Interactions of Lyn with the Antigen Receptor during B Cell Activation*

Signaling through the B cell antigen receptor requires a complex set of interactions involving transmembrane components of the IgM receptor complex and cytosolic protein-tyrosine kinases. We have focused on the nature of these protein-protein interactions, the requirements for their occurrence, as well as the temporal sequence of events during the activation process. We found that cross-linking B cell antigen receptors at 0 "C resulted in the rapid association of the Src-family protein-tyrosine kinase, Lyn, with the antigen receptor complex as judged by the presence of Lyn in anti-IgM and anti-phos- photyrosine immune complexes and the presence of M B - 1 in anti-Lyn immune complexes. Receptor engage- ment also resulted in the rapid association of Lyn with the phosphotyrosine phosphatase, CD4S. This associa- tion of Lyn with receptor components was stable in the detergent Brij 96, but was readily disrupted by Nonidet P-40, suggesting the involvement of hydrophobic interactions in stabilizing formation of the Lyn-receptor complex. The protein-tyrosine kinase, Syk, was also found associated with activated receptor complexes. This association of Syk with components of the antigen recep- tor complex was stable to Nonidet P-40. Antibodies directed against the carboxyl teminus of Syk, but not against the amino-terminal SH2 domain, co-immunopre-cipitated M B - 1 from activated cells, consistent with the binding of Syk through an SH2 domain-phosphotyrosine interaction. Signaling through

Signaling through the IgM receptor complex of B cells requires a complex set of interactions involving a number of transmembrane as well as cytosolic proteins. The nature of these interactions, the requirements for their occurrence, as well as the temporal sequence of events during the activation process have been the focus of intense investigation by a number of laboratories. The membrane IgM molecule, which has a very short cytoplasmic tail, is linked to cytoplasmic effector molecules by its association with heterodimers of MB-1 and B29, which are also glycosylated transmembrane proteins (1-4). These three proteins form the transmembrane unit of the IgM receptor complex and serve to transduce extracellular signals to soluble protein-tyrosine kinases associated with the cytoplasmic face of the IgM receptor complex. The tyrosine phosphorylation initiated by these enzymes provides the earliest signals following IgM receptor-mediated activation (5-7), CA37372 awarded by the National Institutes of Health. The costs of * This research was supported in part by Public Health Service Grant 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  eliciting the cascade of early biochemical responses that accompany receptor cross-linking. A number of cytoplasmic tyrosine kinases have been described in B cells, including the Src-family enzymes p53/56lYn (Lyn), p56lCk, p59@", and ~5 5~" (8,9) and the 72-kDa non-Src-family kinase, p72"Yk (Syk or PTK72) (10,11). These protein-tyrosine kinases are associated with the IgM receptor complex in resting B cells and become activated as a result of receptor cross-linking. The exact mechanism by which this activation occurs is not yet clear. While receptor crosslinking has been reported to transduce signals by mechanisms such as conformational changes, dimerization of receptors and cross-phosphorylation, the requirement for CD45 (12) to activate the Src-family kinases suggests receptor cross-linking may also transduce signals by simple aggregation of receptors, including those not directly bound by the ligand.
The protein-protein interactions that are required for receptor signaling in B cells are still not well defined. The Src-family tyrosine kinases are myristoylated at the amino terminus, a modification required to stabilize membrane association and facilitate interactions with other membrane proteins (13,14). In addition, both the Src-family enzymes as well as Syk possess SH2 domains, which are involved in protein-protein interactions through recognition of specific phosphotyrosine residues (15). The SH3 domains of Src-family enzymes have been shown to be involved in interactions that anchor proteins to the cytoskeletal network (15).
In the work reported here, we have examined protein-protein interactions involving the Src-family tyrosine kinase, Lyn, as it relates to IgM receptor-mediated activation. The results indicate that when B cells are activated by receptor cross-linking, active Lyn rapidly appears in association with CD45 and components of the antigen receptor complex. This association is stable in the detergent Brij 96, but is disrupted by Nonidet P-40. The appearance of active Lyn in the receptor complex precedes by several minutes the appearance of active Syk, which forms an Nonidet P-BO-stable association with the antigen receptor.
MATERIALS AND METHODS Antibodies-Rabbit polyclonal anti-Syk peptide antibody was prepared against a synthetic peptide (Purdue Cancer Center Peptide Synthesis Facility) corresponding in sequence to the carboxyl-terminal 28 amino acids of Syk as deduced from the porcine syk sequence (16). Antibodies were also prepared against the amino-terminal SH2 domain of murine Syk expressed in Escherichia coli as a glutathione S-transferase fusion protein. To generate the fusion protein, total RNA was isolated from the murine B cell line LIO.A using RNAzol B (Cinnal Biotecx Laboratories, Inc.). Random primed first strand cDNA was synthesized using the cDNA cycle kit (Invitrogen). A 515-base pair DNA fragment containing the amino-terminal SH2 domain of murine Syk was amplified by polymerase chain reaction using the following primers: ACTTGCCCTTCTT(CPT)TT(CPT)GG and GAGGACCGTCTGCTCT-GAT. The polymerase chain reaction product was cloned into a pBluescript I1 KS EcoRV site T vector (17). A BamH1-Hind111 fragment containing the insert was subcloned in frame into the BamH1-Hind111 site of the bacterial expression vector pGEX-KG (18). The fusion con-struct was verified by sequencing. Polyclonal antibodies were prepared by immunizing rabbits with the 46-kDa glutathione S-transferase-Syk SH2-domain fusion protein purified from E. coli by chromatography on glutathione-agarose (18) and SDS-PAGE' (Purdue University Cancer Center Antibody Production Facility). Rabbit polyclonal antiphosphotyrosine antibody was prepared as described (11). Rabbit polyclonal anti-Lyn peptide antibody was purchased from Santa Cruz Biotechnology, Inc. Rat monoclonal anti-CD45 antibody was obtained from PharMingen.
Cell Culture and Actiuation"Ball7 B cells were obtained from Dr. Joseph Bolen (Bristol-Myers Squibb) and cultured in RPMI 1640 containing 10% fetal calf serum. 1-3 x lo6 celldsample were resuspended in serum-free RPMI or phosphate-buffered saline and activated on ice with 25 pg/ml goat F(ab)', anti-mouse IgM (Cappel) for the times indicated. The period of activation was terminated by the addition of lysis buffer to the cells. When IgM receptor complexes were immunoprecipitated, the cells were activated with the intact goat anti-mouse IgM (Sigma) used for immunoprecipitation. Cell lysates were prepared by incubating cells for 15 min on ice with buffer containing 50 mM "ris-HCl, pH 7.4, 1% Nonidet P-40, or Brij 96 (Sigma), 150 mM NaCl, 5 mM EDTA, 1 m M sodium orthovanadate, and 10 pg/ml each leupeptin and aprotinin. The lysates were vortexed briefly and centrifuged at 15,000 x g for 5 min at 4 "C.
Immune Complex Kinase Assays-The supernatants from cell lysates were incubated for 1 h at 4 "C with 25 p1 of protein A-Sepharose (Sigma) that had been previously incubated for 1 h at room temperature with 10 p1 of specific antibody. Anti-CD45 antibodies coupled to protein G+agarose (Pierce Chemical Co.) were used for the immunoprecipitation of CD45. Soluble protein G (Zymed) (1 pg/ml) was added to the cell lysates before incubation with anti-CD45-protein G-agarose to block binding of goat F(ab)', to the resin. The immune precipitates were washed twice with 500 pl of wash buffer (25 m M HEPES pH 7.4,0.1% detergent, 150 m~ NaCI, 1 m~ sodium orthovanadate, and 10 pg/ml each leupeptin and aprotinin) and twice with 500 pl of the same buffer without detergent. Phosphorylation reactions were performed for 1 min at 30 "C in kinase buffer containing 10 mM MnCl,, 20 m M p-nitrophenylphosphate, 25 m~ HEPES pH 7.5, and 25 pCi of [y-32PlATP (1 Ci = 37 GBq). The kinase reaction was stopped by boiling the samples for 5 min in Laemmli sample buffer with 5% 2-mercaptoethanol and 1% SDS. The phosphoproteins were separated by SDS-PAGE, transfemed to polyvinylidene difluoride membranes (Immobilon-P) and treated with 1 N KOH at 55 "C for 2 h. Phosphotyrosine-containing proteins were detected by autoradiography.
Metabolic LabeZing"Ball7 cells (5 x lo6) were incubated for 10 h in methionindcysteine-free RPMI medium containing 0.5 mCi of Trans5Slabel (ICN), a mixture of [35Slmethionine and [35Slcysteine (>lo00 Ci/ mmol). Lyn was immunoprecipitated with anti-Lyn antibodies as described above. IgM complexes were immunoprecipitated with anti-IgM antibodies from activated and unactivated cells as described above. The resulting immune complexes were then washed in buffer containing 1% Nonidet P-40. Metabolically labeled Lyn protein was reimmunoprecipitated from the Nonidet P-40 wash with anti-Lyn antibodies, separated by SDS-PAGE, and detected by fluorography.

RESULTS
Stimulation of Protein-Tyrosine Phosphorylation with Anti-IgM Antibodies-The cross-linking of antigen receptors on the surface of B cells with anti-IgM antibodies is accompanied by increases in the phosphorylation on tyrosine of multiple proteins (5,6). This phosphorylation is rapid and transient when cells are activated at 37 "C, but occurs at a slower rate when cells are activated on ice. To look more closely at the sequence of events occurring following receptor engagement, we incu-  Immunoprecipitation of Lyn with Anti-phosphotyrosine Antibodies-Engagement of the antigen receptor on B cells leads to the activation of the receptor-associated protein-tyrosine kinases, Lyn and Syk (8, 11,19,20). We have shown previously that Syk is phosphorylated on tyrosine following receptor cross-linking and can be recovered from activated cells by immunoprecipitation with anti-phosphotyrosine antibodies (10,11,21). The kinase can then be detected by its catalysis of an autophosphorylation reaction in the resulting immune complex. This is shown in Fig. 2 for Ball7 cells. In this experiment, cells were activated on ice by cross-linking the antigen receptor with goat F(ab)', anti-mouse IgM. The recovery of the 72-kDa Syk kinase by immunoprecipitation with anti-phosphotyrosine antibodies from Nonidet P-40 lysates increased as a function of time following the cross-linking of surface IgM.
Although engagement of the antigen receptor leads to the activation of the Src-family tyrosine kinase, Lyn, as well as Syk, we observed little Lyn autophosphorylating activity in anti-phosphotyrosine immunoprecipitates from Nonidet P-40 lysates prepared from activated cells (Fig. 2). We found, however, that when lysates were prepared using the detergent Brij 96 in place of Nonidet P-40, a substantial amount of Lyn autophosphorylating activity was present in anti-phosphotyrosine immune complexes following receptor cross-linking (Fig. 3A).
Phosphorylated Lyn appeared as a doublet due to the existence of two alternatively spliced forms of lyn mRNA (22). The major co-immunoprecipitating substrate of 34 kDa in these complexes is the receptor-associated protein MB-1.
Interaction of L.yn with Drosine-phosphorylated Proteins-Membrane IgM has previously been shown to associate with the Triton X-100-insoluble cytoskeletal matrix following ligand binding (23). Therefore, the appearance of Lyn in immunoprecipitates prepared from Brij 96 lysates and its absence from  Fig. 1 (lanes 1-5 and 6-10]. Cells were lysed in buffers containing Nonidet P-40 or Brij 96 as indicated. Proteins were separated by SDS-PAGE and transferred to polyvinylidine difluoride membranes. Lyn was detected by Western blotting. anti-phosphotyrosine immune complexes were isolated. The immune complexes were then washed with buffers containing either Brij 96 or Nonidet P-40 prior to incubation with [y-32P]ATP. As shown in Fig. 3 A , anti-phosphotyrosine immunoprecipitates from cell lysates prepared in Brij 96 and washed in Brij 96 contained high levels of Lyn autophosphorylating activity. In contrast, when the same immunoprecipitates from Brij 96 lysates were washed with Nonidet P-40, there was a dramatic loss of Lyn activity. To determine if Lyn was simply inhibited by residual detergent in the assay, Nonidet P-40 was added to the immune complex kinase assay a t increasing concentrations. No significant inhibition of Lyn activity was seen until concentrations of Nonidet P-40 reached a level 10-fold over that used in the resin washes (data not shown). Furthermore, tyrosine-phosphorylated Lyn could be selectively reimmunoprecipitated with anti-Lyn antibodies from the Nonidet P-40 wash of an anti-phosphotyrosine immune complex prepared from activated cells (Fig. 3B), indicating that the Lyn protein itself is removed from the complex by Nonidet P-40 and verifying the identity of the radiolabeled protein doublet as Lyn. Taken together, these data suggest that the immunoprecipitation of Lyn by anti-phosphotyrosine antibodies occurs via an association of Lyn with one or more other phosphoproteins rather than by a direct interaction of tyrosine-phosphorylated Lyn with the anti-phosphotyrosine antibody. This association is stable in Brij 96, but is readily disrupted in the presence of Nonidet P-40.
Interaction of Lyn with the Antigen Receptor-Lyn has previously been reported to associate with the B cell antigen receptor (9) and to bind to an expressed, immobilized polypeptide corresponding in sequence to the cytoplasmic tail of MB-1 (26). This suggested the possibility that Lyn was present in antiphosphotyrosine immune complexes due, at least in part, to its association with components of the antigen receptor complex.
To examine this further, receptor complexes were immunoprecipitated with antibodies directed against IgM from Brij 96 lysates of cells prior to or following receptor cross-linking. As shown in Fig. 5 Fig. 6). 3sS-Labeled Lyn was then reimmunoprecipitated with anti-Lyn antibodies from the detergent wash. As shown in Fig. 7, Lyn protein was preferentially recovered from the antigen receptor complex isolated from activated cells. In contrast, anti-Lyn antibodies immunoprecipitated comparable amounts of Lyn protein from the total lysates of unactivated versus activated cells. The intrinsic activity of Lyn, as measured by autophosphorylation, was greater in anti-Lyn immune complexes isolated from activated cells.
These data indicate that the enhanced level of Lyn activity appearing in anti-IgM immune complexes from activated cells arose from both an increase in the level of Lyn protein and an increase in its enzymatic activity. Syk autophosphorylating activity also appeared in anti-IgM immune complexes following receptor cross-linking, although the kinetics of the appearance of Syk activity in the receptor complex were much slower than those for the appearance of Lyn (Fig. 5).
An association of Lyn and Syk with the antigen receptor was also supported by the observation that antibodies directed against each kinase co-immunoprecipitated MB-1 from Brij 96 lysates of anti-IgM-activated Ball7 B cells as detected by in vitro phosphorylation (Fig. 6). This appearance of MB-1 in anti-Lyn or anti-Syk immunoprecipitates was dependent on receptor cross-linking. The forces stabilizing the Lyn and Syk interactions with receptor components could be distinguished by their sensitivities to disruption with Nonidet P-40. The Lynassociated MB-1 could be washed from the anti-Lyn immune complex by buffers containing Nonidet P-40, while the Sykassociated MB-1 was stable in the presence of Nonidet P-40 (Fig. 6). Such a stable association would be consistent with an SH2 domain-phosphotyrosine interaction as has been previously demonstrated to be important for the binding of the ZAP-70 tyrosine kinase to the TCR 5 chain (27). To explore this further, antibodies were generated against a region of Syk en-  receptor complexes (lanes 3 and 4 ) , Lyn (lanes 5 and 6 ) or Syk (lanes 7 and 8) were immunoprecipitated from the cell lysates with the appropriate antibodies (for Syk, the anti-peptide antibody was used), and the immune complexes were washed with buffers containing Brij 96 (lanes 1, 3, 5 The phosphorylation of MB-1 in anti-Lyn and anti-Syk immune complexes suggested that both Syk and Lyn were capable of catalyzing the phosphorylation of MB-1 in vitro. Consistent with this observation, the dissociation of Lyn from anti-phosphotyrosine immune complexes by washing with Nonidet P-40 had little effect on the level of Syk that remained in the complex and reduced, but did not eliminate, MB-1 phosphorylating activity (Fig. 6, lanes 1 and 2).  (lanes I and 3) or anti-IgM activated (lanes 2 and 4 ) Ball7 cells using either the anti-Syk peptide antibody directed against the carboxyl terminus (lanes 1 and 2 ) or the anti-Syk antibody directed against the amino-terminal SH2 domain (lanes 3 and 4 ) . The immune complexes were incubated with [-ps2PlATP, and the phosphoproteins were detected as described in Fig.  2. The migration position of Syk is indicated by the asterisk.
The protein identified as MB-1 that was phosphorylated in both anti-Syk and anti-Lyn immune complexes was indistinguishable by one-dimensional phosphopeptide mapping from MB-1 phosphorylated in anti-IgM immunoprecipitates (data not shown). Likewise, the 72-kDa phosphoprotein identified as Syk in both anti-phosphotyrosine and anti-IgM immune complexes was identical by peptide mapping to the 72-kDa phosphoprotein present in anti-Syk immune complexes. Similar peptide mapping results have been described previously (11,21).
Association of Lyn with CD45-Cross-linked antigen receptor complexes have been reported to co-aggregate with multiple transmembrane proteins including the phosphotyrosine phosphatase, CD45, a likely candidate for a positive regulator of Lyn activity (28). To examine a possible involvement of CD45 in the formation of an active signaling complex, CD45 was immunoprecipitated from Brij 96 lysates of activated cells, and the associated kinases and substrates were visualized by incubation of the immune complex with [y32P]ATP. As shown in Fig.  9, Lyn autophosphorylating activity appeared rapidly in anti-CD45 immune complexes following receptor engagement and peaked a t 45 s. Anti-CD45 immune complexes also contained MB-1, and its phosphorylation followed that of Lyn. The association of Lyn with CD45 was also sensitive to disruption with Nonidet P-40 (data not shown).

DISCUSSION
The activation of B cells can be initiated by the clustering of cell surface IgM with cross-linking antibodies. An early response to this receptor cross-linking is the phosphorylation of multiple proteins on tyrosine resulting from the activation of endogenous protein-tyrosine kinases that include both Lyn and Syk (8)(9)(10)(11). Since the activation of many protein-tyrosine kinases results in an increased rate of autophosphorylation, we used anti-phosphotyrosine antibodies to immunoprecipitate activated tyrosine kinases to compare the rates of activation of Lyn and Syk. As reported previously, Syk becomes tyrosine phosphorylated following receptor cross-linking (10,11,21) and can be recovered by immunoprecipitation with anti-phosphotyrosine antibodies from Nonidet P-40-lysates of activated cells (Fig. 2). Little Lyn, however, is recovered from comparable lysates (Fig. 21, despite the ability of Nonidet P-40 to readily solubilize the bulk of the kinase (Fig. 4). We have also found it difficult to show increases in the in vivo autophosphorylation of Lyn on Western blots using anti-phosphotyrosine antibodies. This is similar to the observations of others who have seen little or no change in the phosphotyrosine content of Lyn following receptor engagement in certain B cell lines (19). Lyn only appears in anti-phosphotyrosine immune complexes following receptor engagement if cell lysates are prepared with Brij 96 rather than Nonidet P-40 (Fig. 3). This, however, is an indirect association of Lyn with the anti-phosphotyrosine antibody since Nonidet P-40 readily dissociates the kinase from the immune complex (Figs. 3 and 6). This indicates that Lyn is either associated or becomes associated with one or more additional proteins that are tyrosine-phosphorylated following receptor engagement.
The identities of the tyrosine-phosphorylated proteins with which Lyn interacts have yet to be determined. Components of the antigen receptor complex are possible candidates since both MB-1 and B29 are phosphorylated on tyrosine following the treatment of cells with anti-IgM antibodies (29) and MB-1 is readily phosphorylated in vitro by the tyrosine kinases present in anti-phosphotyrosine immune complexes (Fig. 3). Furthermore, Lyn autophosphorylating activity is present in anti-IgM immune complexes (Fig. 5), and MB-1 appears in anti-Lyn immune complexes (Fig. 6) following receptor engagement. However, the release of Lyn from the anti-phosphotyrosine immune complexes by washing with Nonidet P-40 not only reduces the level of MB-1 phosphorylation in the complex but also reduces the phosphorylation of additional co-immunoprecipitating substrates (Figs. 3 and 61, including proteins with estimated molecular weights of 120,000 and 150,000. Proteins corresponding to these same apparent molecular weights have also been reported previously to be tyrosine-phosphorylated in several murine B cell lines (30). In human B cells, the transmembrane receptors CD19 and CD22 have been shown to be phosphorylated on tyrosine and to associate with activated antigen receptors (31)(32)(33)(34). In fact, a direct association of Lyn with CD19 has been reported (35). It is not yet known if the 120-and 150-kDa tyrosine-phosphorylated proteins correspond to the murine equivalents of either of these glycoproteins. Thus, the enhanced appearance of Lyn in the antigen receptor complex following receptor engagement could be a function of the direct association of Lyn with components of the antigen receptor or Lyn could be "delivered" to the complex in association with one or more of these other protein substrates.
The cross-linking of surface IgM leads first to the rapid formation of a multi-component complex that minimally includes the antigen receptor, the protein-tyrosine kinase Lyn, and the phosphotyrosine phosphatase CD45 (Fig. 9). Visualization of such a complex requires the use of a mild detergent such as Brij 96. The co-aggregation of Lyn and CD45 may explain the rapid activation of Lyn that occurs following receptor cross-linking. Lyn, like other members of the Src-family of kinases, possesses a negative-regulatory phosphotyrosine located near the carboxyl terminus that is a potential substrate for dephosphorylation by CD45. How CD45 and Lyn are brought together in the receptor complex is not clear. It has been reported that Lyn is associated directly with CD45 in resting B cells (36). In Ball7 cells, the appearance of Lyn activity in anti-CD45 immune complexes is greatly enhanced upon cross-linking IgM (Fig. 9). Since this occurs without any direct manipulation of CD45, it suggests that the aggregation alone is sumcient to bring CD45 into the IgM receptor complex. This would seem feasible since the ratio of CD45 to IgM receptors on the cell surface is estimated to be 1O:l (28). There is also evidence that CD45 comodulates with the IgM receptor when either IgM or CD45 are cross-linked on the cell surface (28). Alternatively, CD45 may maintain a noncovalent association with the IgM receptor complex prior to activation, as has been shown recently (361, and Lyn may be recruited to this complex upon receptor crosslinking. The stability of the Lydreceptor association makes it unlikely that Lyn is attracted to the antigen receptor complex through a phosphotyrosine-SH2 domain interaction. This is based on the observation that Lyn is readily dissociated from components of the antigen receptor complex or from other tyrosine-phosphorylated proteins with Nonidet P-40 (Figs. 3 and  6). Phosphotyrosine-SH2 domain associations are generally high-affinity interactions that are not readily disrupted by nondenaturing detergents (37). Nonidet P-40 has previously been shown to disrupt the antigen receptor complex by dissociating the MB-l.B29 heterodimer and associated Src-family kinases from IgM (25). In resting B cells, this association between the Src-family kinases and MB-l.B29 is stable in the presence of Nonidet P-40 (25). The nature of this interaction, however, appears to be distinct from that observed with the activated receptors since Nonidet P-40 is capable of dissociating the newly recruited Lyn from anti-phosphotyrosine immune complexes (Figs. 3 and 6), which retain MB-1, and of dissociating MB-1 from anti-Lyn immune complexes (Fig. 6). These interactions are similar to those reported by DeFranco and co-workers (38) who observed an activation-dependent association of Lyn and Syk with chimeric receptor proteins constructed with cytoplasmic tails corresponding to the ARH-1 motifs of MB-1 or B29. The association of Syk, but not of the newly recruited Lyn, with the chimeric receptors was stable to Nonidet P-40. The sensitivity of the Lyn-antigen receptor interaction to disruption with Nonidet P-40 suggests that this association may be stabilized by hydrophobic interactions. Consistent with this hypothesis, we have found that replacement of the Lyn amino-terminal myristate with a less hydrophobic fatty acid seriously compromises the ability of Lyn to associate with receptor components.' The association of Syk with MB-1 that is observed following receptor engagement is stable in the presence of Nonidet P-40 (Fig. 6) and may, therefore, represent an SH2 domain-phosphotyrosine interaction. During the activation of T cells, the ZAP70 tyrosine kinase binds to the tyrosine-phosphorylated (39,40) and E (41)  SH2 domains (27). Syk, which is structurally related to ZAF'70, likely binds to tyrosine-phosphorylated MB-1 in an analogous fashion. The inability of Syk to bind MB-1 in the presence of an antibody that interacts with its amino-terminal SH2 domain (Fig. 8) is consistent with this hypothesis.
While receptor-stimulated tyrosine phosphorylation is rapid and transient at 37 "C, it is more delayed (Fig. 1) and prolonged when cells are activated on ice (6). By slowing the rate of receptor-stimulated protein phosphorylation, we have been able to examine more closely the temporal sequence of events that occur following receptor engagement. As discussed above, the data presented here indicate that cross-linking the antigen receptor results first in the aggregation of multiple membrane proteins that remain associated in the presence of Brij 96. The formation of this complex, which contains activated Lyn, precedes any substantial increase in the concentration of cellular tyrosine-phosphorylated proteins (Fig. 1). The rate of accumulation of phosphotyrosine-containing proteins parallels the rate of activation of Syk and the appearance of Syk activity in anti-IgM complexes. Since the recruitment of Lyn to the receptor precedes the recruitment and activation of Syk, it is possible that Lyn-catalyzed tyrosine phosphorylation is required for or promotes Syk activation. This is consistent with the observation that the activation of Syk is considerably reduced in lynnegative DT40 chicken B cells (42). In T cells lacking antigen receptors, aggregation of an expressed Syk-CD16 chimera elevates the levels of cellular tyrosine-phosphorylated proteins and triggers Ca2+ mobilization (43). Thus, the binding of Syk to phosphorylated receptor components may provide a mechanism for its aggregation and activation, perhaps through transphosphorylation. Syk may then catalyze the phosphorylation of many of the downstream effectors of the activation response.