THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 268, No. 21, Issue of July 25, pp. 15900-15905 1993 Printed in 6.S.A.

Involvement of p7ZSyk, a Protein-Tyrosine Kinase, in Fcr Receptor Signaling*

(Received for publication, March 12, 1993)

Alka Agarwal, Patrick Salem, and Keith C. RobbinsS From the Laboratory of Cellular Development and Oncology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892

Previous studies have demonstrated that multichain immune recognition receptors, such as the T-cell recep- tor, signal their occupancy by inducing tyrosine phos- phorylation of cellular protein substrates. Type I and 11 receptors for the Fc portion of IgG are single-chain immune recognition receptors having external, trans- membrane, and cytoplasmic domains. In the present study, we have investigated the possibility that, upon engagement, Fcr receptors induce protein-tyrosine phosphorylation. Our findings reveal increased phos- phorylation of a number of proteins on tyrosine resi- dues after cross-linking of either high (FcyRI) or low (FcrRII) affinity receptors expressed on HL60 cells. Engagement of FcrRII induced rapid tyrosine phos- phorylation that decayed to basal levels by 40 min. In contrast, phosphorylation induced by Fc-yRl cross- linking was more delayed, peaking at 5-10 min, and returning to basal levels by 60 min. Kinase assays of cellular proteins immunoprecipitated from lysates of activated cells by antibody to phosphotyrosine re- vealed phosphorylation of a 72-kDa molecule that was not present in lysates of resting cells. This phosphopro- tein was identified as ~ 7 2 " ~ ~ by immunoprecipitation with antibodies directed against two different regions of the eyk gene product. Immunoprecipitation with antibodies against ~ 7 2 ' ~ " followed by immunoblotting with anti-phosphotyrosine antibodies revealed an ac- tivation-dependent tyrosine phosphorylation of ~72'~' . Thus, our present findings demonstrate induction of protein-tyrosine phosphorylation following engage- ment of monomeric immune recognition receptors and identify ~ 7 2 ~ ~ ' as a tyrosine kinase substrate involved in signaling by FcrRI and FcrRII.

Protein kinases with specificity for tyrosine residues were initially discovered as the products of certain oncogenes. The normal counterparts of such oncogenic kinases can be divided into two major classes: those that contain extracellular ligand binding domains and the cytoplasmic class that localizes to the inner face of the plasma membrane (Hunter and Cooper, 1985; Yarden and Ullrich, 1988). Upon ligand binding, recep- tor kinases transduce proliferative signals by activating their cytoplasmic kinase domains which in turn phosphorylate an array of substrates on tyrosine residues. Normal activators for the cytoplasmic class of kinases are not known. However, such kinases are most highly expressed in hematopoietic cells

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

2 To whom correspondence and reprint requests should be ad- dressed.

and are thought to be involved in signaling mature cell func- tions.

Previously, we have shown that protein-tyrosine phos- phorylation functionally links Fc receptors for IgE and his- tamine granule release in mast cell lines (Benhamou et al., 1990). Signaling via tyrosine phosphorylation has also been shown after activation of mIgM in B-cells (Campbell and Sefton, 1990), engagement of the antigen receptor complex in T-cells (Klausner and Samelson, 1991; Samelson et al., 1990), and cross-linking of Fcy type I11 receptors in NK cells (Ein- spahr et al., 1991; O'Shea et al., 1991; Vivier et al., 1991). Each of the receptors shown to induce tyrosine phosphorylation consists of multiple gene products that together constitute a functional receptor complex. Keegan and Paul (1992) have recently designated these complexes as multichain immune recognition receptors. A number of studies have implicated non-receptor protein kinases as mediators of intracellular tyrosine phosphorylation following multichain immune rec- ognition receptor cross-linking (Campbell and Sefton, 1992; Cook et al., 1991; Eiseman and Bolen, 1992; Samelson et al., 1990). In contrast to multichain immune recognition recep- tors, type I and I1 Fcy receptors consist of single polypeptide chains. They possess high sequence homology with each other in their extracellular domains, but their cytoplasmic tails are unrelated by amino acid sequence (Ravetch and Kinet, 1991). Recent reports have shown that activation of FcyRI or FcyRII in monocytes and U937 monocytic leukemia cells, respec- tively, leads to increases in intracellular calcium (Macintyre et al., 1989; Vandewinkel et al., 1990) and tyrosine phosphoryl- ation of phospholipase Cy (Liao et al., 1992). FcyRIIA has also been shown to induce tyrosine phosphorylation in plate- lets (Huang et d., 1992). These findings have suggested that tyrosine phosphorylation might play a role in the coupling of these receptors to second messenger pathways.

In the present study, we have investigated the possibility that Fcy receptors I and I1 are coupled to protein-tyrosine kinases of the non-receptor class in myelomonocytic cells. Our findings demonstrate tyrosine phosphorylation of cellular substrates upon FcyRI or -RII engagement. We also identify the protein-tyrosine kinase subclass represented by p7YYk as a tyrosine kinase substrate involved in signaling by Fcy receptors.

EXPERIMENTAL PROCEDURES

Cells and Antibodies-The human myelomonocytic cell line HL60 (Collins et al., 1977) was maintained at 5% COa in RPMI 1640 supplemented with 10% fetal bovine serum. Human IgG, goat anti- human IgG F(ab)'* antibody and rabbit anti-mouse IgG F(ab)'2 were obtained from Cappel Organon Teknika Corp. (Durham, NC). Mono- clonal antibodies 32.2, 197, 22 (Anderson et al., 1986; Guyre et al., 1989), and IV.3 (Loony et al., 1986) were obtained from Medarex Inc.

15900

p7.Tyk in Fcy Receptor Signaling 15901

(W. Lebanon, NH). Anti-phosphotyrosine antibody (anti-PY)’ alone or coupled to horseradish peroxidase was obtained from ICN. En- hanced chemiluminescence (ECL) reagent used for Western blotting was purchased from Amersham. [y-3ZP]ATP (3000 Ci/mmol) was from Du Pont-New England Nuclear.

Cell Actiuation-For activation experiments, cells were suspended at a concentration of 1 X 107/ml in serum-free RPMI 1640 containing 20 mM HEPES, pH 7.5,0.1% bovine serum albumin, 5 pg/ml selenous acid, 10 pg/ml transferrin, and 50 pM sodium vanadate. Cells were treated with 10 pg/ml human IgG or anti-receptor antibodies on ice for 20 min and were washed once with phosphate-buffered saline. Cell pellets were suspended in serum-free RPMI medium warmed to 37 “C and incubated with either 20 pg/ml goat anti-human IgG F(ab) ’2

or with 20 pg/ml rabbit anti-mouse IgG F(ab)’* for periods indicated. Cells were lysed in a buffer containing 50 mM Tris-HC1, pH 8.0, 5 mM EDTA, 150 mM NaCl, 1% Nonidet P-40,50 p~ sodium vanadate, and a mixture of protease inhibitors which included 2 mM diisopro- pylfluorophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF, 10 mM iodoacetamide, 20 pg/ml aprotinin, and 20 pg/ml leu- peptin. Lysates were clarified at 14,000 X g for 20 min at 4 “C, and their protein concentrations were determined by the method of Brad- ford (1976) using commercially prepared reagents (Bio-Rad). Post- nuclear supernatants were processed for either Western blotting or immunoprecipitation.

Western Blotting-Cell lysates were fractionated by polyacryl- amide (8%) gel electrophoresis, and proteins were transferred to nitrocellulose membranes (Schleicher & Schuell). Filters were prein- cubated for 3 h with 4% bovine serum albumin, prepared in a buffer containing 50 mM Tris-HC1, pH 7.6, 0.9% NaC1, and 0.05% Tween 20 (TTBS). Anti-phosphotyrosine antibody linked to horseradish peroxidase was diluted in TTBS containing 0.5% bovine serum al- bumin and was used to replace preincubation buffer. After l h of incubation with antibody at room temperature, the blot was washed 6 times with TTBS. Proteins were visualized with the aid of ECL.

Immunoprecipitation and in Vitro Kinase Assay-Cell extract, pre- pared from 1 X 10’ cells, was incubated for 1 h on ice with the antibodies indicated and for an additional 30 min with protein A- Sepharose beads (Pharmacia LKB Biotechnology Inc.) which were, at times, coated with rabbit anti-mouse IgG. Beads were washed twice with ice-cold phosphate-buffered saline containing 1 mM vanadate and 1% Nonidet P-40 and once with a buffer containing 10 mM Tris- HCl, pH 7.2, 1 M NaCl, and 1% Triton X-100. For in uitro kinase assays, beads were washed again with 10 mM Tris, pH 7.2, and 10 mM MnCL Reactions were initiated by the addition of 10 pCi of [y- 32P]ATP at room temperature and were stopped after 5 min by the addition of an equal volume of 2~ sample buffer. Samples were boiled, fractionated by polyacrylamide gel electrophoresis, and dried. Pro- teins phosphorylated in uitro were visualized by autoradiography.

Reimmunoprecipitation-Pro~ins phosphorylated during in uitro kinase reactions were treated by the addition of 50 pl of 50 mM Tris, pH 8.0,150 mM NaC1,5 mM EDTA, 1% Nonidet P-40, and 3% SDS. Samples were boiled for 10 min, clarified by centrifugation, and diluted 10-fold with the same buffer lacking SDS. Proteins were immunoprecipitated and analyzed as described above.

Phosphoamino Acid Analysis-Lysates from activated or control cells were immunoprecipitated with anti-PY and scored for immune complex kinase activity. Products of the kinase reaction were evalu- ated by phosphoamino acid analysis as described (Gutkind and Rob- bins, 1989). Briefly, proteins were fractionated by polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride mem- branes, and digested with 6 N HCl. Hydrolysates were mixed with unlabeled standards and analyzed by thin layer electrophoresis, fol- lowed by thin layer chromatography.

RESULTS

Engagement of Fcy Receptors Induces Tyrosine Phosphoryl- ation in HMO Cells-To determine whether Fcy receptors couple to protein-tyrosine phosphorylation, HL60 cells were treated sequentially with human IgG and goat anti-human IgG F(ab)’2 to cross-link IgG-bound Fcy receptors. Tyrosine phosphorylation of cellular substrates was evaluated by sub- jecting cell lysates to immunoblot analysis using anti-phos- photyrosine antibody (anti-PY) as a probe. As shown in Fig.

The abbreviations used are: PY, phosphotyrosine; mAb, mono- clonal antibody.

1, a readily detectable increase in the tyrosine-phosphorylated state of a 115-kDa protein was observed after 30 s of cross- linking. The intensity of tyrosine phosphorylation increased slowly to a maximum at 10 min and, by 40 min of treatment, tyrosine phosphorylation had returned to basal levels. The appearance and decay of a 60-kDa protein followed a pattern similar to that of p115, and peak phosphorylation of a 72-kDa species was at 5 min. Control cells treated with either IgG or anti-IgG alone over the same time period did not display increased tyrosine phosphorylation (Fig. 1). We conclude that enhanced tyrosine phosphorylation of cellular substrates is induced by Fcy receptor cross-linking in HL60 cells.

FcyRI and FcyRII Both Participate in Signaling through Protein-Tyrosine Phosphorylation-HL6O is a promyelomon- ocytic leukemia cell line that expresses high affinity (FcyRI) as well as low affinity (FcyRII) receptors for IgG. Only when induced to differentiate do they display the FcyRIII low affinity receptor. To determine whether FcyRI, FcyRII, or both were mediators of the tyrosine phosphorylation observed, we took advantage of monoclonal antibodies that specifically recognize each receptor type. Furthermore, any contributions from Fc portions were eliminated by utilizing only their F(ab)’:, fragments. As shown in Fig. 2, tyrosine phosphoryla- tion of a 115-kDa protein was again detected by immunoblot- ting with anti-PY when either high or low affinity receptors were engaged individually. However, initial tyrosine phos- phorylation of p115 was more rapid after FcyRII activation. Engagement of FcyRI was followed by delayed p115 phos- phorylation which subsequently declined from its peak at 10- 20 min to basal levels by 60 min. In addition, a prominent band at 68 kDa was observed after FcyRI but not FcyRII cross-linking. The time course for tyrosine phosphorylation after FcyRI activation was nearly identical with that observed when using human IgG to bind Fcy receptors. Thus, tyrosine phosphorylation after FcyRII activation was more rapid, whereas engagement of high affinity receptors led to a more delayed phosphorylation.

To determine whether differences in the kinetics of phos- phorylation were intrinsic to each receptor or were attribut- able to differences in monoclonal antibodies used, three dis- tinct antibodies against FcyRI were tested in parallel. Each antibody is known to recognize different epitopes of the receptor (Anderson et al., 1986; Guyre et al., 1989) and the Fc portion of one of them (mAb 197) but not the others (32.2 or 22) is also capable of binding FcyRI receptors (Guyre et al., 1989). As shown in Fig. 3, the extent of total tyrosine phos- phorylation as well as the time of its appearance and decay were similar for each antibody tested. These findings strongly suggest that the receptor, not the binding antibody, dictates the time course for tyrosine phosphorylation of cellular sub- strates. The lack of additional monoclonal antibodies recog- nizing FcyRII without also being capable of binding FcyRI, precluded similar experiments for that receptor. Nevertheless, we conclude that upon activation, both high and low affinity receptors are coupled to tyrosine phosphorylation and that the kinetics for this response differs between receptor types I and 11.

Fcy Receptor Cross-linking Induces Tyrosine Phosphoryla- tion of ~ 7 2 ” : a Protein-Tyrosine Kinase-Activation of pro- tein-tyrosine kinases often involves their autophosphoryla- tion on tyrosine residues (Hunter and Cooper, 1985). To investigate the possibility that changes in protein-tyrosine kinase activity might be detectable upon Fcr receptor cross- linking, phosphotyrosine-containing proteins were immuno- precipitated from lysates of control or treated cells and as- sayed for kinase activity in vitro. As shown in Fig. 4, a protein

15902 ~ 7 2 ’ ~ ~ in F c r Receptor Signaling 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

FIG. 1. Engagement of Fcy recep- tors induces protein-tyrosine phos- phorylation. Protein extracts (50 pg/ lane) from untreated HL60 cells (lane I ) or cells incubated in the presence of hu- man IgG (BindingAb) (lanes 7-11), goat anti-human IgG F(ab)’2 (Crosslinking Ab) (lanes 2-6), or both (lanes 12-18), were fractionated by polyacrylamide gel electrophoresis, transferred to nitrocel- lulose filters, and analyzed with antibody to phosphotyrosine. The duration of treatment is shown in minutes. Electro- phoretic mobility of molecular size standards is indicated in kilodaltons.

, I

Time (min) 0 0.5 2 10 40 60 0.5 2 10 40 60 0.5 2 5 10 20 40 60

Binding Ab Crosslinking Ab I I

F5 kDa

Time (rnin)

A

B

Binding Ab Crosslinking Ab

0 0.5 10 60 0.5 10 60 d.5 2 5 10 20 40 60 k~~ “

r2O0 LI

- -Lt: - f

‘L

- 200

c l 1 5 -97

-68

. FIG. 2. Engagement of FcyRI or FcyRII induces protein-

tyrosine phosphorylation. Protein extracts (50 &lane) from un- treated HL60 cells (lanes 1 ) or cells incubated in the presence of anti- FcyRI (panel A ) or anti-FcyRII (panel R ) (Binding A b ) (lanes 5-7), rabbit anti-mouse IgG F(ab)’2 (Crosslinking A b ) (lanes 2-4), or both (lanes 8-14), were fractionated by polyacrylamide gel electrophoresis, transferred to nitrocellulose filters, and analyzed with antibody to phosphotyrosine as described under “Experimental Procedures.” The duration of treatment is shown in minutes. Electrophoretic mobility of molecular size standards is indicated in kilodaltons.

of 72 kDa was detectable when either of the Fcy receptors was activated, but was not observed in lysates of control cells. In contrast, an 80-kDa band was observed in lysates of control as well as stimulated cells. These findings raised the possibil- ity that p72 was a protein tyrosine kinase activated by en- gagement of Fcy receptors. A recent report has described the cloning of a gene, designated syk, that encodes a 72-kDa product with protein-tyrosine kinase activity (Taniguchi et al., 1991). Our interest in this molecule derived from obser- vations that a 72-kDa protein was the major phosphotyrosine- containing species detectable in basophilic leukemia cells after engagement of their Fc,RI receptors (Benhamou et al., 1990). Thus, we had prepared antibodies against peptides represent- ing residues 13-27 (anti-sykl) and residues 116-130 (anti-

1 2 3 4 5 6 7 8 9 1011 1 2 1 3 1 4 ”-

Time(min) 0 10 0 0.5 10 60 0 0.5 10 60 0 0.5 10 60 kDa

r 2 O 0

m I Binding Ab L I

Crosslinking Ab u - - - FIG. 3. Tyrosine phosphorylation response to binding dif-

ferent monoclonal antibodies against Fc-yRI. HL60 cells were treated with rabbit anti-mouse F(ah)’, (lanes 2, 4-6, 8-10, and 12- 14), mAb 32.2 (lanes 3-6), mAb 197 (lanes 7-10), or mAb 22 (lanes 11-14). Untreated cells (lane 1 ) served as a control. Cells were lysed, fractionated by polyacrylamide gel electrophoresis, and blotted onto nitrocellulose filters. Phosphotyrosine-containing proteins were de- tected by anti-phosphotyrosine antibody. The electrophoretic mobil- ity of molecular size standards is indicated in kilodaltons.

syk2) of the syk gene product, ~ 7 2 ~ ~ . A characterization of these antibodies and their use in exploring the involvement of p7‘Zyk in signaling by Fc,RI receptors will be described elsewhere.’ The availability of these antibodies made it pos- sible to determine whether the 72-kDa protein detected by in vitro kinase assays after immunoprecipitation with anti-PY might be p7TYk.

After immune complex kinase reactions, phosphorylated proteins were denatured and immunoprecipitated a second time. As shown in Fig. 5A, anti-PY immunoprecipitated pro- teins of 72 and 60 kDa from kinase reactions containing lysate from activated cells but not from control cells. Thus, both p72 and p60 were phosphorylated i n vitro by a protein- tyrosine kinase. Similarly, when reaction mixtures were re- immunoprecipitated with anti-sykl antibody, 72- and 60-kDa proteins were again detected only from lysates of activated cells. Furthermore, the peptide serving as the immunogen that gave rise to anti-sykl blocked the detection of p72 but not p60. Thus, detection of the 72-kDa protein was dependent upon anti-sykl antibodies, whereas detection of p60 was not. Thus, the 60-kDa protein was nonspecifically detected and not associated with p7Yyk (Fig. 5 A ) . Furthermore, the detect- ability of the 60-kDa molecule in other similar experiments was variable. Confirmation that p72 was indeed ~ 7 2 ~ ~ was

* P. Salem and K. C. Robbins, manuscript in preparation.

p W y k in Fcr Receptor Signaling 15903 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Time (min) 0 0.5 5 IO 40 0.5 5 10 40 60 0.5 2 5 IO 20 40 60 ”-

FIG. 4. Kinase activity in anti- phosphotyrosine immune com- plexes. HL60 cells were treated with goat anti-human IgG F(ab)’? (panel A ) or rabbit anti-mouse F(ab)’p (panels B and C) (lanes 2-5) and with human IgG (panelA) , anti-FcyRI ( p a n e l B ) , or anti- FcyRII (panel c) (lanes 6-10), or both (lanes 11-17). Untreated HL60 cells (lanes I ) served as a control. Cells were lysed in a buffer containing 1% Nonidet P-40 and subjected to immune complex kinase analysis using anti-phosphotyro- sine as described under “Experimental Procedures.” Proteins labeled in &TO

were visualized by autoradiography after fractionation by electrophoresis. The electrophoretic mobility of molecular size standards is indicated in kilodaltons.

. . . - ” . - A

B

\ C

\ \ \ \ I I I I

Binding Ab

Cross- linking Ab

obtained upon immunoprecipitation of the same reaction mixture with anti-syk2 (Fig. 5 A ) . Again, p72 and p60 were detected but only p72 was blocked by preincubation of anti- syk2 with its original peptide immunogen. These findings demonstrate immunoprecipitation of an active form of p7Ysk by antibody against phosphotyrosine after Fc-y receptor en- gagement.

p7.Yyk Is Phosphorylated on Tyrosine in Vivo and in Vitro- T o determine whether the kinase activity detected in vitro was specific for tyrosine residues, the p7Yyk band was excised from a preparative gel and subjected to phosphoamino acid analysis. As shown in Fig. 5B, only phosphotyrosine was detectable in the p7Yyk sample analyzed. Thus, the kinase activity responsible for phosphorylation of p72 in vitro dis- played a specificity for tyrosine residues.

As an independent means of determining whether p7Yyk was phosphorylated on tyrosine in vivo after receptor engage- ment, cells were lysed a t varying time periods after activation, immunoprecipitated with anti-syk2, and immunoblotted using anti-PY as a probe. As shown in Fig. 6, tyrosine phos- phorylation of ~ 7 2 ~ ~ was readily detected at 5 min, but was no longer observed 40 min after activation. Thus, ~ 7 2 ~ ~ is transiently phosphorylated in vivo on tyrosine residues within minutes after receptor engagement.

DISCUSSION

The present study demonstrates protein-tyrosine phos- phorylation of intracellular substrates in signal transduction

I l l l l l l

I / / / / I I

e

I j:: p115

P72

I

- I I

c p 1 1 5 - 97

P72 - 68

mediated by Fc-yRI and -RII. Previous studies have linked tyrosine phosphorylation to signaling via other immune rec- ognition receptors including high affinity Fc receptors for IgE in basophils (Benhamou et al., 1990), mIgM in B-lymphocytes (Campbell and Sefton, 1990), the T-cell receptor complex (Klausner and Samelson, 1991), and Fc-yRIIA receptors in platelets (Huang et al., 1992). Thus, our present findings reinforce the idea that the signal generated by engagement of immune recognition receptors is transduced across the cell membrane by protein-tyrosine phosphorylation of intracellu- lar molecules. The specific functions signalled by these recep- tors in different cell types as well as the identity of the molecules involved in these processes are currently under investigation in a number of laboratories.

The relative contribution of protein-tyrosine kinases and phosphatases to the increased tyrosine phosphorylation ob- served in response to Fcy receptor engagement is not ad- dressed here. Other studies have suggested that phosphatase as well as kinase activities are involved in functional signaling (Rankin et al., 1993). Nevertheless, it appears that ~ 7 2 ” ~ plays a central role in the signaling process mediated by Fc-yRI and -RII. Previous studies have shown that ~72”~” is a protein- tyrosine kinase distinct from either the src family or ab1 family of enzymes (Taniguchi et al., 1991). Our findings demonstrate changes in p7Yyk concomitant with enhanced tyrosine phos- phorylation of a number of cellular proteins. Increased tyro- sine phosphorylation of p7Yyk in activated cells accounted for

15904 p W y k in Fcy Receptor Signaling

A 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 nn"

Time(min) 0 10 0 10 0 0 10 10 0 0 10 10

c* I 0

.)'

. . * Fp7* P60

Anti-PY - Anti-SYK I I

SYK peptide u u u u

B

". TLE

FIG. 5. A, tyrosine phosphorylation of ~ 7 2 ~ ' upon engagement of F c r receptors. HL60 cells were treated with human IgG and goat anti-human IgG F(ab)'2 for the time indicated in the panel. Lysates were subjected to immune complex kinase reactions using anti-phos- photyrosine antibody (lanes 1 and 2) . Portions of the reaction mixture were re-immunoprecipitated with anti-phosphotyrosine antibody (lanes 3 and 4 ) , anti-sykl (lanes 5-8), or anti-syk2 (lanes 9-12). In some cases (lanes 6,8, 10, and 12), antibodies were preincubated with homologous peptide. Proteins phosphorylated in uitro were visualized by autoradiography after fractionation by polyacrylamide gel electro- phoresis. Molecular size standards are indicated in kilodaltons. B, protein-tyrosine kinase activity of ~ 7 2 ~ ' . ~ 7 2 * ~ ' , labeled in the im- mune complex kinase assay, was excised from a gel and subjected to phosphoamino acid analysis as described under "Experimental Pro- cedures." Locations for phosphotyrosine ( P - Y ) , phosphothreonine (P-T), and phosphoserine ( P - S ) standards are indicated by dotted ouals.

its detection in kinase assays after immunoprecipitation with anti-PY. Thus, our present results implicate a protein-tyro- sine kinase outside of the src and ab1 families as a major player in signaling tyrosine phosphorylation in hematopoietic cells. Very recently, Taniguchi et al. (1992) have shown an increase in ~ 7 2 " ~ ~ enzymatic activity in uitro upon stimulation of porcine platelets with thrombin. Establishing functional relationships among ~ 7 2 " ~ ~ and other protein-tyrosine kinases in the initiation of particular cellular responses will require further studies, possibly relying on genetic approaches.

The multimeric antigen receptors previously shown to en- hance protein-tyrosine phosphorylation all consist of mole- cules displayed on the cell surface as well as subunits which include one or more members of a small family of cytoplasmic proteins linked as dimers by disulfide bridges (Cambier, 1992; Keegan and Paul, 1992). These intracellular chains contain a consensus sequence known as antigen receptor homology 1 (ARH1) which is characterized by a precise spacing of 6 particular amino acids over a stretch of around 26 residues (Keegan and Paul, 1992). Recent studies using chimeras be- tween the IL2 receptor CY chain and the cytoplasmic domain of CD3-t have shown that 22 residues which comprise the bulk of the CD3-t ARHl domain are sufficient to induce

Time (rnin)

Binding Ab Crosslinking Ab

SYK Ip

1 2 3 4 5 6 7 8 9

0 10 0.5 2 5 10 20 40 60 p5 + + + + + + + +

+ + t + + + +

+ + + + + + + + + FIG. 6. Tyrosine phosphorylation of ~ 7 2 " ~ in vivo upon

engagement of Fcy receptors. HL60 cells were treated with human IgG (Binding A b ) and goat anti-human IgG F (ab)'2 for the time indicated. Lysates were immunoprecipitated with anti-syk2, fraction- ated by polyacrylamide gel electrophoresis, transferred to nitrocellu- lose filters, and analyzed with antibody to phosphotyrosine. The duration of treatment is shown in minutes. The electrophoretic mobility of molecular size standards is indicated in kilodaltons.

tyrosine phosphorylation (Letourneur and Klausner, 1992). Thus, the presence of an ARHl motif appears to correlate with the capacity to activate tyrosine phosphorylation path- ways.

Cytoplasmic portions of human FcyRIIA and RIIC contain the consensus ARHl motif, whereas FcyRIIB gene products contain only a portion of the consensus sequence. Since all three FcyRII genes are expressed in HL60 cells (Brooks et al., 1989; Stengelin et al., 1988; Stuart et al., 1989), we cannot determine whether the limited motif of FcyRIIB receptors is sufficient to signal tyrosine phosphorylation. In contrast to the FcyRII proteins, FcyRI does not possess any of the ARHl motif. Thus, our demonstration that tyrosine phosphorylation is induced by engagement of FcyRI or -RII receptors strongly argues that FcyRI is coupled to protein-tyrosine phosphoryl- ation in a manner that is independent of ARH1. Based on the delayed increase in tyrosine phosphorylation induced by FcyRI and the similar array of proteins phosphorylated upon engagement of either receptor, it seems likely that the path- way utilized by FcyRII overlaps with the one activated by FcyRI. Thus, we propose the involvement of a molecule not required for FcyRII signaling but responsible for linking FcyRI activation to protein-tyrosine phosphorylation. The requirement for such a molecule might also account for the delayed tyrosine phosphorylation observed after engagement of the high affinity FcyRI.

Investigations aimed at identification of the protein-tyro- sine kinase(s) involved in signaling by immune recognition receptors have uncovered a 72-kDa protein-tyrosine kinase, termed PTK72 (Hutchcroft et al., 1992). Tyrosine phos- phorylation of this molecule is increased upon cross-linking of membrane IgM on B-lymphocytes (Hutchcroft et al., 1991). In T-lymphocytes, a 70-kDa tyrosine phosphoprotein (ZAP- 70) has been described (Chan et al., 1991; Wang et al., 1992). Increased tyrosine phosphorylation of ZAP-70 parallels its association with the T-cell receptor { chain following T-cell receptor stimulation. More recently, the ZAP-70 molecule has been shown to be related to but distinct from p7YYk (Chan et al., 1992). These findings have suggested a family of ~ 7 2 " ~ - related protein-tyrosine kinases involved in signaling by he- matopoietic cells.

In several cell types, including basophils (Benhamou et al.,

p7.Fyk in Fcr Receptor Signaling 15905

1990) as well as B- and T-lymphocytes (Chan et al., 1991; Hutchcroft et al., 1991, 1992; Wang et al., 1992), molecules of 70-72 kDa become heavily tyrosine-phosphorylated upon ac- tivation of their immune recognition receptors. Our present findings that ~ 7 2 ~ ~ is tyrosine-phosphorylated in vivo after Fcy receptor engagement suggests that such 70-kDa sub- strates consist of p7YYk or one of its family members. We have also shown that the predominant tyrosine phosphopro- tein observed after Fcr receptor cross-linking is a molecule of 115 kDa. We are currently engaged in attempts to identify this molecule. If p7ZSyk is enzymatically stimulated upon Fcr receptor cross-linking by tyrosine phosphorylation, then p115 becomes a likely physiologic substrate for the ~ 7 2 ’ ~ ~ protein- tyrosine kinase in myelomonocytic cells.

Acknowledgments-We thank Dr. Silvio Gutkind and Dr. Oliver Sartor for valuable discussions.

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