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The Journal of Immunology, 1999, 163: 1110-1114.
Copyright © 1999 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: Extracellular Signal-Regulated Kinase Activates Syk: A New Potential Feedback Regulation of Fc{epsilon} Receptor Signaling1

Rong Xu*, Rony Seger{dagger} and Israel Pecht2,*

Departments of * Immunology and {dagger} Biological Regulation, The Weizmann Institute of Science, Rehovot, Israel


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
The protein tyrosine kinase Syk is an essential element in several cascades coupling Ag receptors to cell responses. Syk and the mitogen-activated protein kinase extracellular signal-regulated kinase 1 (ERK1) were found to form a tight complex in both resting and Ag-stimulated rat mucosal-type mast cells (rat basophilic leukemia 2H3 cell line RBL-2H3). A direct serine phosphorylation and activation of Syk by ERK was observed in in vitro experiments. Moreover the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) kinase (MEK) inhibitors markedly decreased the Ag-induced phosphorylation of the tyrosyl residues of Syk and its activation as well as suppressed the degranulation of the cells. These results suggest a positive feedback regulation of Syk by ERK in the cascade coupling the type 1 Fc{epsilon} receptor to the secretory response of mast cells; hence, the existence of a novel type of cross-talk between protein serine/threonine kinases and protein tyrosine kinases is suggested.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Clustering of the type 1 Fc{epsilon} receptor (Fc{epsilon}RI)3 on mast cells initiates the biochemical cascades, which culminate in the secretory response of these cells. Subsequent steps involve the activation of the protein tyrosine kinases Lyn and Syk (1, 2, 3, 4). Activated Syk further couples this cascade to downstream events, leading to the secretion of granule-stored mediators by these cells as well as to de novo synthesis and to the secretion of leukotrienes and cytokines (5). Syk-deficient mast cells fail to degranulate, to synthesize and secrete these leukotrienes and cytokines when stimulated by Fc{epsilon}RI clustering (5, 6). Transfection of Syk into these cells reconstitutes Fc{epsilon}RI-mediated secretion (5). The activation of Syk was shown to involve the phosphorylation of serine residues in addition to tyrosyl phosphorylation (7, 8), but its mechanism and functional role are unknown. Syk activation is also known to be essential for the Fc{epsilon}RI-induced activation of mitogen-activated protein (MAP) kinases (MAPKs) (6), probably through phosphorylation of Shc and triggering of the Grb2/Sos/Ras cascade (9). Earlier studies have shown MAPK/extracellular signal-regulated kinase (ERK) involvement in mast cell synthesis and secretion of arachidonic acid and TNF-{alpha}; however, a role for MAPK/ERK in the induction of degranulation has been excluded (10, 11). Here, we report results that suggest that ERK is involved in the degranulation of mast cells due to its interactions with and regulation of Syk. This finding is expressed by the phosphorylation of Syk by ERK on serine residues, causing its enhanced activity, as well as by the direct association of the two enzymes as evidenced by their coisolation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Reagents

DNP11-BSA (Ag consisting of 11 molecules of dinitrophenol (DNP) conjugated with 1 molecule of BSA) was prepared in our laboratory from BSA (fraction V) (Sigma, St. Louis, MO) by reaction with fluoro-2,4 dinitrobenzene. The mouse IgE class, DNP-specific mAb A2IgE preparation has been described previously (12). Lyn- and Syk-specific polyclonal Abs were generous gifts of Dr. J. Cambier (National Jewish Center, Denver, CO). These Abs were raised in rabbits immunized with the 1–131 domain of Lyn and the 257–352 linker domain of Syk, respectively. The plasmid encoding the hemopoietic-lineage cell-specific protein (HS1) fused with GST (GST-HS1) (13) was kindly provided by Dr. Ulrich Blank (Immuno-Allergie, Institute Pasteur, Paris, France), and the protein was expressed in Escherichia coli and was affinity purified on GST beads. GST-mSyk recombinant baculovirus was kindly provided by Dr. R. Geahlen (Purdue University, West Lafayette, IN). Recombinant GST murine Syk (rSyk) was expressed in Spodoptera frugiperda insect cells and purified as described previously (14). All other reagents were purchased from commercial sources as indicated.

Immunoprecipitation and Western blotting

Rat basophilic leukemia 2H3 (RBL-2H3) cells were plated (7 x 106/15 ml DMEM plus 10% FCS in 100-mm tissue culture dishes), cultured overnight, and saturated with a DNP-specific monoclonal IgE. Following the addition of Ag (50 ng/ml DNP11-BSA or as indicated), cells were lysed at the indicated times in 0.7 ml of Triton X-100 lysis buffer (50 mM HEPES (pH 7.4), 100 mM NaF, 10 mM EDTA, 10% glycerol, and 1% Triton X-100) containing protease and phosphatase inhibitors (1 mM sodium orthovanadate and 50 mM {beta}-glycerophosphate) on ice for 20 min. Digitonin lysis buffer (1% Digitonin, 150 mM NaCl, and 10 mM triethanolamine (pH 7.8)) containing the same inhibitors was used for coimmunoprecipitation. Lysates were centrifuged for 15 min at 15,000 x g at 4°C, and the postnuclear supernatants were reacted with the relevant Ab bound to protein G-Sepharose beads (Pharmacia, Uppsala, Sweden). After 2 h of equilibration (4°C), the beads were washed once with 0.5 M LiCl, three times with ice-cold lysis buffer (for the coimmunoprecipitation, 0.2% Digitonin was used instead of 1%), and once with lysis buffer without detergent; the bound proteins were then eluted at 95°C for 5 min with SDS sample buffer plus 0.5% 2-ME. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes blocked by 0.1% gelatin/0.05% Tween 20 in Tris-buffered saline before incubation with specific Abs. Proteins were detected with peroxidase-conjugated secondary Abs (Jackson ImmunoResearch Laboratories, West Grove, PA) and chemiluminescence reagents (Amersham, Little Chalfont, U.K.).

In vitro kinase assays and phosphoamino acid analysis

Syk was immunoprecipitated from RBL-2H3 cell lysates. Syk-carrying beads were washed further with a buffer of 20 mM HEPES and 100 mM NaCl (pH 7.5). Wild-type Syk (1 x 107 cells) or rSyk (0.5 µg) were incubated with or without active ERK (Sigma, Israel) in 21 µl of kinase buffer B (25 µM {beta}-glycerophosphate, 0.5 µM DTT, 1.27 µM EGTA, 0.05 µM NaVO4, 10 µM MgCl2, and 0.04 µM ATP) for 20 min at 30°C. Syk activity was assayed by the addition of 10 µl of kinase assay buffer A (0.02 µM ATP instead of the 0.04 µM of ATP used in buffer B) containing 10 µCi of [{gamma}-32P]ATP, 5 mM MnCl, and 1.5 µg of Syk substrate (GST-HS1) incubated for 8 min at 30°C. The reaction was stopped by SDS sample-buffer plus 0.5% 2-ME. Samples were analyzed by SDS-PAGE, autoradiography, and Western blotting. The phosphorylation of Syk by ERK was attained by incubation with active ERK (70 ng) in 21 µl of kinase buffer A containing 10 µCi of [{gamma}-32P]ATP for 20 min at 30°C and was subsequently analyzed by SDS-PAGE, followed by autoradiography. Syk-containing bands were cut out and analyzed for phosphoamino acid (15).

Secretory response assay

RBL-2H3 cells were plated in 96-well plates (7 x 104/well/100 µl DMEM), incubated overnight at 37°C, saturated with DNP-specific monoclonal IgE, washed three times with Tyrode’s buffer (16), and incubated in 50 µl of Tyrode’s buffer with the indicated amounts of MAPK/ERK-activating kinase (MEK) inhibitors (PD98059 (Biomol, Plymouth Meeting, PA) or U0126 (DuPont, Wilmington, DE)) or without inhibitors for 20 min at 37°C. Next, 50 µl of Tyrode’s buffer containing different Ag concentrations was added, and the incubation continued for 50 min at 37°C. Supernatant aliquots (15 µl) were then taken and transferred to a separate plate, and 40 µl of {beta}-hexosaminidase substrate solution (1.3 mg/ml p-nitrophenyl-N-acetyl-{beta}-D-glucosamine in 0.1 M citrate (pH 4.5)) was added to the samples. The plates were then incubated for 60 min at 37°C, and the reaction was terminated by the addition of 150 µl of "stop" solution (0.2 M glycine (pH 10.7)). The absorbency change caused by substrate hydrolysis was measured at 405 nm in an ELISA reader. Degranulation was calculated as the percentage of the total enzyme activity measured in 1% Triton X-100-lysed cells; data are the average of three independent experiments.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
MEK inhibitors suppress Ag-induced tyrosyl phosphorylation of Syk and cell degranulation

RBL-2H3 cells were saturated with a monoclonal, DNP-specific IgE (A2) and incubated with the MEK inhibitors PD98059 (17) or U0126 (18) for 20 min at 37°C followed by stimulation with Ag; next, the state of tyrosyl phosphorylation of Lyn and Syk in the cells was examined. The protein tyrosine kinases were immunoprecipitated from lysates by the appropriate specific polyclonal Abs followed by SDS-PAGE separation and Western blotting with a phosphotyrosine-specific mAb (PY-20); Although Ag-induced tyrosyl phosphorylation of Lyn was found to be unaffected, that of Syk was markedly suppressed in a dose-dependent manner by up to ~74% by both inhibitors (Fig. 1GoA). Both inhibitors also reduced ERK activation (Fig. 1GoB and data not shown) and the Ag-induced activation of Syk (data not shown). Degranulation assays, in which the activity of secreted {beta}-hexosaminidase was monitored, showed that both MEK inhibitors caused up to a 40% decrease in Ag-induced secretion (Fig. 1GoC). These results indicate that although MEK/ERK were shown to act downstream of Lyn, they may cause regulation of Syk activity and thereby control certain elements of the stimulus-response coupling in these cells.



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  		FIGURE 1. MEK inhibitors suppress the Ag-induced tyrosyl phosphorylation of Syk and cell degranulation. RBL-2H3 cells were pretreated (+) with 100 µM of PD98059 or 25 µM of U0126 or left as a control (-) and were stimulated by the indicated amounts of Ag (DNP11-BSA): A, Syk and Lyn were immunoprecipitated from cell lysates separated by SDS-PAGE, Western blotted by a phosphotyrosine specific mAb (PY-20) (Transduction Laboratories, Lexington, KY), and reblotted with Syk- or Lyn-specific polyclonal Abs. B, A total of 70 µg of protein of WCLs was loaded and analyzed by SDS-PAGE followed by Western blotting with an activated ERK-specific mAb (19 ). C, The secretory response was assayed by monitoring the {beta}-hexosaminidase activity in the cell supernatants. Degranulation is calculated as the percentage of the total enzyme activity measured in 1% Triton X-100-lysed cells; data are the average of three independent experiments made in duplicates. Inset: secretion induced by 3 ng of Ag from the cells pretreated by the indicated concentration of MEK inhibitors.

 
Syk is phosphorylated and activated by ERK

To better define the above relationships, Syk was affinity isolated from resting RBL-2H3 cells by specific polyclonal Abs and the activity of Syk was examined. It was found that Syk underwent autophosphorylation even without incubation with ERK; however, its phosphorylation was enhanced by incubation with active ERK (Fig. 2GoA). Phosphoamino acid analysis showed that this additional phosphorylation was confined to serine residues (Fig. 2GoB). Furthermore, using the Syk substrate GST-HS1 (13), the enzymatic activity of Syk was found to be markedly enhanced following incubation with active ERK (Fig. 2GoA). This activation of Syk exhibited a clear concentration dependence upon active ERK (Fig. 2GoC). Because there are no serine/threonine residues in the HS1 peptide and because GST-HS1 was not phosphorylated by ERK (Fig. 2GoC), these results indicate that Syk can be directly phosphorylated and activated by ERK. Active Syk neither phosphorylated recombinant ERK nor affected its activation (data not shown).



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  		FIGURE 2. Syk is phosphorylated and activated by ERK. Syk was immunoprecipitated from 1 x 107 resting RBL-2H3 cell lysates. A, Protein G beads carrying Syk were washed with a buffer of 20 mM HEPES and 100 mM NaCl (pH 7.5) and subsequently incubated with (+) or without (-) 70 ng of active ERK followed by the addition of SDS sample buffer (samples 1 and 2) or by continued incubation with 3 µg of GST-HS1 (samples 3 and 4). Proteins were analyzed by SDS-PAGE followed by autoradiography. B, Phosphoamino acid analysis of the radiolabeled Syk from samples 1 and 2 in A. The positions of phosphoserine (PS), phosphothreonine (PT), and phosphotyrosine (PY) are indicated by arrows. C, Dose-dependent activation of Syk by ERK. The indicated amounts of active ERK were incubated with or without protein G beads carrying Syk for 20 min followed by the addition of Syk substrate (1.5 µg GST-HS1). Proteins were eluted, separated, and detected by autoradiography. Upper panel, Results of autoradiography. Lower panel, Results of Western blotting of the same membrane by polyclonal Syk-specific Abs.

 
rSyk is phosphorylated and activated by ERK

To exclude the possibility that traces of coimmunoisolated proteins are responsible for the capacity of ERK to phosphorylate and activate Syk, rSyk was employed in in vitro kinase assays. It was found that rSyk underwent efficient autophosphorylation and also phosphorylated GST-HS1 when incubated with [{gamma}-32P]ATP (Fig. 3GoA). Still, rSyk incubation with active ERK further enhanced its activity (Fig. 3GoB) in a dose- (Fig. 3GoB, left panel) and time-dependent manner (Fig. 3GoB, right panel). Coincubation of nonphosphorylated recombinant ERK with rSyk did not cause any changes in Syk activity (data not shown), suggesting that the above activity enhancement depends upon phosphorylation by ERK and not only association of ERK to Syk (see below). Purified rSyk was shown to have a relatively high basal phosphorylation and activity (Fig. 3GoA). To reduce this basal phosphorylation, rSyk (in its GST bead-bound form) was dephosphorylated by incubation with Shrimp alkaline phosphatase (1 U per 1 µg rSyk) at 37°C for 20 min, washed three times with PBS, eluted by reduced glutathione, incubated with active ERK, and subsequently assayed for its phosphorylation and activity. Comparison between untreated rSyk and dephosphorylated rSyk showed that the autophosphorylation and activity of the latter were abolished (Fig. 3GoC). Although active ERK does phosphorylate this dephosphorylated rSyk (Fig. 3GoC, compare samples 5 and 6) on serine residue(s) (Fig. 3GoD), the activity of rSyk was only slightly increased (Fig. 3GoC, compare samples 4 and 8 and data not shown). This may indicate that the basal phosphorylation of rSyk is essential for attaining its full activity. Taken together, these results may also suggest that the basal tyrosyl phosphorylation observed in Ag-stimulated RBL-2H3 cells, in which Syk is activated by binding to the phosphorylated {gamma} subunits of Fc{epsilon}RI (3) or upon phosphorylation by Lyn (7), is important for the activity of Syk, which is yet further activated by ERK.



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  		FIGURE 3. rSyk is phosphorylated and activated by ERK. A, rSyk kinase activity assay. Indicated amounts of rSyk were incubated in 30 µl of kinase buffer B containing 10 µCi of [{gamma}-32P]ATP with or without GST-HS1. Proteins were separated by SDS-PAGE, and autoradiograms are presented. B, rSyk (500 ng) was incubated with the indicated amounts of active ERK for 20 min (left panel) or with 70 ng of active ERK for the indicated times (right panel) in 21 µl of kinase buffer B, followed by an in vitro kinase assay using GST-HS1 as the substrate. The vertical axis presents the densitometric analysis of 32P-labeled GST-HS1 intensity. C, rSyk (500 ng) was dephosphorylated (+) or not (-) by Shrimp alkaline phosphatase and subjected to an in vitro kinase assay with (+) or without (-) activated ERK. In samples 3, 4, 7, and 8, the activity of rSyk was examined by adding GST-HS1 as the substrate. D, Phosphoamino acid analysis of the radiolabeled Syk from samples 5 and 6 in C.

 
ERK1 and Syk are coisolated from RBL-2H3 cell lysates

Further studies of Syk-ERK relationships provided clear evidence that these two enzymes directly associate in RBL-2H3 cells. Both resting or Ag-stimulated RBL-2H3 cells were lysed in 1% Digitonin lysis buffer, and ERK was immunoprecipitated by polyclonal ERK-specific Abs. Immunocomplexes were washed twice in 0.2% Digitonin lysis buffer, once in 0.5 M LiCl, and once in the lysis buffer without detergent and were subjected to Western blotting with Syk-specific polyclonal Abs. Syk was found to be associated with ERK in resting as well as in Ag-stimulated cells (Fig. 4GoA). Significantly, ERK1 was also found in Syk immunocomplexes (Fig. 4GoB). When analyzed by Western blotting with a mAb specific for activated ERK (19), the associated ERK1 (Fig. 4GoC, upper panel) was already found to be active at 1 min after Ag stimulation and remained so as long as 20 min afterward. This activation pattern was similar to that of the Ag-induced activation of cytoplasmic ERK1 in these cells (data not shown). Because no Shc or ribosomal S6 kinases were detected bound to Syk under these conditions (data not shown), we assume that there is at least partial ERK1 association with Syk, and that most of the ERK1 molecules (including the cytoplasmic molecules) can be activated upon Ag stimulation. The whole cell lysate (WCL) shown in Fig. 4Go, A and B, exhibited a different pattern of ERK1/2 due to a different source of ERK-specific Abs. Moreover, when RBL-2H3 cells were stably transfected with dominant-negative MEK (K97A-MEK) (20), the Fc{epsilon}RI-induced {beta}-hexosaminidase secretion was suppressed by 50% compared with the responses of cells transfected with constitutively active MEK ({Delta}N-EE-MEK) (21) (data not shown).



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  		FIGURE 4. ERK1 coisolates with Syk from RBL-2H3 cells. RBL-2H3 cells were stimulated by Ag (50 ng/ml) for the indicated times and lysed in 1% Digitonin lysis buffer. A, ERK was immunoprecipitated (IP) from the indicated cell lysates by specific polyclonal Abs (Sigma) and analyzed by SDS-PAGE followed by Western blotting with Syk-specific polyclonal Abs (upper panel) and reblotting with HRP coupled to ERK-specific mAbs (Transduction Laboratories) (lower panel). B, Syk was immunoprecipitated from the indicated cell lysates, and samples were analyzed by SDS-PAGE followed by Western blotting with ERK-specific polyclonal Abs (Sigma) (upper panel) and reblotting with Syk-specific polyclonal Abs (lower panel). The WCL shows the position of ERK1 and ERK2. C, Syk was immunoprecipitated from the indicated cell lysates; samples were analyzed by SDS-PAGE and Western blotting with a mAb specific for activated ERK (19 ) (upper panel) and reblotting with Syk-specific polyclonal Abs (lower panel).

 
In conclusion, the above results show that the MEK/ERK pathway is involved in the regulation of the secretory response of mast cells to their immunological stimulus by the phosphorylation of Syk on serine residues (probably on one of its Ser-Pro couples), leading to its activation. Part of ERK1 was found to be associated with Syk in RBL-2H3 cells. Therefore, among the different Fc{epsilon}RI-activated MAPKs, ERK1 might have the role of phosphorylating Syk, thereby positively regulating the Fc{epsilon}RI coupling cascade.


   Acknowledgments
 
We thank Dr. R. Geahlen for providing the GST-mSyk virus, Dr. Ulrich Blank for the plasmid encoding GST-HS1, DuPont-Merck Pharmaceutical Company for the MEK inhibitor U0126, and Sigma (Israel) for the active ERK. The continuous devoted help by Arieh Licht, Tami Hanoch, Martin Schlee, and other members of our laboratories with reagents, experiments, and discussions is greatly appreciated.


   Footnotes
 
1 This work was supported by a grant from the Israel National Science Foundation founded by the Israel Academy of Sciences and Humanities (to I.P.) and by a Minerva grant (to R.S.). Back

2 Address correspondence and reprint requests to Dr. Israel Pecht, Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel. E-mail address: Back

3 Abbreviations used in this paper: Fc{epsilon}RI, the type 1 Fc{epsilon} receptor (for IgE); ERK, extracellular signal-regulated kinase; HS1, hemopoietic lineage cell-specific protein; DNP, dinitrophenol; MAP, mitogen-activated protein; MAPK, MAP kinase; MEK, MAPK/ERK kinase; RBL-2H3, rat basophilic leukemia 2H3 cell line; WCL, whole cell lysate; rSyk, recombinant GST murine Syk. Back

Received for publication April 26, 1999. Accepted for publication May 25, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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Y. Yung, Z. Yao, D. M. Aebersold, T. Hanoch, and R. Seger
Altered Regulation of ERK1b by MEK1 and PTP-SL and Modified Elk1 Phosphorylation by ERK1b Are Caused by Abrogation of the Regulatory C-terminal Sequence of ERKs
J. Biol. Chem., September 14, 2001; 276(38): 35280 - 35289.
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