunizing rabbits with the EPTGGAWGPDRGLC peptide coupled to keyhole limpet hemocyanin as described previously (12). The anti-Syk2 antibodies were prepared with the peptide CAVELRL- RNYYYDVVN, which corresponds to the carboxyl-terminal amino acids. The IgG fractions of the anti-Syk antibodies were purified by

16902

p72Vk in FceRI Signaling 16903 GammaBind Plus Sepharose (Pharmacia Biotech, Inc.). The anti-Sykl antibodies were also affinity purified as described previously (12). Both anti-Syk antibodies immunoprecipitated p72qk, but only anti-Sykl an- tibodies bound to ~ 7 2 ~ ~ ' in immunoblots (data not shown). In lysates depleted with anti-Syk2 antibodies the anti-Sykl antibodies did not detect any protein in immunoblots, demonstrating that the antibodies recognized the same molecule. In the reciprocal experiments, there was no immune complex kinase activity in immunoprecipitates with anti- Syk2 antibodies if the lysates had been depleted with anti-Sykl anti- bodies (data not shown). Anti-HS1 (SPY75) antibodies were prepared as described previously (36). All other antibodies have been described pre- viously (37-39).

Cell Culture and Activation-The RBL2H3 cells were maintained as monolayer cultures and activated as described previously (4). The cells were stimulated with 30 ng/ml antigen (dinitrophenyl coupled to hu- man serum albumin), 0.5 p~A23187, or 40 nM phorbol myristate acetate (PMA). After stimulation for the indicated times, the monolayers were rinsed twice with ice-cold phosphate-buffered saline and solubilized in RIPAlysis buffer (10 mM phosphate buffer, pH 7.4, containing 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 50 mM NaF, 1 mM Na3V0,, and 1 mM phenylmethylsulfonyl fluoride, 50 pg/ml leupeptin, 0.5 unit/ml aprotinin, 2 mM pepstatin A) or with Triton X-100 lysis buffer (same components as RIPA lysis buffer except for the elimination of the SDS and sodium deoxycholate). The plates were left on ice for 20 min, and the supernatants were collected after centrifugation for 15 min a t 14,000 rpm, 4 "C. In some experiments the cells were activated in the absence of extracellular calcium by washing in Ca2+-free Pipes-buffered saline containing 4 mM EDTA and then stimulated with antigen in the presence of 50 p~ EDTA.

Immunoblotting-Cell lysates (io5 cellsflane) or immunoprecipitates (2 x 106/lane) were separated by SDS-PAGE, electrotransferred to ni- trocellulose membranes, and the immunoblotted proteins were visual- ized with the enhanced chemiluminescence (ECL) kit from Amersham Corp. In the SDS-PAGE (8% gels), to separate the 50-80-kDa proteins optimally, the current was stopped when the 49.5-kDa prestained marker reached the bottom of the gel. Immunoblotting with the horse- radish peroxidase-conjugated anti-phosphotyrosine antibody PY-20 was described previously (21). Immunoblotting with anti-Sykl antibody was visualized by horseradish peroxidase-conjugated donkey anti-rabbit an- tibody. In some experiments, antibodies were stripped and the mem- branes reprobed with other antibodies as recommended by the manu- facturer.

Immunoprecipitation and in Vitro Kinase Assay-The 1% Triton X-100-solubilized lysates were precleared by mixing for 1 h a t 4 "C with preimmune rabbit IgG prebound to protein A beads and then immuno- precipitated with anti-Syk2 antibodies (3 pg) or polyclonal rabbit anti- phosphotyrosine antibodies (5 pg) prebound to protein A beads. Follow- ing immunoprecipitation, in vitro kinase assay was as described previously with 10 pCi of [-p3'P1ATP (3,000 Ci/mmol, DuPont NEN) (40). In some experiments the membranes were incubated for 1 h a t 60 "C in 1 M KOH to hydrolyze preferentially phosphoserine and phos- phothreonine, and then the alkali-resistant phosphotyrosine residues were visualized by autoradiography. Phosphoamino acid analysis was performed as described previously (12). In the experiments to deplete p720k, cell lysates were first incubated for 2 h a t 4 "C with either preimmune rabbit IgG or anti-Sykl antibodies coupled to Sepharose 4B beads, and the supernatants were then used for immunoprecipitation.

Metabolic Labeling of Cells-Cells were metabolically labeled by cul- ture in medium containing [35Slmethionine and [35Slcysteine (Tran35S- label, ICN, Irvine, CA) as described previously (37).

V8 Protease Digestion and Two-dimensional Gel Electrophoresis- These were carried out as described previously (22,40).

RESULTS

Activation 0 f p 7 2 ~ ~ by FcaIAggregation in RBL-2H3 Cells- Recently, we found that FceRI activation results in tyrosine phosphorylation of p72"Yk (12). To investigate whether ~ 7 2 " ~ ~ is activated after FceRI stimulation, immunoprecipitates with anti-Syk2 antibodies were subjected to in vitro kinase assay. FceRI stimulation resulted in dramatic activation of p72Wk as demonstrated by the autophosphorylation activity of this ki- nase (Fig. IA), detectable within 30 s, reaching a maximum at 10 min, and there was still increased kinase activity at 30 min (data not shown). By phosphoamino acid analysis there was only phosphorylation on tyrosine residues in p72Wk (data not shown). This time course of p72"yk activation paralleled its ty-

1 2 3 4 5 6

(') Time 0 0.5 1 5 10

. mom Anti-PTyr

t- -_ Anti-Syk . .

1 2 3 4 5 FIG. 1. Aggregation of FcsRI results in the activation of p72vk.

Panel A, in vitro kinase assay of anti-Syk immunoprecipitates. Cells were stimulated with antigen for the indicated times (in min), solubi- lized in 1% Triton X-100 lysis buffer, and immunoprecipitated with anti-Syk2 antibodies (lanes I d ) or preimmune rabbit IgG (lane 6). After in vitro kinase assay, proteins were separated by SDS-PAGE (10% gels), transferred to Immobilon P membrane, washed in KOH, and visualized by autoradiography. Panel B , immunoblotting of the anti-Syk immunoprecipitates with anti-phosphotyrosine antibodies. In parallel, samples from the same washed immunoprecipitates were eluted, sepa- rated by SDS-PAGE (10% gel), and analyzed by immunoblotting with either anti-phosphotyrosine (upper bund) or anti-Sykl antibodies (lower bund).

rosine phosphorylation that was detectable within 30 s and reached a maximum at 10 min after stimulation (Fig. lB). The in vitro autophosphorylation of p72"Yk induced a shift in elec- trophoretic mobility with a slight decrease in migration. How- ever, there was no obvious shift in the electrophoretic mobility of the in vivo tyrosine-phosphorylated p72Wk (Fig. lB). Never- theless, FceRI aggregation results in tyrosine phosphorylation and activation of p72"yk.

p 7 P k Is a Minor Component but a Major nrosine Kinase of pp72"Aggregation of the FceRI on RBL-2H3 cells results in rapid tyrosine phosphorylation of - 72-kDa proteins (pp72). Therefore, antibody depletion experiments were used to clarify the relationship of p72"yk to pp72. Cell lysates were either in- cubated with preimmune rabbit IgG or depleted of ~ 7 2 ~ ~ and then proteins were affinity purified with anti-phosphotyrosine antibodies. As we have shown previously, depletion of p72Wk did not decrease the intensity of pp72 in anti-phosphotyrosine im- munoblots (Fig. 2A) . The anti-Sykl antibody completely de- pleted p72wk from these lysates of FceRI-activated cells (data not shown, also see Fig. 6B). In 35S metabolically labeled cells, there was only a slight decrease in the intensity of pp72 pre- cipitated with anti-phosphotyrosine antibodies after depletion of Syk (Fig. 2C). These results confirm our previous finding that p72vk is a minor component of pp72 (12).

There are 72-kDa protein tyrosine kinase(s), called PTK72, activated after FceRI aggregation (6, 13). Therefore, in vitro immune complex kinase assays were used to investigate the relationship of p72"Yk to the FccRI-activated 72-kDa protein tyrosine kinase. Anti-phosphotyrosine antibodies precipitated a pp72 kinase activity only from the FceRI-activated cell ly- sates (Fig. 2B ). Separation of the proteins under different con- ditions, e.g. on SDS-PAGE (8% gels), did not further resolve this pp72 phosphoprotein. By phosphoamino acid analysis the pp72 contained only phosphotyrosine (data not shown). The in- creased pp72 tyrosine kinase activity correlated with the in- creased immunoprecipitation of the tyrosine-phosphorylated proteins (compare Fig. 2, panel A, with panel B). Similar re- sults were obtained in experiments using another monoclonal anti-phosphotyrosine antibody, 1G2 (data not shown). Deple- tion of p72vk from the cell lysates markedly diminished the pp72 tyrosine kinase activity (Fig. 2B). These results suggested

16904 p72qk in Fc&I Signaling

(A) P-Tyr Blot SYk

depletion n

DNP - - + + + + Time 1 20 1 20 1 20

kDa

106 - 80 -

49.5 -

32.5 -

1 2 3 4 5 6

(B) Kinase Assay

DNP Time

kDa 106 - 80 -

49.5 -

32.5 -

SYk depletion n - - + + + +

1 20 1 20 1 20

t

1 2 3 4 5 6

(C) Metabolic Labeling

DNP - + + LL ,+ .

7 8 9 FIG. 2. p72Wk is a minor component but a mqjor protein tyro-

sine kinase of pp72. Panel A, anti-phosphotyrosine immunoblotting of proteins affinity purified with anti-phosphotyrosine antibodies from FccRI activated cells. Cells were either not stimulated or stimulated with antigen (dinitrophenyl (DNP)) for 1 or 20 min as indicated and then solubilized in 1% Triton X-100 lysis buffer. The lysates were incu- bated with rabbit IgG (lanes 1 4 ) or anti-Sykl antibody (lanes 5 and 6) coupled to Sepharose 4B beads. The lysates were then immunoprecipi- tated with rabbit polyclonal anti-phosphotyrosine antibodies, the pro- teins eluted, separated by SDS-PAGE (10% gel), and analyzed by im- munoblotting with anti-phosphotyrosine antibody PY-20. Panel B, in vitro kinase assay of the anti-phosphotyrosine immunoprecipitates. Parallel immunoprecipitation samples as in panel A were subjected to in vitro kinase assay, separated by SDS-PAGE (10% gel), and visualized by autoradiography after treatment with KOH. Panel C, depletion of ~ 7 2 ~ ~ from anti-phosphotyrosine immunoprecipitates of 35S metaboli- cally labeled cells. Metabolically labeled cells (lo7) were immunopre- cipitated with anti-phosphotyrosine antibodies, separated by SDS- PAGE (10% gel), and visualized by autoradiography. Cell lysates were from nonstimulated (lane 7) or activated cells (lanes 8 and 9) . Lysates were also depleted of ~ 7 2 ' ~ ' (lane 9) . p72'Yk was well labeled under these experimental conditions (data not shown). Arrows indicate pp72.

that the major pp72 protein tyrosine kinase was p72Wk. There- fore, one-dimensional phosphopeptide mapping was used to compare p72vk with the pp72 tyrosine kinase activity immuno- precipitated with anti-phosphotyrosine antibodies. The 32P-la- beled digestion products of both p72Wk and pp72 tyrosine kinase activity were identical (Fig. 3). Therefore, these results dem- onstrate that p72"Yk is the major pp72 protein tyrosine kinase in FceRI-activated cells.

Multiple Drosine-phosphorylated Proteins Migrate in the 69- 75-kDa Size Range-Previous experiments and the present re- sults demonstrate that p72"Yk is only one component of pp72 (12). To examine in more detail the major tyrosine-phospho- rylated proteins that migrated in this size range, we used a lower percentage polyacrylamide gel with the electrophoretic separation optimized to detect 50-80-kDa proteins. Instead of the single 72-kDa band seen in the SDS-PAGE 10% gels (Fig. 4A) at least three bands in this size range were detectable in the 8% gels (Fig. 4B). These tyrosine-phosphorylated proteins had a molecular mass of 69,71, and 72 kDa. In the 8% gels, the 72-kDa protein was the major component of pp72, whereas the 69- and 71-kDa proteins were much fainter. In time course experiments (Fig. 5A), FceRI activation resulted within 30 s in the tyrosine phosphorylation of these proteins and reached a maximum within 5-10 min (Fig. 5A). There was also tyrosine phosphorylation of a 75-kDa protein that was detectable by 1 min of stimulation. Similar results were obtained when the cells were solubilized with different detergents including Triton X-100, Nonidet P-40, CHAPS, or RIPA (data not shown). Thus, what was described as pp72 is actually accounted for by mul- tiple tyrosine-phosphorylated proteins that migrate in this size range.

Stimulation of RBL-2H3 cells with A23187 induces an in- crease in intracellular calcium and results in protein tyrosine phosphorylations (17, 22, 41). Therefore, we investigated what effect the calcium ionophore has on the tyrosine phosphoryla- tions of the 69-75-kDa proteins. As reported previously, incu- bation with A23187 induced the tyrosine phosphorylation of ppll0 (17, 22). Here the calcium ionophore induced the weak tyrosine phosphorylation of 69- and 71-kDa proteins (Fig. 5B). Protein kinase C also plays a role in secretion in RBG2H3 cells (3). Furthermore, the activation of protein kinase C with PMA results in protein tyrosine phosphorylation of ppll0 (17, 22). Here PMA induced the tyrosine phosphorylation of not only ppll0 but also of the 69- and 71-kDa proteins (Fig. 5B). These experiments suggest that tyrosine phosphorylation of 69- and 71-kDa proteins was secondary to the calcium influx andor protein kinase C activation.

The tyrosine phosphorylation of some proteins (e.g., ppll0) in RBL-2H3 cells requires the presence of extracellular calcium (17, 22). In the absence of extracellular calcium FceRI activa- tion still induced the tyrosine phosphorylation of 72- and 75- kDa proteins (Fig. 5C). After longer exposures, very weak ty- rosine phosphorylation of 69-kDa proteins could be detected, but there was no phosphorylation of 71-kDa proteins (Fig. 5C). These differences were observed at all time points of stimula- tion between 0 and 20 min (data not shown). Therefore, there were differences in the requirement of extracellular calcium for the tyrosine phosphorylation of 69-75-kDa proteins.

p72Wk Migrates as a 69-kDa Tyrosine-phosphorylated Protein-Experiments then determined which of these 69-75- kDa bands was p7ZWk. In cell lysates separated by 10% gels the anti-Sykl antibody immunoblotted proteins that migrated at the lower region of pp72 (Fig. 6A). In contrast, in 8% gels p72Wk migrated parallel to the tyrosine-phosphorylated 69-kDa pro- teins (Fig. 6A). Depletion of p72Wk from the cell lysates did not completely deplete the tyrosine-phosphorylated 69-kDa pro- teins (Fig. 6B). Therefore, p72Wk is also a minor component of

p72"yk in Fc&I Signaling 16905

Undigested FIG. 3. Identification of the major pp72 protein tyrosine kinase as ~729'~. Cells (5 x lofi) were either not ac- tivated or stimulated with antigen for 20 min and solubilized in 1% Triton X-100 lysis buffer. Lysates from FceRI-activated cells were immunoprecipitated with anti- phosphotyrosine antibodies and non- stimulated cell lysates with anti-Sykl an- tibodies. Both immunoprecipitates were subjected to in vitro kinase assay, eluted, and separated by SDS-PAGE (10% gel). Each lane was cut out and placed on top of a second dimension SDS-PAGE (620% linear gradient gel) and overlayed with 1 pg of V8 protease. The digestion was per- formed overnight in the gel. After separa- tion and transfer to Immobilon P mem- brane the polypeptides were visualized by autoradiography.

kDa kDa

106 - 80 - - PP72

80-

PP72

4 8

1

*

49.5

32.5 - . . - 1 2

"

P-Tyr

U " * 75

/

49.5- =

1 2

FIG. 4. pp72 is composed of multiple tyrosine-phosphorylated proteins. Cells were either not stimulated (lane 1 ) or stimulated for 10 min with antigen (lane 2 ), solubilized in RIPA lysis buffer, separated by either 10% (punel A ) or 8% (panel B) SDS-PAGE, and analyzed by immunoblotting with the anti-phosphotyrosine antibody PY-20. Arrows indicate pp72 and the different 69-75-kDa tyrosine-phosphorylated proteins.

the 69-kDa tyrosine-phosphorylated proteins. Previously we have reported that cell activation with either PMA or A23187 did not result in tyrosine phosphorylation of p72" (12). In the present studies we observed that in cell lysates the 69-kDa protein was weakly phosphorylated on tyrosine residues by PMA or A23187 stimulation (Fig. 5B). Cell lysates were de- pleted of p729yk, separated by two-dimensional gel electrophore- sis, and immunoblotted with anti-phosphotyrosine antibodies. The results confirmed that p72"YR was completely depleted, but there were still some 69-kDa tyrosine-phosphorylated proteins that migrated with a more acidic PI than this kinase (data not shown and also see Fig. 7). Altogether, these results indicate that p72'.@ migrated at 69 kDa and that besides ~72'3'~ there were other 69-kDa proteins that were tyrosine-phosphorylated after cell activation.

Separation of Tyrosine-phosphorylated Proteins by Tho-di- mensional SDS-PAGE and the Identification of pp75 as the Tyrosine-phosphorylated Protein ~ 7 5 ~ ~ ' (SPY75)"The analysis of the proteins phosphorylated on tyrosine by SDS-PAGE (8%) suggested that there were different proteins migrating in simi- lar positions in these polyacrylamide gels. Therefore, we sepa- rated cell lysates by two-dimensional gel electrophoresis to re- solve these proteins better. Solubilization with the urea lysis buffer used for two-dimensional analysis resulted in similar anti-phosphotyrosine immunoblotting patterns when analyzed by one-dimensional SDS-PAGE (Fig. 7 versus Fig. 4). By two-

kDa - 106 - - 80 - - 49.5 - - 32.5 - - 27.5 - - 18.5 -

I P

(A) Time kDa

106-

80 -

49.5 -

~ 7 2 ' ~ ~ -

I V8 Protease

I digested

0

SYk

DNP-HSA

0 0.5 1 3 5 10 20

F-

kDa kDa

106 - t 110 106-

F - * r: 80 - 80 - t 75 -+ 72 t 69

. ".- - c " - 7gp " 49.5 - 49.5 -

FIG. 5. Qrosine phosphorylation of the 69-75-kDa proteins af- ter FceRI, PMA, or calcium ionophore A23187 stimulation. Cells were stimulated with different secretagogues and then solubilized in RIPA lysis buffer, separated by SDS-PAGE (8% gels), and analyzed by immunoblotting with anti-phosphotyrosine antibodies. Panel A, time course of the FceRI-mediated protein tyrosine phosphorylations. DNP- HSA, dinitrophenyl-human serum albumin. Panel B, cell activation for 15 min with A23187 (0.5 PM), PMA (40 nM), or buffer alone. Panel C, cell activation in the absence of extracellular calcium was for 15 min with PMA, antigen (dinitrophenyl (DNP)) , or buffer alone. Arrows indicate the different 69-75-kDa tyrosine-phosphorylated proteins.

dimensional analysis, pp72 consisted of multiple tyrosine-phos- phorylated 69-72-kDa proteins (Fig. 7B). These results suggest that the accumulation of several tyrosine-phosphorylated pro- teins can form the single bands observed in the one-dimen- sional analysis. For example, when separated by SDS-PAGE (8% gels), the 72-kDa tyrosine-phosphorylated protein ap- peared as a single band, but by two-dimensional gel electro- phoresis it was composed of at least four proteins that migrated with different PI. These results demonstrate that FceRI aggre- gation results in tyrosine phosphorylation of a number of 69- 75-kDa proteins.

Immunoblotting of either the total cell lysates or of affinity- purified proteins was used to identify some of these tyrosine- phosphorylated proteins on the two-dimensional gels (Fig. 7, C

16906 p72sYk in Fc&I Signaling

( 4 1 0%

kl

106 80

+ 49.5

32.5

8% kDa

80 -

49.5 - 1 2 3

(6) Time kDa

106 - 80 -

49.5 - Blot: Anti-Syk

SYk depletion

0 1 2 0 0 1 2 0 n

t

.. . . 1 2 3

FIG. 6. p72qb migrates at 69 kDa. Panel A, detection of ~ 7 2 " ~ in total cell lysates. Cells were either not stimulated (lane I ) or stimulated with antigen (lane 2 ) for 10 min and then solubilized in 1% Triton X-100 lysis buffer. Total cell lysates were separated by SDS-PAGE (10 and 8% gels), and tyrosine-phosphorylated proteins were detected by immunoblotting with anti-phosphotyrosine antibodies. After stripping the antibodies, the same membrane (lane 2 ) was immunoblotted with anti-Sykl antibodies (lane 3). Panel B, depletion of ~729"' from total cell lysates. Cells were stimulated with antigen for the indicated times (in min) and solubilized in Triton X-100 lysis buffer. Lysates were then precleared with rabbit IgG or anti-Sykl antibody, and supernatants were separated by SDS-PAGE (8% gels) and analyzed for tyrosine-phosphorylated proteins by immuno- blotting (upper section). After stripping the antibodies, the same membrane was immunoblotted with anti-Sykl antibodies (lower section). Arrows indicate p72'Yk.

.. -

and D). These include p72'Yk, p53/56'Yn, and the 71-80-kDa pro- teins with PI of - 4.8 which coprecipitates with anti-Lyn anti- bodies (40). p72"Yk was again a minor component in immuno- blotting with anti-phosphotyrosine antibodies and detectable only after long exposures (data not shown). Furthermore, it was different from the major 72-kDa tyrosine-phosphorylated pro- teins (Fig. 70).

Recently, a 75-kDa protein was reported to be a major sub- strate of protein tyrosine kinases in B cell receptor-mediated stimulation and was identified as the product of the HS1 gene (42, 43). This protein is found not only in B cells but also in mouse bone marrow-derived mast cells, where it was found to be a major substrate for tyrosine kinases in FceRI-mediated signaling (36). Therefore, we investigated whether the 75-kDa tyrosine-phosphorylated protein expressed in RBL-2H3 cells was ~ 7 5 ~ ' ' (SPY75). By immunoblotting of proteins separated by SDS-PAGE, a 75-kDa protein was recognized by anti-HS1 (SPY75) antibodies (Fig. 7C). Because this antibody did not immunoprecipitate the native protein from RBL-2H3 cells, cell lysates separated by two-dimensional gel electrophoresis were analyzed by anti-phosphotyrosine immunoblotting and then stripped, and the same membrane was reprobed with anti-HS1 (SPY75) antibodies. There was a protein strongly phosphoryl- ated on tyrosine residues, PI approximately 5.4, and the same protein was also immunoblotted with anti-HS1 (SPY751 anti- bodies (Fig. 7C). Although p75*" (SPY75) is not a component of pp72, it was tyrosine phosphorylated by the aggregation of FceRI and may be involved in the signal transduction in RBL- 2H3 cells.

DISCUSSION The aggregation of FceRI resulted in p72Vk activation, and

this paralleled the in vivo tyrosine phosphorylation of this ki- nase. Thus, FceRI aggregation may regulate intracellular en- zymes that result in the phosphorylation of p72"Yk on tyrosine residues. The tyrosine phosphorylation of p72"yk would then regulate its kinase activity. Besides the Src family ki- nase p53/56'Yn and p60'"" are expressed in the RBL-2H3 cells and are activated by FceRI aggregation (11). The activation and phosphorylation of both ~ 7 2 9 ~ and p53156'Y" are early events that are independent of the influx or increase in intracellular calcium. In T cells the activation of ZAP-70 requires the pres- ence of an Src family protein tyrosine kinase (23). Thus, it is

possible that the activation of p72"Yk could depend on p53156'Y" and/or p60'-sm. The association of ~ 7 2 " ~ ~ with tyrosine-phospho- rylated FceRI subunits suggests that this kinase plays an im- portant role in signal transduction in cells.

The protein tyrosine kinase PTK72 is associated with FceRI in RBL-2H3 cells, and it has been suggested that it is activated by FceRI aggregation (13). PTK72 and p72"yk share several characteristics such as their apparent molecular weight, tissue distribution, and sensitivity to proteolysis with the generation of a - 40-kDa fragment. Furthermore, both kinases associate with the antigen receptor on B cells and with FceRI (12,13,31, 35). By protease digestion PTK72 and the 72-kDa protein ty- rosine kinase were identical (13). Here by using anti-Syk anti- bodies and protease digestion we observed that p72'Yk was the major pp72 protein tyrosine kinase. In immunoblots anti- PTK72 antibodies bound to immunoprecipitated p72"Yk, demon- strating that these molecules share antigenic determinants2 Altogether, these results show that PTK72 and p72Wk are prob- ably the same protein.

In our prior observations on pp72 we found that it was rap- idly tyrosine phosphorylated after FceRI aggregation, and this was not a result of calcium influx or protein kinase C activation (22). In studies with anti-Syk antibodies we found that the tyrosine phosphorylation of p72ek had the same properties (12). However, ~ 7 2 9 ~ is a minor component and migrates at 69 kDa. Here we also found a 72-kDa protein that was the major com- ponent of pp72 and had the same characteristics. Recently it was reported that RBL cells may contain mRNA but not protein coding for ZAP-70, a p72"yk-related tyrosine kinase (23). Several other 70-77-kDa protein tyrosine kinases have been reported in T and B cells, ~ 7 2 ' ' ~ (also known as ~ 7 2 ' " ~ or p72'"'), p77*lk (also known as ~77'"'~), p72fyn-related kinase (27,28,44). Thus, although other tyrosine kinases could participate in FceRI- mediated signaling, the results of immune complex kinase as- says indicate that the pp72 protein tyrosine kinase is p72"Yk. Most of the other tyrosine-phosphorylated proteins are prob- ably kinase substrates.

The detailed analysis suggested that several proteins to- gether account for the pp72 detected in immunoblots. Under the optimal conditions of separation of the proteins by one- dimensional SDS-PAGE there were tyrosine-phosphorylated

M. Benhamou and R. P. Siraganian, unpublished observations.

p729yk in Fc&I Signaling 16907

kDa

106- & * 80 -

cn 0 v)

1

kDa

106 - 80 -

- 4

Anti-PTyr

49.5 -

kDa I

PH 8.2 * 4.3 v)

I ' 4 0 (n

49.5 -

kDa

106- ,'

80 -

Antl-r I yr

v) 0 v)

Anti-HS1 (SPY75) Anti-Syk FIG. 7. Separation of tyrosine-phosphorylated proteins by two-dimensional SDS-PAGE and identification of ~ 7 5 ~ ~ ' (SPY75). Cells

were either not stimulated (panel A) or stimulated for 10 min with antigen (panel B ) and solubilized in urea lysis buffer. Total lysates from 4 x lo5 cells were separated by both one- and two-dimensional gel (6%) electrophoresis (in the same gel) and analyzed by immunoblotting with anti-phosphotyrosine antibody PY-20. After stripping the antibodies (from panel B ) the membrane was immunoblotted with anti-HS1 antibodies (anti-SPY75) (panel C). The same membrane was also reblotted with anti-Syk antibodies (panel Dl. Boxes indicate the proteins that have been identified; 1, ~ 7 5 ~ " (SPY75); 2, p72Vk; 3, 71-80-kDa p53/56'yn-associated proteins; 4, p53156'Y".

bands of 69, 71, and 72 kDa. The two-dimensional polyacryl- amide gel electrophoresis followed by anti-phosphotyrosine immunoblot analysis suggests that there are several spots with different PI which correspond to each of these bands. For ex- ample, the strongly phosphorylated 72-kDa band seen in one- dimensional 8% gels is probably the summation of at least four proteins. Thus, it appears that several different proteins col- lectively account for single bands in immunoblots of proteins separated by one-dimensional electrophoresis.

The Src family kinase p53/56'Yn associates with several pro- teins in B cells (43,45), platelets (46,471, and in RBL-2H3 cells (11, 48, 49). Previously we observed the coprecipitation of 71- 80-kDa acidic PI 4.8 tyrosine-phosphorylated proteins by anti- Lyn antibodies (40). These proteins were tyrosine phospho- rylated in vivo in nonstimulated cells, and this phosphorylation was increased by FceRI aggregation. In the in vitro anti-Lyn immune complex kinase assay these proteins were tyrosine phosphorylated, suggesting that they may be endogenous Lyn substrates. The same 71-80-kDa proteins, p53/56'Y" and FceRI, were precipitated by monoclonal antibody AA4, which recog- nizes the unique a-galactosyl derivatives of the GDlb ganglio- sides present on rat mast cells (40). Thus, there is a complex containing FceRI, these 71-80-kDa proteins, gangliosides, and p53/56'?" which could play a role in receptor-mediated signal transduction. These 71-80-kDa proteins are among the compo- nents of pp72 and also contribute to the increased tyrosine

phosphorylation observed in the 71-80-kDa range after FceRI aggregation.

These experiments identified ~ 7 5 ~ " (SPY751 as the 75-kDa tyrosine-phosphorylated protein in RBL-2H3 cells. This protein is the major tyrosine-phosphorylated protein after cross-link- ing of cell surface antigen receptors in B cells (43) and FceRI aggregation in mouse mast cells (36). I t was reported that the ~ 7 5 ~ " coprecipitated with anti-Lyn antibodies and that the tyrosine-phosphorylated form associates with the Src homology 2 domain of Lyn (43). However, we could not detect this Lyn- HS1 association in mast cells or basophils (36). The ~ 7 5 ~ " protein has three copies of a 37-amino acid repeating motif near its amino-terminal region, a helix containing an amphipathic region, and an Src homology 3 domain near its carboxyl-termi- nal region. It is mostly a cytosolic protein, although it is also present in the nucleus. Structurally it could be a DNA-binding protein. I t could also function by interacting with other cy- toskeletal molecules through Src homology 3 domains or alter- natively in transducing signals into the nucleus to activate some of the late events after FceRI aggregation such as the generation of cytokines.

In summary, the present studies demonstrate that p72Vk is the major pp72 protein tyrosine kinase. However, pp72 was found to be composed of multiple tyrosine-phosphorylated pro- teins, a minor component of which was p72"Jk. The aggregation of FceRI induces the activation of p72"Yk and other protein ty-

16908 p72"Yk in F c d I Signaling

rosine kinases, tyrosine phosphorylation of substrates, and the interaction of these molecules.

Acknowledgments-We thank Dr. Marvin Karten and Dr. J. E. Rivier for the purified carboxyl-terminal peptide and Elsa Berenstein for help in the phosphoamino acid analysis. We also thank Drs. M. Hamawy and W. Hook for reviewing the manuscript and giving helpful comments.

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