Structural requirements of Syk kinase for Fcγ receptor– phagocytosis

Experimental Hematology 27 (1999) 875–884

0301-472X/99 $–see front matter. Copyright © 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc.PII S0301-472X(99)00025-9

Structural requirements of Syk kinase for Fc

g

receptor– phagocytosis

Sharon Hunter

a

, Norihito Sato

a

, Moo-Kyung Kim

a

,Zhen-Yu Huang

a

, David H. Chu

b

, Jong-Gu Park

c

, and Alan D. Schreiber

a

a

Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA;

b

Department of Microbiology and Immunology, University of California, San Francisco, CA;

c

Institute of Medical Sciences, Dongsan Medical Center, Taegu, Republic of Korea

(Received 12 October 1998; revised 21 December 1998; accepted 4 January 1999)

The tyrosine kinase Syk plays a critical role in the phagocyticpathway mediated by Fc

g

receptors (Fc

g

R). In transfectedCOS1 cells co-expression of Syk enhances Fc

g

R mediatedphagocytosis. The other member of the Syk kinase family, thehighly homologous tyrosine kinase Zap70, also plays a role insignaling by immunoglobulin gene family receptors, but doesnot increase the phagocytic efficiency of Fc

g

Rs. The homolo-gous tandem SH2 and kinase domains of Syk and Zap70 areseparated by a nonhomologous region referred to as the uniquedomain. Zap70’s inability to enhance phagocytosis was not dueto unique domain tyrosine 292, previously implicated in nega-tive regulation of Zap70 function. We determined the regionsof Syk important for its interaction with the phagocytic path-way. An intact kinase domain was required for Syk’s effect onphagocytosis. Furthermore, the Syk variant SykB, lacking 23amino acids in the unique region, signaled for phagocytosis asefficiently as did Syk. We then constructed exchange chimerasbetween Syk and Zap70 and determined the contributions ofthe SH2, unique and kinase domains to phagocytic signaling.Our data suggest that the Syk kinase domain, which has highintrinsic kinase activity, is important for facilitating phago-cytic signaling by Fc

g

RI and Fc

g

RIIIA. © 1999 InternationalSociety for Experimental Hematology. Published by ElsevierScience Inc.

Keywords:

Receptors—Signaling—Tyrosine kinases—Phago-

cytosis

Introduction

Fc

g

receptors (Fc

g

Rs) that bind the Fc

g

domain of IgG playan important role in several immunologic systems, for re-cent reviews [1–3]. A major function of Fc

g

Rs in the mono-cyte/macrophage lineage is the induction of phagocytosis of

IgG coated cells. Using COS1 cells expressing Fc

g

Rs as amodel system, we have demonstrated that each of the threeclasses of human Fc

g

Rs (Fc

g

RI, Fc

g

RII, and Fc

g

RIII) canmediate a phagocytic signal [4–6]. Fc

g

RIIA is unique amongthe Fc

g

R family in that it can mediate phagocytosis as a sin-gle chain. Fc

g

RI and Fc

g

RIIIA also transmit a phagocyticsignal but require the co-expression of an accessory

g

chain(Fc

g

RI/

g

and Fc

g

RIIIA/

g

) [5]. Chimeric receptors consist-ing of the extracellular domain (EC) of Fc

g

RI or Fc

g

RIIIA

a

chain and the transmembrane (TM) and cytoplasmic(CYT) domains of the

g

chain (designated I-

g

-

g

and

a

-

g

-

g

,respectively) are also efficient at mediating a phagocyticsignal when transfected into COS1 cells [7,8].

For Fc

g

RIIA-, Fc

g

RI/

g

- or Fc

g

RIIIA/

g

-mediated phago-cytosis, a cytoplasmic amino acid motif known as the ITAM(immunoreceptor tyrosine based activation motif), which isalso present in the B-cell, T-cell and Fc

«

receptor com-plexes, is essential. The

g

chain ITAM consists of two cop-ies of a YXXL sequence separated by 8 amino acids (12amino acids separate the YXXLs in the case of Fc

g

RIIA).Current evidence suggests that crosslinking Fc

g

Rs at thecell surface leads to a series of signaling events that are gen-erally similar in ITAM containing receptors [3,9,10]. Evi-dence suggests that the two YXXL tyrosines in the ITAMbecome phosphorylated largely as a result of activation ofthe Src related family of tyrosine kinases (SRTKs). Thesedoubly phosphorylated ITAMs recruit SH2 (Src homology2) containing molecules including protein tyrosine kinases,such as Syk or Zap70, and tyrosine phosphatases leading tovarious downstream activation events including, in the caseof Fc

g

Rs, phagocytosis.An early signaling event following crosslinking of Fc

g

Rsis an increase in the tyrosine phosphorylation of substrateswithin the cell. Phosphorylation of the cytoplasmic ITAMtyrosines is essential for function, and maintenance of theintegrity of these conserved sequences is required for effi-cient Fc

g

RIIA-, Fc

g

RI/

g

- and Fc

g

RIIIA/

g

-mediated phago-

Offprint requests to: Sharon Hunter, Ph.D., 7 Silverstein, University ofPennsylvania School of Medicine, 3400 Spruce Street, Philadelphia PA19104; E-mail: [email protected]

876

S. Hunter et al./Experimental Hematology 27 (1999) 875–884

cytosis and signaling [11,12]. We have previously defined arole for the tyrosine kinase Syk in Fc

g

R mediated phago-cytic signaling. Using antisense oligonucleotides we haveshown that ablation of Syk expression significantly reducesFc

g

R mediated phagocytosis in cultured human monocytes[13]. Also, overexpression of Syk kinase in COS1 cells sig-nificantly enhances phagocytosis mediated by Fc

g

Rs, nota-bly Fc

g

RI/

g

and Fc

g

RIIIA/

g

, and Syk deficient macro-phages do not signal for Fc

g

R mediated phagocytosis [8,14].The related tyrosine kinase Zap70, on the other hand, has noeffect on the phagocytic efficiency of Fc

g

RI/

g

or Fc

g

RIIIA/

g

when co-transfected into COS1 cells [8,12]. Similarly, co-transfection of members of the family of Src related ty-rosine kinases such as Lyn, Fyn, Fgr, Lck, and Src do notenhance the

g

chain mediated phagocytic signal.Syk and Zap70 are members of the same family of ty-

rosine kinases and their overall structures are highly homol-ogous [15]. Both kinases contain two SH2 domains that are55% and 46% homologous in amino acid sequence, respec-tively, and a kinase domain that is 64% homologous. Inboth Syk and Zap70, these domains are separated by alinker or unique region that shares little homology. WhileZap70 expression is confined to T cells and natural killer(NK) cells, Syk is found in most cells of the hematopoieticlineage including B cells, mast cells, and monocytes/mac-rophages [16]. Syk is also expressed in T-cell lineages andboth Zap70 and Syk are activated following T-cell receptoractivation [17]. However, despite the highly conservedstructures of Syk and Zap70, their ability to bind similarsubstrates [18] and their ability to substitute for each otherin some receptor signaling pathways [19], evidence isemerging that Syk and Zap70 play very distinct roles in sig-naling cascades [8,12,14,20].

Our observation that Syk, but not Zap70, enhances thephagocytic signal in Fc

g

R expressing COS1 cells, promptedus to study the structural basis for this process. To addressthis issue, we used Syk kinase mutants as well as exchangechimeras between Syk and Zap70, and determined their ef-fects on Fc

g

R mediated phagocytosis. We also studied anaturally occurring splice variant of Syk, SykB, which ishomologous to Syk but lacks a 23 amino acid insertion inthe unique domain, and determined its ability to enhanceFc

g

R mediated phagocytosis.

Materials and methods

DNA and construction of chimeras

The chimeric Fc

g

Rs, I-

g

-

g

(Fc

g

RI-

g

-

g

) and

a

-

g

-

g

(Fc

g

RIIIA

a

-

g

-

g

),were engineered by two-step overlap extension polymerase chainreaction (PCR) using Fc

g

RI, Fc

g

RIIIA, and FcR gamma subunitcDNAs as templates as previously described [7,8]. cDNAs for hu-man Syk, porcine Syk and Y518F, Y519F, and K395R mutants ofporcine Syk were kindly provided by Clement Couture (La JollaInstitute for Allergy and Immunology, San Diego, CA) and previ-ously described [21,22]. cDNAs for the SykB variant, the Zap70

Y292F mutant and the exchange mutants between human Syk andZap70 were also created by overlap PCR using human Syk andZap70 cDNAs (kindly provided by Andrew Chan, Howard HughesMedical Institute, Washington University School of Medicine, St.Louis, MO) as templates and subcloned into the eucaryotic expres-sion vector pCDNA3 (Invitrogen, San Diego, CA). Again usingoverlap extension PCR, human Syk, human Zap70 and all ex-change chimeras between Syk and Zap70 were tagged at theC-terminus with the myc epitope EQKLISEEDL sequence, whichis recognized by mAb 9E10 [23]. SykB cDNA was also created byoverlap PCR. All of the mutants created by PCR were subjected toDNA sequencing for verification.

Cell culture and COS cell transfection

COS1 cells were cultured and maintained in Dulbeco’s modifiedEagles medium (DMEM) containing glucose (4.5 mg/mL),glutamine (2 mM), streptomycin (100

m

g/mL), penicillin (100 U/mL)and 10% heat inactivated fetal bovine serum. COS1 cells weretransiently transfected at 80% confluency (3

3

10

5

cells/35 mmwell in a six well plate or 1

3

10

6

cells/100 mm plates) in DMEMculture medium containing DEAE-Dextran (750 mg/mL), chloro-quin chloride (100 mM) and 4.0

m

g plasmid DNA (at a concentra-tion of 500 ng/

m

L) per milliliter. Following 3.5 hours incubation at37

8

C the transfection medium was removed and the cells incu-bated in a 10% solution of dimethyl sulphoxide (DMSO) in phos-phate buffered saline (PBS) for 2 minutes at room temperature.The cells were then washed twice with serum-free DMEM, over-laid with fresh medium and incubated for 48 hours before analyz-ing. To create the COS1 cell line stably expressing the

g

chain chi-mera I-

g

-

g

, COS1 cells were transfected with I-

g

-

g

using thecalcium phosphate transfection method recommended by the man-ufacturers (5

9

Prime-3

9

Prime, Boulder CO) and I-

g

-

g

expressingclones were selected for neomycin resistance with G418 (500

m

g/mL) using limiting dilutions.

Binding and phagocytosis of IgG sensitized red blood cells

Antibody coated sheep erythrocytes (Rockland, Gilbertsville, PA)(EA) were prepared in magnesium- and calcium-free PBS by incu-bating 10

9

sheep red blood cells (RBCs) per milliliter with an equalvolume of the highest subagglutinating concentration of IgG rab-bit-anti-sheep RBC antibody (Cappel Laboratories, West Chester,PA) as previously described [4]. The medium was removed fromCOS1 transfectants and the cells overlaid with EA (10

8

cells/mL)and incubated at 37

8

C for 30 minutes. Unbound EA were washedaway with PBS, and EA binding to the transfected cells was deter-mined by light microscopy. Extracellularly bound EA was then re-moved by a short (20 seconds) hypotonic wash. The cells werestained with Wright-Geimsa and the number of COS1 cells withmore than one internalized EA was determined in a blinded fashionby light microscopy. Phagocytosis was determined by measuringthe phagocytic index (PI), the number of EA ingested per 100COS1 cells, and approximately 300 cells were counted per experi-ment. I-

g

-

g

or

a

-g-g expressing cells were determined by flow cy-tometry. In the experiments presented, the percent of Fcg receptorexpressing cells and the mean fluorescence intensity were similarfor transfectants within each experiment.

Flow cytometryThe 105 cells were incubated on ice for 30 minutes with anti-FcgRImAb 32.2 [for staining the chimeric I-g-g receptor] or anti-FcgRIIImAb 3G8 [for staining the a-g-g receptor]. The cells were washed

S. Hunter et al./Experimental Hematology 27 (1999) 875–884 877

and labeled with fluorescein isothiocyanate (FITC)-conjugatedgoat anti-mouse F(ab9)2 IgG (TAGO Inc, Burlingame, PA) for 30minutes on ice. Following washing, the cells were fixed in a solu-tion of 4% paraformaldehyde. Isotype controls were used for allantibodies and fluorescence was measured on a FACSTAR cytom-eter (Becton Dickenson, Mountain View, CA).

Immunoprecipitation and Western blottingFollowing stimulation of transfected COS1 cells with EA at 378Cfor 30 minutes, the cells (in 100 mm plates) were incubated on iceto halt further phagocytosis. Externally bound EA were removedby hypotonic lysis and the COS1 cell lysate obtained by the addi-tion of 1.0 mL lysis buffer (1% NP40, 150mM NaCl, 25mMTris HCl, 1mM EGTA containing the following protease inhibi-tors: 1mM NaVO4, 1mM PMSF, 10 mg/mL aprotinin, 50 mg/mLleupeptin and 100 mg/mL soybean trypsin inhibitor), followed byincubation on ice for 30 minutes. Cleared lysates were immuno-precipitated with 10 mg/mL anti-myc mAb, 9E10 [23]. Immuno-precipitates were analyzed by SDS/PAGE and immunoblots wereperformed with anti-myc mAb 9E10. Immunoblots were devel-oped with horseradish peroxidase-conjugated goat anti-mouse IgG(BioRad, Richmond, Va) and were visualized by enhanced chemi-luminescence (ECL) (Amersham Corp., Arlington Heights, Il).

In vitro immune complex kinase assayTransfected COS cells (in 100 mm plates), were lysed with 1.0 mL1% NP40 lysis buffer at 08C, for 30 minutes as previously de-scribed [7]. Immunoprecipitations were performed by mixing celllysates with anti-myc mAb 9E10. The immune complexes were in-cubated with [g32P]ATP for 10 minutes at room temperature to al-low autophosphorylation of the kinases and phosphorylation of theexogenously added substrate GST-erythrocyte band 3 and werethen separated by SDS-PAGE. The gel was washed with 1 N KOHat 558C for 2 hours to remove serine/threonine phosphorylation be-fore drying and exposing to Kodak XAR-5 film (Rochester, NY).

Results

Syk kinase, but not Zap70 kinase, increases the efficiency of phagocytosis mediated by the human FcgRI and FcgRIIIA g chainMembers of all three classes of Fcg receptors (FcgR) areable to induce a phagocytic signal. FcgRI and FcgRIIIAtransmit a phagocytic signal that requires co-expression oftheir accessory g chain subunit (FcgRIIIA/g and FcgRI/g)[4–6]. We constructed two chimeric receptors (EC-TM-CYT) that contain the extracellular (EC) domains of eitherFcgRIIIA (a-g-g) or FcgRI (I-g-g) and the transmembrane(TM) and cytoplasmic (CYT) domains of the g chain. InCOS1 cells both chimeras induce efficient phagocytosis,comparable to that observed when the g chain is co-trans-fected with either FcgRI or FcgRIIIA [7,8].

Syk and Zap70 tyrosine kinases are members of a uniquefamily of kinases that have a similar overall structure con-sisting of two tandem SH2 domains and a C-terminal cata-lytic kinase domain that are highly homologous in amino

acid sequence [15,24,25]. The linker region, separating theSH2 and kinase domains, is referred to as the “unique” do-main due to the marked lack of homology between Syk andZap70 in this region (Fig. 1A). The SH2 domains of Sykand Zap70 bind doubly tyrosine phosphorylated ITAMs inthe cytoplasmic domains of Ig superfamily receptors [18,26]and it has been suggested that Syk and Zap70 perform sim-ilar functions. Zap70, for example, successfully restores B-cellreceptor signaling in Syk deficient B lymphocytes [19] andSyk can restore thymic development in Zap-70 deficientmice [27]. Furthermore, in a T-cell line expressing Fcg re-ceptors, both Syk and Zap70 are activated following crosslink-ing of the Fcg receptor [28] and both Syk and Zap70 havebeen implicated in T-cell signaling pathways [17,29]. How-ever, evidence is accumulating to indicate that Syk andZap70 kinases play distinct functional roles in the signaltransduction pathways of cells of hematopoietic lineage[9,14,20].

We have previously shown that the tyrosine kinase Syksubstantially enhances phagocytosis mediated by the humanFcgRI and FcgRIIIA g chain [8]. In addition, Syk kinaseenhances phagocytosis by FcgRIIA. In COS1 cells this ef-fect of Syk on phagocytic efficiency is more pronounced inco-transfectants of FcgRI and the g chain and FcgRIIIA andthe g chain than in cells expressing FcgRIIA. Phagocytosismediated by the g chain chimeric receptors I-g-g and a-g-gwas also increased by co-expression of Syk kinases (a-g-gshown in Figs. 2 and 3). Co-transfection of Zap70 howeverhad no significant stimulatory effect on phagocytosis medi-ated by the FcgRI/g and FcgRIIIA/g receptors. In addition,Syk, but not Zap70, also increased phagocytosis by Fcg-RIIA (data not shown). The data thus demonstrate a func-tional difference between Syk and Zap70 kinases. Using theCOS1 cell system as a model for studying phagocytosis weexamined the structures that might account for the ability ofSyk to enhance the phagocytic signal.

The Syk variant SykB increases phagocytosis mediated by human FcgRI/g and FcgRIIIA/gSince the definition of the Syk family of kinases, a secondisoform of Syk, SykB (also referred to as Syk11) has beenidentified in human, mouse, and rat cells [15,30,31]. SykBis identical to Syk except for a 23 amino acid deletion in theunique domain (Fig. 1A), a result of alternative splicing ofSyk mRNA [31]. When the amino acid sequences of Syk,SykB and Zap70 are compared, SykB and Zap70 are morehomologous in primary protein structure than Syk andZap70 in that both lack this 23 amino acid sequence in theirunique domains. We examined whether these 23 amino ac-ids play a role in coupling Syk kinase to the phagocytic sig-naling pathways in our model system.

We deleted these 23 amino acids from Syk kinase, thuscreating SykB. Co-transfection of a-g-g with SykB re-vealed that Syk and SykB enhanced phagocytosis similarly

878 S. Hunter et al./Experimental Hematology 27 (1999) 875–884

Figure 1. Schematic structure of the Syk family of tyrosine kinases and the Syk/Zap70 exchange chimeras. All kinases and exchange chimeras contain aC-terminal myc derived epitope. (A) The structure of Syk, the Syk variant SykB, and Zap70. The location of the SH2 domain, the unique domain, and the kinasedomain are indicated at the top of the figure. Syk SH2 and kinase domains are indicated with shaded boxes; Zap70 SH2 and kinase domains are indicated withsolid boxes. The Syk unique domain is indicated with a solid line; the Zap70 unique domain is indicated with a bold broken line. The amino acid number indi-cating the beginning and end of the unique domain is shown. The location of the 23 amino acid deletion in SykB, which is also absent from Zap70, is indicatedby a gap in the unique domain. The asterisk (*) indicates the position of tyrosine 292 in Zap70. (B) The structures of the Syk/Zap70 chimeras SZS, ZSZ, ZZS,SSZ, SZZ, and ZSS are shown. The numbers indicate the amino acid positions where the domains were exchanged. (C) The structure of the ZSZ-2 chimera. Theamino acid sequence of the border between Syk and Zap70 is noted.


(Fig. 2). These experiments suggest that Syk and SykB donot differ functionally in this FcgR phagocytic model sys-tem. Furthermore, the inability of Zap70 to augment phago-cytosis is not due to the lack of these 23 amino acids andthis region of Syk kinase does not appear to be important forthe function of Syk in the phagocytic signaling pathway.

In the Zap70 unique region, a tyrosine residue (Y292)(Fig. 1A), has been implicated in the negative regulation ofZap70 function [32]. Changing this tyrosine to phenylala-nine (Y292F) results in a mutant Zap70 kinase that, in aT-cell line, allows NF-AT production in the absence of TCRstimulation. The Y292F mutation can also enhance the abil-ity of Zap70 to reconstitute Syk deficient B cells [32]. Wetherefore constructed a Zap70 Y292F mutant. Figure 3 dem-onstrates that Zap70 Y292F did not enhance a-g-g medi-ated phagocytosis when studied in our COS1 cell modelsystem (Fig. 3). Therefore, the proposed inhibitory Y292 inZap70 does not contribute to the inability of Zap70 to en-hance the phagocytic signaling pathway by the FcgRs.

Exchange chimeras between Syk and Zap70 kinasesTo further dissect the structural basis for the observed func-tional difference between Syk and Zap70, we constructedseveral exchange chimeras between Syk and Zap70 (Fig.1B) and determined their ability to enhance phagocytosismediated by I-g-g. The amino acid sequences of Syk andZap70 differ most dramatically within the unique domain.

Because this domain contains several potential regulatoryregions such as Vav and PLCg1 binding sites, as well as pu-tative tyrosine phosphorylation sites [33,34], we first inves-tigated the effect of exchanging Zap70 and Syk unique re-gions (Fig. 1B). When the Syk unique domain is insertedinto Zap70, the resulting chimera, ZSZ (Zap70 SH2 do-mains, Syk unique domain, and Zap70 kinase domain), stillfunctions in a Zap-like fashion in that it does not increasethe efficiency of I-g-g mediated phagocytosis (Fig. 4). Sim-ilarly, insertion of the Zap70 unique region into Syk (chi-mera SZS) does not abolish the ability of Syk to enhancephagocytosis mediated by I-g-g (Fig. 4). These results sug-gest that despite the dramatic differences in the unique do-mains of these kinases, this region alone is not responsiblefor the ability of Syk to enhance the phagocytic signal of theFcgRI/g chain.

We next exchanged the kinase domains of Syk andZap70 to create the chimeras ZZS and SSZ (Fig. 1B). ZZS,which contains the Zap70 SH2 and unique regions and theSyk kinase domain, increases I-g-g mediated phagocytosis.In contrast, the SSZ chimera, which contains the Syk SH2and unique regions and the Zap70 kinase domain, is ineffec-tive in enhancing I-g-g mediated phagocytosis (Fig. 4).

To ensure constant expression of I-g-g in our assays, wealso examined the effect of Syk and Zap70 chimeras inCOS1 cells stably expressing I-g-g (COSI-g-g) (Table 1).Similar to the results described above, transfection of wildtype Syk into COSI-g-g induced an increase in I-g-g medi-

Figure 2. Phagocytosis mediated by the chimeric receptor a-g-g (EC-TM-CYT) following co-transfection with wild type Syk, Zap70, or SykBtyrosine kinases. Both Syk wild type and SykB, in contrast to Zap70,increased the efficiency of a-g-g mediated phagocytosis. Two representa-tive experiments are shown. SykB kinase is identical to wild type Sykexcept for a 23 amino acid deletion in the unique region of the tyrosinekinase. Phagocytosis in Figs. 2 and 3 is expressed as fold increase inphagocytic index compared to transfectants alone.

Figure 3. Phagocytosis mediated by the chimeric receptor a-g-g (EC-TM-CYT) following co-transfection with wild type Syk, Zap70, or the Zap70Y292F mutant tyrosine kinase. Wild type Syk, but not Zap70 or the Zap70Y292F mutant tyrosine kinase, increased the efficiency of a-g-g mediatedphagocytosis. Two representative experiments are shown.


ated phagocytosis. Furthermore, the effects of the Syk-Zapchimeras paralleled those observed in COS1 cells co-trans-fected with I-g-g and the Syk/Zap chimeras. Only those chi-meras that contain the Syk kinase domain (ZZS and SZS)consistently enhanced the I-g-g mediated phagocytic signalwhereas the SSZ and ZSZ chimeras did not enhance phago-cytosis (Table 1). We also examined the activity of two ad-ditional chimeras, ZSS (Zap70 SH2 domains, Syk uniqueand kinase domains) and SZZ (Syk SH2 domains, Zap70unique, and kinase domains). ZSS, containing the Syk ki-

nase domain, increased the efficiency of I-g-g mediatedphagocytosis whereas SZZ had no significant effect (Table 1).

In in vitro kinase assays the chimeras containing the Sykkinase domain (e.g. ZZS, ZSS, and SZS) expressed in COS1cells, were more efficient than those chimeras containingthe Zap70 kinase domain (e.g. SSZ, SZZ, and ZSZ) in phos-phorylation of the exogenous substrate human erythrocyteband 3 (Figs. 5a and 5c). WT Zap70 had low kinase activity,only slightly above control levels, in the in vitro kinase as-says and all chimeras containing the Zap70 kinase domainbehaved in a similar manner to WT Zap70 (Figs. 5a and 5c).In contrast, WT Syk kinase and those chimeras containingthe Syk kinase domain had high levels of kinase activity inthese assays. These results suggest that the higher intrinsickinase activity of the Syk kinase domain contributes toSyk’s ability to enhance I-g-g mediated phagocytosis.

The importance of Syk kinase activity in the phagocyticprocess was further demonstrated using a kinase negativemutant of porcine Syk, K395R [21]. This mutant kinase, inwhich lysine at position 395 is replaced with arginine,thereby disrupting the ATP binding site, did not increase gchain mediated phagocytosis (Fig. 6). Thus, this mutantdemonstrates that the kinase activity of Syk is important forits effect on phagocytosis.

A second Syk mutant, Y518F/Y519F [21,22], in whichtwo tyrosine residues in the activation loop of Syk haveeach been changed to phenylalanine was also examined forits effect on phagocytosis. This Syk mutant that has beenshown to decrease FcgRI signaling in mast cells also de-creased the ability of Syk to augment the phagocytic signal(Fig. 6). Individual mutation of Y518 or Y519 to phenylala-nine did not have as dramatic an effect on phagocytosis asdid the double mutation, similar to the results observed inFc«RI signaling. The in vitro catalytic activity of Syk doesnot appear to be substantially affected by the mutation of ty-rosines 518 and 519, although in vivo, Y518F/Y519F, incontrast to wild type Syk, fails to induce tyrosine phosphor-ylation of multiple intracellular proteins [21,22]. While thesestudies do not rule out the possibility that these kinase do-main tyrosines play a role in the catalytic activity of Syk invivo, Y518 and Y519 may play a role in the association ofSyk with other molecules in the signaling cascade [21,22].

A Zap/Syk/Zap chimera that increases phagocytosis mediated by I-g-gWe also constructed an additional ZSZ chimera containingZap70 SH2 and kinase domains and the Syk unique domain(ZSZ-2, Fig. 1C). This chimera differs from ZSZ in that theborder between the Zap70 SH2 domain and the Syk uniquedomain contains additional amino acids derived from theZap70/Syk unique domains. Surprisingly, this ZSZ-2 chi-mera enhanced phagocytosis mediated by I-g-g (Fig. 7).ZSZ-2 resembled Zap70 with regard to in vitro kinase activ-ity, and in contrast to wild type Syk and the chimeras con-

Figure 4. Phagocytosis mediated by the chimeric receptor I-g-g (EC-TM-CYT) following co-transfection with wild type Syk, Zap70, or the chimericreceptors SSZ, ZZS, SZS and ZSZ. Increased phagocytic efficiency wasobserved with co-transfection of I-g-g with either Syk kinase or the chime-ras ZZS and SZS. Co-transfection of I-g-g with Zap70 kinase or the chime-ras SSZ and ZSZ did not appreciably increase phagocytosis. Phagocytosisis expressed as fold increase in phagocytic index compared to I-g-g trans-fectants alone.

Table 1. Phagocytic index of COS cells

PIExperiment 1

PIExperiment 2

#1 Control vector 74 752 1 Syk 115 1163 1 Zap70 65 674 1 ZZS 102 1185 1 SZS 109 1056 1 ZSS 116 1267 1 SSZ 79 838 1 ZSZ 70 709 1 SZZ 61 64

Phagocytic index of COS cells stably expressing the chimeric receptor I-g-gfollowing transfection with either control vector, wild type Syk, Zap70, orthe Syk/Zap70 chimeras ZZS, SZS, ZSS, SSZ, ZSZ, SZZ. Only wild typeSyk kinase and those chimeras containing the Syk kinase domain, ZZS, SZS,and ZSS increased the efficiency of I-g-g mediated phagocytosis.


taining the Syk kinase domain, ZSZ-2 only minimally phos-phorylated erythrocyte band 3 (Fig. 5a). This observationindicates that alteration in the unique domain can create aSyk-Zap70 molecule that can increase phagocytosis.

DiscussionSeveral studies have suggested the importance of the ty-rosine kinase Syk in the phagocytic process mediated byFcg receptors. Our laboratory has observed that the ablationof Syk kinase in primary human monocytes/macrophageseliminates their ability to phagocytose IgG coated cells [13].Furthermore, we have shown that transfection of humanSyk enhances the phagocytic signal mediated by FcgRs inour COS1 cell model system [8]. Others have shown thatmacrophages obtained from Syk deficient mice do not me-diate the phagocytosis of IgG coated cells [14].

The Syk family of tyrosine kinases comprises two mem-bers, Syk and Zap70, and both members of this tyrosine ki-nase family share substantial structural homology (Fig. 1A)[15]. In a B-cell line it has been demonstrated that Zap70can substitute for Syk in signaling through the BCR sug-gesting an overlap in their signaling capabilities [19]. In ad-dition, both Syk and Zap70 are activated following T-cellreceptor stimulation and have similar substrate specificities[18]. However, we had observed that despite these similari-ties, Zap70, which is largely expressed in nonphagocyticcells, differs from Syk in its inability to enhance phagocyto-sis mediated by FcgRI/g and FcgRIIIA/g in transfectedCOS1 cells [12]. Differences in the function of Syk andZap70 have also been observed in other systems [20,35,36].For example, in a monocytic cell line, Zap70, unlike Syk ki-nase, is not phosphorylated upon Fcg receptor stimulationwhereas in a T-cell line both kinases were tyrosine phos-

Figure 5. In vitro kinase assay of myc-tagged Syk, Zap70 and the Syk/Zap chimeras. (a) COS cells were co-transfected with I-g-g and a control vector (lanes1, 2), wild type Syk (lanes 3, 4), wild type Zap70 (lanes 5, 6), or the Syk/Zap70 chimeras (lanes 7–20) as indicated. In the absence (2) or presence (1) ofreceptor crosslinking, the kinases were immunoprecipitated with anti-myc mAb and in vitro kinase activity determined using GST-band 3 as a substrate asdescribed in the Materials and methods section. The arrow indicates the position of phosphorylated GST-band 3. (b) Anti-myc immunoblot of whole celllysates from 5a confirming the presence of Zap70 (lanes 5, 6) and the Syk/Zap70 chimeras containing the Zap70 kinase domain, SZZ (lanes 7, 8), SSZ (lanes9, 10), ZSZ (lanes 17, 18), and ZSZ-2 (lanes 19, 20) in the COS cell co-transfectants. Zap70 and the Syk/Zap70 chimeras containing the Zap70 kinase domain(lanes 5–10 and 17–20) were expressed well compared to the chimeras containing the Syk kinase domain (lanes 11–16), but had no effect on the phagocyticefficiency of I-g-g. (c) In vitro kinase assay of myc-tagged Syk, Zap70, and the Syk/Zap chimeras. COS cells were co-transfected with I-g-g and a controlvector (lane 1), wild type Syk (lane 2), wild type Zap70 (lane 3) or the Syk/Zap70 chimeras (lanes 4–9) as indicated. In the presence of receptor crosslinking,the kinases were immunoprecipitated with anti-myc mAb and in vitro kinase activity determined using GST-band 3 as a substrate as described in the Materialsand methods section.


phorylated following T-cell receptor stimulation. Similarly,in T-cells Syk can signal through the TCR independently ofCD45 and Lck, whereas Zap70 requires both CD45 and Lckto function. Evidence is, therefore, accumulating to suggestthat Syk and Zap70 play distinct roles in receptor signalingpathways.

In this study we sought to determine the structural re-quirements of Syk responsible for enhancing phagocytosismediated by these Fcg receptors. Because Zap70 has no ef-fect on phagocytosis in COS1 cell FcgR transfectants, theapproach we chose was to construct chimeric Syk-Zap70 ty-rosine kinases to determine the contribution of the Syk SH2and kinase domains as well as the contribution of the uniquedomain which separates these two regions (Fig. 1B).

We used single chain FcgRs containing the extracellular(EC) domain of FcgRI (I-g-g) or FcgRIIIA (a-g-g), and thetransmembrane (TM) and cytoplasmic (CYT) domains ofthe g chain. When transfected into COS1 cells these chi-meric Fcg receptors, I-g-g and a-g-g (EC-TM-CYT), medi-ate phagocytosis as efficiently as co-transfection of the twoFcgR subunits. In addition, co-transfection of I-g-g or a-g-gwith Syk, but not Zap70, enhances phagocytosis (Fig. 2).

Comparison of the primary structure of Syk and Zap70demonstrates that these kinases exhibit less homology in theunique domain than in the SH2 and the kinase domains [15].The most striking difference between Syk and Zap70 is thatSyk contains an extra 23 amino acids in this region. How-

ever, the presence of this 23 amino acid stretch does not ex-plain the ability of Syk to enhance phagocytosis. Syk B, anaturally occurring splice variant of Syk, lacks these 23amino acids. Syk B and Syk however, are equally effectivein enhancing the phagocytic signal of a-g-g (Fig. 2). Thisobservation suggests that this 23 amino acid region is notresponsible for Syk mediated enhancement of phagocytosis.Others have shown, however, that this 23 amino acid regionplays a role in signaling through other ITAM containing re-ceptor pathways such as Fc«RI in rat basophilic leukemiccells (RBL) and antigen receptor signaling in a murine T-cellline [37]. These conflicting results may be due to cell-typespecific differences between our system and certain murinecells. Alternatively, these observations may be due to differ-ences in FcgRI/g and FcgRIIIA/g signaling, which is inde-pendent of the b chain involved in signaling by Fc«RI inRBL cells, and the z chain involved in T-cell receptor sig-naling.

Study of the function of our Syk-Zap70 chimeras in phago-cytosis demonstrated that the kinase domain of Syk and it’sassociated kinase activity is important for the increase inI-g-g mediated phagocytosis (Fig. 4 and Table 1). The dataalso suggest that the Zap70 SH2 domains can interact withthe Fcg receptor as efficiently as the Syk SH2 domains be-cause the chimeras ZZS and ZSS, containing the SH2 do-mains of Zap70, enhanced the I-g-g mediated phagocyticsignal. Only those chimeras containing the Syk kinase do-main (ZZS, ZSS and SZS) increased the FcgR phagocyticsignal, whereas those chimeras containing the Zap70 kinase

Figure 6. Effect of wild type Syk kinase and the Syk mutants SykK395R,SykY518F, SykY519F, and SykY518F/Y519F on phagocytosis mediatedby FcgRI. COS cells were co-transfected with FcgRI and the g chain; orFcgRI, the g chain and wild type Syk, SykK395R, SykY518F, SykY519F,or SykY518F/Y519F. Phagocytosis is expressed as fold increase in phago-cytic index compared to FcgRI 1 g chain transfectants alone.

Figure 7. Enhancement of phagocytosis mediated by I-g-g following co-transfection with the ZSZ chimera, ZSZ-2. Increased phagocytosis wasobserved following co-transfection of I-g-g with Syk and with ZSZ-2.Phagocytosis is expressed as fold increase in phagocytic index compared toI-g-g transfectants alone.


domain had no substantial effect and behaved in a mannersimilar to WT Zap70. These results were observed both inco-transfection experiments of I-g-g or a-g-g with the Syk-Zap70 chimeras as well as in cells that were stably express-ing I-g-g (Fig. 4 and Table 1).

In the stably expressing cell line COSI-g-g, the effect ofSyk kinase was less dramatic than that observed with co-transfected cells. In this permanent cell line essentially allcells express I-g-g. In our COS1 transfection experimentswe routinely observe a 20%–25% transfection efficiency.Therefore, following transfection of COSI-g-g with Syk ki-nase, only a fraction (,25%) of cells expressing I-g-g alsoexpress Syk kinase. Thus, in our stably expressing COSI-g-gcell line, the overall effect of Syk kinase on phagocytosis isonly observed in a fraction of the phagocytosing cells. Theresulting Syk mediated increase in phagocytosis is essen-tially diluted by the remaining I-g-g expressing cells, whichdo not express Syk kinase and are phagocytosing low levelsof EA. In contrast, in the experiments co-transfecting COS1with I-g-g and Syk kinase, virtually all of the I-g-g express-ing cells also express Syk kinase and the effect of Syk ki-nase on phagocytosis is observed in virtually all the phago-cytic I-g-g expressing cells.

In vitro kinase assays demonstrated that Syk and Zap70differ considerably in their ability to phosphorylate exoge-nous substrates in COS1 cells (Fig. 5). Syk appears to havea higher intrinsic kinase activity than Zap70 and this higheractivity was also observed with the chimeras noted to en-hance the phagocytic signal (ZSS, ZZS, SZS) (Fig. 5a and5c). This was observed even though WT Syk and the chime-ras containing the Syk kinase domain were expressed lessefficiently than WT Zap70 and the chimeras containing theZap70 kinase domain (Fig. 5b). This suggests that the highintrinsic kinase activity of Syk is associated with the activ-ity of its kinase domain in increasing the phagocytic signal.Our finding that the kinase inactive Syk mutant (K395R)does not enhance phagocytosis supports this observation.Despite the low in vitro kinase activity of the Zap70 kinasedomain chimeras, the chimeras are functional as they re-store antigen receptor signaling in a Syk deficient B cell linein a manner similar to wild type Zap70 (D. Chu, unpub-lished observations). Furthermore the Zap70 kinase domainchimeras are comparable in their in vitro kinase activity towild type Zap70.

The observations with the exchange chimeras, taken to-gether with the in vitro kinase assays and Syk kinase mutantdata, strongly suggest that Syk’s ability to enhance FcgRI/gand FcgRIIIA/g mediated phagocytosis is primarily associ-ated with the kinase domain of Syk and with its high intrin-sic kinase activity. These results are consistent with otherwork which demonstrates that antibody mediated clusteringof Syk kinase domains is sufficient to mediate a phagocyticsignal while clustering of Zap70 kinase domains was lessefficient [38]. It has also been suggested [39] that mouseSyk has superior intrinsic kinase activity than does mouse

Zap70 and that the kinase domain of mouse Syk rendersZap70 more Syk-like with respect to kinase activity andability to phosphorylate exogenous substrates.

The Syk mutant kinase Y518F/Y519F decreases the abil-ity of Syk to augment the phagocytic signal in COS1 cells,consistent with the importance of the Syk kinase domain incoupling the signal derived from receptor crosslinking todownstream phagocytic machinery. In vitro evidence indi-cates that in this case the decrease in signaling is not due toa decrease in the catalytic activity of Syk [22], suggestingthat these tyrosine residues may couple Syk to other down-stream signaling molecules [22]. Taken together the datasuggest that aspects of the Syk kinase domain, other than itscatalytic activity, also play a role in signaling for phagocy-tosis.

We observed that a Syk/Zap70 chimera (ZSZ-2) thatlacks the Syk kinase domain but contains additional aminoacids in the border between the SH2 and unique domains,surprisingly also enhanced the FcgRI/g mediated phago-cytic signal. In vitro kinase assays demonstrated that thischimera has the same low level of tyrosine kinase activity asthe original ZSZ chimera and Zap70 itself (Fig. 5a). It ispossible that there is a change in the conformation of thismutant chimera which allows an interaction(s) with a down-stream mediator(s) of phagocytosis or that it induces an al-ternative phagocytic pathway. We have also observed that asimilar mutant Syk kinase with three additional amino acidsat the border between the SH2 and unique domains is con-stituitively active in T cells (D. Chu, unpublished data).Syk, therefore, may be sensitive to changes in this borderarea between the SH2 and the unique domains.

In summary, we have observed that the ability of Syk,but not Zap70, to facilitate phagocytosis by the Fcg recep-tors FcgRI and FcgRIIIA largely resides in its kinase do-main. In addition, it appears that structural aspects of theSyk kinase domain are important for the phagocytic signaland that alterations in the region between the SH2 andunique domains can affect phagocytic efficiency.

AcknowledgmentsSupported by NIH Grants AI-22193 and HL-27068. We thank Dr.Arthur Weiss (University of California, San Francisco) for his ad-vice and helpful suggestions with the manuscript.

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Structural requirements of Syk kinase for Fcγ receptor– phagocytosis