©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction between lck and syk Family Tyrosine Kinases in Fc Receptor-initiated Activation of Natural Killer Cells (*)

Adrian T. Ting , Christopher J. Dick , Renee A. Schoon , Larry M. Karnitz , Robert T. Abraham , Paul J. Leibson (§)

From the (1)Department of Immunology, Mayo Clinic and Foundation, Rochester, Minnesota 55905

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Ligation of the FcR on natural killer (NK) cells results in the tyrosine phosphorylation of multiple substrates critical for intracellular signaling and activation of NK cell effector functions. However, it remains unclear which nonreceptor protein-tyrosine kinases (PTK) participate in this process. In this report we demonstrate that FcR ligation induced the tyrosine phosphorylation and increased the catalytic activities of both syk family PTKs, ZAP-70, and syk. The phosphorylation of ZAP-70 and syk was enhanced markedly by overexpression of wild-type lck but not by a kinase-inactive mutant, suggesting that early FcR-initiated activation of lck results in the subsequent regulation of syk family PTKs. The regulatory interplay between src and syk family PTKs was emphasized further by the observation that lck overexpression enhanced the association of ZAP-70 with the chain of the FcR complex. Additional analyses indicated that lck induced the subsequent tyrosine phosphorylation of phospholipase C (PLC)-2. Interestingly, the regulatory effects of lck on ZAP-70, syk, and PLC-2 could not be replaced by overexpression of either fyn or src, demonstrating a selective role for lck in effectively coupling FcR stimulation to critical downstream signaling events. Taken together, our results suggest not only that FcR stimulation on NK cells is coupled to the intracellular activation of both ZAP-70 and syk, but that the src family member, lck, can selectively regulate this tyrosine kinase cascade.


INTRODUCTION

Exposure to foreign antigens elicits an antibody response from the immune system. The subsequent interaction of Fc receptor-bearing effector cells with antibodies complexed to soluble or cell-bound antigens initiates a variety of effector functions. Natural killer (NK)()cells represent a distinct subpopulation of lymphocytes which express the IgG Fc receptor type IIIA, hereafter referred to as FcR(1) . The FcR on human NK cells is a multimeric receptor complex consisting of the ligand-binding subunit (i.e. CD16), which associates noncovalently with dimers of and chains(2) . Although none of the components of the receptor complex possesses intrinsic kinase activity, stimulation of the FcR rapidly activates a protein-tyrosine kinase (PTK) signaling pathway that results in the tyrosine phosphorylation of substrates critical for cellular activation, including the chain and phospholipase C (PLC)- isoforms(3, 4, 5, 6, 7) . This FcR-initiated PTK signaling pathway appears to be requisite for the activation of NK cell-mediated cytotoxicity and lymphokine production(8, 9, 10) .

Elegant studies by several groups have demonstrated that ligation of chimeric receptors containing the intracellular portions of either the or chains is sufficient to activate intracellular PTKs as well as mediate downstream cytolytic function and lymphokine production (11-14). Additional studies have identified a conserved motif in the and chains, with the consensus amino acid sequence YXXL-X-YXXL, which is sufficient to mediate intracellular signaling(15, 16) . src family members are candidates for the FcR-associated PTKs. Recent reports have shown that lck, a member of the src family, is detectable in anti-FcR immunoprecipitates and that ligation of FcR increases the in vitro catalytic activity of lck(10, 17, 18) . Nonetheless, the precise regulatory function of lck during FcR-initiated signaling remains unclear. The syk family PTKs represent an additional class of cytoplasmic PTKs that have been implicated in lymphoid cell signal transduction. T cell antigen receptor (TCR) ligation has been shown to induce the tyrosine phosphorylation of ZAP-70 and its association with the chains of activated TCR complexes(19, 20) . Furthermore, a deficiency in ZAP-70 expression severely impairs TCR-mediated signaling(21, 22) . Likewise, stimulation of the B cell antigen receptor (BCR) results in the tyrosine phosphorylation of the receptor-associated syk(23, 24) . In contrast to T and B cells, the function of syk family PTKs in NK cell activation is less clear. Ligation of the FcR on NK cells can induce the association of a 70-kDa phosphotyrosyl protein with the receptor complex(25) , but the precise roles of ZAP-70 and/or syk during FcR signal transduction and the nature of their potential interaction with src family PTKs are unknown.

We focused our initial examinations of the PTKs involved in FcR signaling on lck and its role in coupling FcR stimulation to subsequent tyrosine phosphorylation events. Using the vaccinia virus expression system, we overexpressed wild-type lck in cloned human NK cells. Overexpression of wild-type active lck, but not a kinase-deficient mutant of this PTK, markedly enhanced the FcR-induced tyrosine phosphorylation of ZAP-70, syk, and PLC-2. Furthermore, only lck effectively coupled the FcR to downstream PTKs, as neither fyn nor src could substitute for lck. Taken together, our data strongly implicate a role for the src family PTK, lck, in the FcR-initiated regulation of ZAP-70, syk, and PLC-.


EXPERIMENTAL PROCEDURES

Cell Lines

Human CD16 NK cell lines were isolated and passaged as described previously(26) . The cell surface phenotype of these NK cell lines was monitored by flow cytometry. All NK cell lines used in these studies were >90% CD16. In addition, all CD16 NK cells expressed the following additional phenotypic markers: CD56, CD11b, CD2, and HLA-DR.

Chemical Reagents and Antibodies

All chemicals and drugs, unless otherwise noted, were obtained from Sigma. Fluorescein- and phycoerythrin-conjugated monoclonal antibodies (mAb) were obtained from Becton-Dickinson Monoclonal Center (Mountain View, CA). The anti-FcR mAb (3G8) has been described previously(27) . Immunoprecipitating ZAP-70 and syk rabbit antisera and immunoblotting anti-phosphotyrosine mAb 4G10 were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Immunoblotting ZAP-70 and syk antisera were raised in rabbits injected with keyhole limpet hemocyanin-conjugated synthetic peptides (ZAP-70 residues 326-341; 28 C-terminal amino acids of porcine p72)(20, 28) . The generation and characterization of the lck and PLC-2 rabbit antisera have been described previously(7, 29) . The antiserum was generously provided by Augusta Ochoa (National Cancer Institute).

Vaccinia Viruses

Recombinant vaccinia viruses encoding wild-type or mutant src family PTK were generated essentially as described(30) . Blunt ended cDNA fragments were obtained from the following sources. A a StuI fragment of c-lck was excised from the plasmid NT18 (31) and a EcoRV-SmaI fragment of c-fyn was excised from the plasmid pMTFR(32) , both of which were generously provided by Roger Perlmutter (University of Washington, Seattle). A StuI fragment of lck with a lysine to arginine mutation at position 273 was excised from a plasmid generously provided by Bart Sefton (Salk Institute, San Diego). A HindIII fragment of c-src was obtained from the plasmid pM5H (33) kindly provided by Sarah Parsons (University of Virginia, Charlottesville). These blunt ended cDNAs were inserted into the SmaI cloning site of the vector pSC11 (34) and introduced into WR strain vaccinia by homologous recombination. The recombinant vaccinia viruses were characterized by infection of CV-1 cells and subsequent detection by immunoblotting with antisera specific for the different src family PTKs. In vitro autophosphorylation assays were also performed on immunoprecipitated src family PTKs to confirm their catalytic activities. Viruses were propagated in HeLa cell cultures and released by lysing infected HeLa cells with a probe sonicator. The lysate was then layered over a cushion of 36% sucrose solution in 10 mM Tris-HCl, pH 9.0, and centrifuged in a Beckman SW 28 rotor (13,500 rpm, 2 h) to purify the virus. Viruses were subsequently titered on confluent BSC-1 monolayers using the method described previously(35) . NK cells (2 10 cells/ml in serum-free RPMI) were infected for 1 h at 37 °C at a multiplicity of infection of 20. Cells were then incubated for an additional 3-5 h at 1 10 cells/ml in RPMI with 10% bovine calf serum. Infected NK cells were washed twice and resuspended in RPMI with 0.5% bovine serum albumin for stimulation.

Immunoblotting

100-µl aliquots of NK cells (2 10 cells/sample) were incubated at 4 °C for 3 min with 10 µl of anti-FcR mAb (3G8) (final concentration, 100 µg of goat F(ab`)2 fragment anti-mouse IgG (Organon Teknika Corp., West Chester, PA) was then added to the cell suspension, mixed, and briefly pelleted. After incubating at 37 °C for the indicated time, pelleted cells were lysed with buffer containing 10 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA, 50 mM NaF, 30 mM NaPO, 500 µM NaVO, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 10 µg/ml leupeptin, and 1% Triton X-100, pH 7.4. Insoluble material was removed by centrifugation at 15,000 g for 10 min, and detergent-soluble proteins resolved by 8.5% SDS-polyacrylamide gel electrophoresis (PAGE). Resolved proteins were electrophoretically transferred to Immobilon-P membranes (Millipore, Bedford, MA), and immunoblotting with the anti-phosphotyrosine mAb 4G10 was performed as described previously (7).

Immunodetection with rabbit antisera specific for ZAP-70, PLC-2, and lck were performed as described previously(7) . Briefly, membranes containing resolved proteins were blocked overnight in Tris-buffered saline containing 2% milk and 0.2% polyoxyethylene sorbitan monolaurate (Tween 20) and incubated for 1 h with the antiserum diluted in Tris-buffered saline containing 2% bovine serum albumin, 0.2% Tween 20, and 0.05% NaN. After three washes with 0.2% Tween 20 in Tris-buffered saline, immunoreactive proteins were detected with protein A-horseradish peroxidase and the ECL detection system from Amersham Corp.

Immunoprecipitations

200-µl aliquots of NK cells (2 10/sample) were incubated at 4 °C for 3 min with 10 µl of anti-FcR mAb (3G8) (final concentration, 10 µg/ml). The cells were pelleted gently (700 g, 30 s, 4 °C) and resuspended in 200 µg of goat F(ab`) fragment anti-mouse IgG. After incubating at 37 °C for the indicated time, reactions were terminated with 1 ml of ice-cold lysis buffer containing 20 mM Tris-HCl, 40 mM NaCl, 5 mM EDTA, 50 mM NaF, 30 mM NaPO, 0.1% bovine serum albumin, 500 µM NaVO, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 10 µg/ml leupeptin, and 1% Triton X-100, pH 7.4. After 10 min at 4 °C, the samples were centrifuged (15,000 g, 10 min) to remove nuclear and cellular debris. Postnuclear supernatants were immunoprecipitated for 1-2 h at 4 °C with rabbit antisera bound to protein A-Sepharose beads. The immunoprecipitates were washed three times and bound proteins eluted with 50 µl of SDS-sample buffer and resolved by SDS-PAGE. Anti-phosphotyrosine immunoblotting was performed as described above.

In Vitro Kinase Assay

Anti- and anti-syk immune complex kinase reactions were performed using a modification of the method described previously(36) . Briefly, cell stimulation was terminated in a Brij 96 lysis buffer (1% Brij, 25 mM Tris, pH 7.6, 150 mM NaCl, 1 mM NaVO, 5 mM EDTA, and 10 µg/ml aprotinin and leupeptin). Immunoprecipitates were washed two times, and the beads were incubated at 25 °C for 5 min in 20 mM Tris, pH 7.6, 10 mM MnCl, 1 µM ATP, 0.25 µg of cfb3 (cytoplasmic fragment of band 3; generously provided by Lawrence E. Samelson, NIH), 10 µCi of [-P]ATP. The radiolabeled 43-kDa cfb3 was quantitated after scanning the membrane using the Radioanalytic Imaging System (model 4000, AMBIS, Inc., San Diego).


RESULTS

Vaccinia Virus Infection of Human NK Cells

Studies on NK cells have been hampered by the lack of a suitable methodology to genetically manipulate these cells. We report here the use of the vaccinia virus expression systems to overexpress efficiently src family PTKs in this cell type. Vaccinia viruses encoding wild-type src, fyn, lck, and a kinase-inactive lck mutant (lysine to arginine mutation at position 273) were generated by homologous recombination. The absence of kinase activity in the lck mutant was verified by infection of CV-1 cells with the different viruses followed by a lck-specific immune complex autophosphorylation assay (data not shown). After a 4-h infection of cloned NK cells with recombinant viruses encoding either wild-type or kinase-inactive lck, high levels of lck expression were detected by immunoblotting with a lck-specific antiserum (Fig. 1). Likewise, infection of NK cells with recombinant viruses encoding src or fyn resulted in the efficient overexpression of catalytically active PTKs (data not shown). Immunoblot analyses in multiple experiments revealed that infection of NK cells with the lck- and fyn-encoding viruses resulted in approximately 2-5-fold increases in both lck and fyn protein relative to endogenous levels. Furthermore, infection with either the lck-encoding or fyn-encoding viruses resulted in 2-3-fold increases in the total catalytic activity of anti-lck + anti-fyn immunoprecipitates for their shared substrate, enolase (data not shown). Whereas there was no detectable src in uninfected NK cells, infection with the src-encoding virus resulted in high levels of src expression (data not shown). The secretory and cytotoxic functions of NK cells remained intact during the 4-6-h infection with the vaccinia viruses.


Figure 1: Overexpression of lck in NK cells. Cloned NK cells (5 10/sample) were left uninfected for 4 h with either the control nonrecombinant WR strain vaccinia virus, the vaccinia virus encoding wild-type lck, or the vaccinia encoding the mutant kinase-inactive lck. Detergent-soluble proteins were resolved by SDS-PAGE, transferred to Immobilon-P membrane, and probed with a lck-specific antiserum.



lck Participates in the FcR-initiated Tyrosine Phosphorylation of Multiple Proteins

lck can physically associate with the FcR complex and exhibits enhanced in vitro catalytic activity after FcR ligation in NK cells(10, 17, 18) . Furthermore, we demonstrated in NK clones that FcR ligation induced a transient 2-3-fold increase in lck-specific in vitro kinase activity, whereas no change in fyn-specific activity was detected (data not shown). We next examined the effects of lck overexpression on FcR-mediated signal transduction. As shown in Fig. 2, overexpression of lck in unstimulated NK cells led to the increased tyrosine phosphorylation of several intracellular proteins (fifth lane). Interestingly, the immunoreactive proteins migrating at molecular masses of approximately 150, 120, 116, 85, and 75 kDa displayed electrophoretic mobilities identical to those induced by stimulation of uninfected NK cells with anti-FcR mAb (second and fifth lanes). FcR ligation of lck-overexpressing NK cells led to further increases in the tyrosine phosphorylation levels of these substrates (sixth lane). Moreover, infection of NK cells with either the control wild-type WR strain vaccinia (third and fourth lanes) or the recombinant virus expressing the kinase-inactive lck (seventh and eighth lanes) did not alter the FcR-induced tyrosine phosphorylation events. We next tested whether other members of the src family PTKs can induce the same effects as lck. In contrast to the dramatic effects observed with lck, overexpression of fyn had only minimal effects on the tyrosine phosphorylation levels of both resting and FcR-stimulated NK cells (Fig. 3, seventh and eighth lanes). Furthermore, overexpression of src had no detectable effects on the FcR-induced tyrosine phosphorylation of proteins (Fig. 3, ninth and tenth lanes). In Fig. 3, both fyn (seventh and eighth lanes) and src (ninth and tenth lanes) are detectable as phosphotyrosyl proteins migrating at molecular mass of 60 kDa. These observations are consistent with a specific role for lck in FcR-initiated tyrosine phosphorylation events.


Figure 2: lck-induced tyrosine phosphorylation in NK cells. NK cells (2 10/sample) were either uninfected or infected as described in Fig. 1. Cells were subsequently left unstimulated (-) or stimulated for 1 min with cross-linked anti-FcR mAb 3G8 (+). Detergent-soluble proteins were resolved by SDS-PAGE, transferred to Immobilon-P membrane, and sequentially probed with the anti-phosphotyrosine mAb 4G10 (upper panel) and with the lck antiserum (lower panel).




Figure 3: Effects of src family PTK overexpression on tyrosine phosphorylation in NK cells. NK cells (2 10/sample) were either uninfected or infected with control WR, lck-, fyn-, or src-encoding vaccinia virus. FcR stimulation and phosphotyrosine detection of cellular lysates were performed as described in Fig. 2.



FcR Ligation Induces the Tyrosine Phosphorylation and Increases the Catalytic Activities of ZAP-70 and syk

We next investigated whether members of the syk family PTK are involved in FcR signaling. NK cells were stimulated with cross-linked anti-FcR mAb, and then either ZAP-70 or syk was immunoprecipitated with its respective antiserum. Precipitated proteins were resolved by SDS-PAGE, transferred to Immobilon-P membranes, and blotted with the anti-phosphotyrosine mAb, 4G10. Stimulation of FcR rapidly elevated the phosphotyrosine levels of both ZAP-70 (Fig. 4A) and syk (Fig. 4B). Both phosphorylation events exhibited similar kinetics, with phosphorylation peaking at 1 min and returning to basal level by 30 min. Although the 21-23-kDa tyrosine-phosphorylated isoforms of associated with ZAP-70 after FcR ligation or after pervanadate-induced(37, 38) stimulation (Fig. 5), similar associations with syk were not detected (data not shown). These results are consistent with an earlier report of the FcR complex in activated NK cells associating with a 70-kDa phosphotyrosyl protein that displayed a peptide map similar to that of ZAP-70(25) .


Figure 4: Kinetics of FcR-induced tyrosine phosphorylation of ZAP-70 and syk. NK cells (2 10/sample) were stimulated with cross-linked anti-FcR mAb 3G8 for the indicated time (minutes). ZAP-70 (panel A) or syk (panel B) immunoprecipitates were resolved by SDS-PAGE, transferred to Immobilon-P membrane, and probed with the anti-phosphotyrosine mAb 4G10.




Figure 5: Kinetics of FcR-induced association of phospho- with ZAP-70. NK cells (2 10/sample) were stimulated for the indicated time (min) with either cross-linked anti-FcR mAb 3G8 or pervanadate (PV). ZAP-70 immunoprecipitates were resolved by SDS-PAGE (12.5% gel), transferred to Immobilon-P membrane, and probed with the anti-phosphotyrosine mAb 4G10.



We extended this analysis to determine whether the FcR-induced modifications of ZAP-70 and syk were associated with any changes in their catalytic activity. Recent reports have demonstrated that a peptide fragment of the human erythrocyte band 3 (i.e. cfb3) is an in vitro substrate for both ZAP-70 and syk(36, 39) . Using this exogenous substrate in an in vitro kinase assay, we demonstrated that FcR ligation induced rapid and kinetically similar increases in the catalytic activities of ZAP-70 and syk, with maximal increases by 1 min and returns toward base line by 30 min (Fig. 6). The pervanadate-induced(37, 38) tyrosine-phosphorylated forms of ZAP-70 and syk also had increased in vitro catalytic activity (Fig. 6).


Figure 6: FcR-induced increases in the in vitro catalytic activities of both ZAP-70 and syk. Panel A, NK cells (1 10/sample) were stimulated for the indicated times (min) with either cross-linked anti-FcR mAb 3G8 or pervanadate (PV). ZAP-70 or syk immunoprecipitates were incubated in an in vitro kinase assay that included the 43-kDa exogenous substrate, cfb3 (marker in right margin). Panel B, radiolabeled cfb3 was quantitated after scanning the membrane using an AMBIS 4000 radioanalytic imaging system.



Effects of lck Overexpression on ZAP-70 and syk

The data presented thus far implicate both src and syk family PTKs in FcR signaling. However, the precise regulatory role of lck in the FcR-initiated PTK pathway, including its potential interaction with syk family PTKs, is not known. To evaluate these questions, NK cells were first infected with either control WR vaccinia virus or the recombinant lck-encoding vaccinia virus and then stimulated with cross-linked anti-FcR mAb (Fig. 7). Similar to our previous observation with uninfected NK cells (Fig. 4), cross-linking of the FcR on control WR-infected cells induces the tyrosine phosphorylation of ZAP-70 (first and second lanes) and syk (fifth and eighth lanes). Significantly, overexpression of lck markedly enhanced the FcR-induced tyrosine phosphorylation of both ZAP-70 (third and fourth lanes) and syk (seventh and eighth lanes). When the FcR-induced tyrosine phosphorylation levels of ZAP-70 and syk in lck-overexpressing cells were compared with their respective counterparts in control cells, the enhancement was consistently greater for ZAP-70 (approximately 3-fold) than for syk (approximately 1.5-fold) (Fig. 7). This quantitative difference in the augmentation of the phosphorylation of ZAP-70 and syk by lck was observed reproducibly in three separate experiments. These effects caused by lck overexpression required an active kinase as they were not seen with the kinase-inactive mutant (data not shown). Furthermore, neither fyn nor src overexpression enhanced the FcR-induced tyrosine phosphorylation of ZAP-70 and syk (Fig. 8). Taken together, these results suggest that lck can selectively regulate ZAP-70 and syk during FcR signaling. In addition, the quantitative difference in lck's effect on the two related members of the syk family may reflect a difference in their requirement for src family PTKs.


Figure 7: lck-mediated tyrosine phosphorylation of ZAP-70 and syk. NK cells (2 10/sample) were infected with either the control WR or the lck-encoding vaccinia virus. Cells were subsequently left unstimulated (-) or stimulated for 1 min with cross-linked anti-Fc R mAb 3G8 (+). ZAP-70 (first four lanes) or syk (fifth through eighth lanes) immunoprecipitates were resolved by SDS-PAGE, transferred to Immobilon-P membrane, and probed with the anti-phosphotyrosine mAb 4G10.




Figure 8: Effects of src family PTK overexpression on ZAP-70 and syk tyrosine phosphorylation. NK cells (2 10/sample) infected with either the control WR, lck-, fyn-, or src-encoding vaccinia virus were stimulated (+), or not (-), for 1 min with cross-linked anti-FcR mAb 3G8. Phosphotyrosine detection of ZAP-70 (upper panel) or syk (lower panel) immunoprecipitates was performed as described in Fig. 7.



We next investigated whether lck-mediated events regulate the association of ZAP-70 with the FcR complex. NK cells overexpressing lck were stimulated with cross-linked anti-FcR mAb, lysed in a buffer containing 1% Triton X-100, and the chain immunoprecipitated. Precipitated proteins were resolved by SDS-PAGE and analyzed by immunoblotting. Detection with the anti-phosphotyrosine mAb revealed a phosphotyrosyl-containing protein migrating at approximately 70 kDa which associated with the chain after FcR stimulation in NK cells overexpressing lck (Fig. 9, upper panel). ZAP-70 was identified among these -associated proteins by reblotting the membrane with ZAP-70-specific antiserum (Fig. 9, lower panel). In parallel analyses, we were unable to detect syk among these -associated proteins, but we cannot exclude the possibility that this reflects decreased sensitivity of the syk-specific antiserum used for detection. Overexpression of src, fyn (Fig. 9), or the kinase-inactive lck (data not shown) did not have any effect on the association of phosphotyrosyl proteins with . Thus, the result in Fig. 9suggests that lck can function to recruit ZAP-70 to the FcR complex and is consistent with the notion that lck couples the FcR complex to syk family PTK activation in NK cells.


Figure 9: lck-induced association of a phosphotyrosyl protein with . NK cells (2 10/sample) were infected and stimulated as described in Fig. 6. Detergent-soluble proteins were immunoprecipitated with anti- antiserum, resolved by SDS-PAGE, and transferred to Immobilon-P membrane. Phosphotyrosine-containing proteins (upper panel) and ZAP-70 (lower panel) were detected with mAb 4G10 and anti-ZAP70 antiserum, respectively.



lck Overexpression Augments FcR-induced Tyrosine Phosphorylation of PLC-2

Our group and others(5, 6, 7, 8, 9, 10) have demonstrated that tyrosine phosphorylation events, including those involving the PLC- isoforms, are essential for the initiation of FcR-mediated NK cell cytotoxicity. However, it remains unclear what role src family PTK, in particular lck, may play in mediating the activation of PLC- isoforms. We addressed this question by examining the effect of lck overexpression on FcR-induced tyrosine phosphorylation of PLC-2 in NK cells. Similar to that seen previously in uninfected NK cells(7) , stimulation with cross-linked FcR induced the tyrosine phosphorylation of PLC-2 in either uninfected or control WR vaccinia virus-infected NK cells (Fig. 10, first four lanes). Upon overexpression of lck, the FcR-induced tyrosine phosphorylation of PLC-2 was enhanced dramatically (fifth and sixth lanes). In contrast, overexpression of the kinase-inactive lck did not produce the same effect (seventh and eighth lanes). We extended our analysis to test whether other src family PTKs may function in the same manner as lck in regulating PLC-2 phosphorylation. Neither fyn nor src overexpression had the same effect as lck (data not shown). Thus, our results implicate a possible role for lck in the regulation of PLC- isoforms during FcR-initiated activation of NK cells.


Figure 10: lck-mediated tyrosine phosphorylation of PLC-2. NK cells (2 10/sample) were either uninfected or infected with the vaccinia viruses as described in Fig. 1. Cells were subsequently stimulated (+) or not (-) for 1 min with cross-linked anti-FcR mAb 3G8. PLC-2 immunoprecipitates were resolved by SDS-PAGE and transferred to Immobilon-P membrane. The membrane was sequentially probed with the anti-phosphotyrosine mAb 4G10 (upper panel) and PLC-2 antiserum (lower panel).




DISCUSSION

Biochemical studies have yielded valuable insights into the signaling machinery triggered by the FcR complex in human NK cells (40-42). Nonetheless, although the use of molecular techniques has greatly expanded our understanding of signal transduction in other lymphoid cell types, the study of NK cell signaling has been restricted by the lack of a suitable system to manipulate this cell population genetically. We report here the successful use of the vaccinia virus expression system to investigate the role of lck in mediating FcR signal transduction. Specifically, our analyses strongly suggest that lck can selectively regulate downstream syk family PTKs and PLC-2 during the course of FcR activation.

The role of lck in signal transduction has been studied extensively in T cells. The seminal observations that lck associates with the CD4 and CD8 coreceptors in T cells and that cross-linking of these coreceptors increases lck kinase activity suggest a critical function for lck during antigen-induced T cell activation(43, 44, 45, 46, 47) . Aside from its interaction with CD4 and CD8, lck appears to be important as well in CD4/CD8-independent TCR signaling as emphasized by studies with genetic mutants of T cell lines. lck-deficient variants of the human Jurkat and the mouse CTLL-2 T cell lines exhibit impaired TCR signaling and effector functions(29, 48) . Conversely, overexpression of an activated mutant of lck enhances TCR-mediated signaling in a T cell hybridoma (49). In contrast to T cells, the role of lck in FcR-initiated signal transduction in NK cells has not been as well characterized. Although recent reports have demonstrated the physical association of lck with the FcR complex in NK cells and an induction in the catalytic activity of lck(10, 17, 18) , the functional significance of these observations remains to be elucidated. Thus, we conducted a detailed examination of the involvement of lck in FcR-mediated signaling in NK cells. Our preliminary characterization demonstrated that lck overexpression led to the tyrosine phosphorylation of select substrates, and these were enhanced further by FcR cross-linking. Interestingly, most of these substrates corresponded to those that were tyrosine-phosphorylated after FcR stimulation of normal NK cells, suggesting that lck is functionally linked to the FcR.

Since several different members of the src family PTKs are expressed in NK cells(50) , we tested whether these related PTKs exhibited any specificity in mediating FcR signaling. Our evaluation showed that lck overexpression, but not that of fyn or src, resulted in the marked elevation of tyrosine phosphorylations in NK cells. Minimal detectable effects on the total pool of detergent-soluble phosphotyrosyl proteins were induced by fyn overexpression, and none was detected with src overexpression. Further examination of specific phosphorylation events, including that of ZAP-70, syk, and PLC-2, indicated that neither fyn nor src could induce the same effects as lck. These observations underline the specificity of lck in coupling the FcR to the PTK signaling pathway. A recent study by Salcedo et al.(18) shows that lck, but not fyn, yes, or src, exhibits a Nonidet P-40 detergent-stable association with both the and chains of the FcR complex. Thus, our observation that only lck effectively coupled the FcR to the tyrosine phosphorylation of downstream substrates in NK cells extends the data obtained from coprecipitation analysis by Salcedo et al.(18) to a functional level. Moreover, in an avian B cell system, the lack of lyn can be replaced by lck and fyn, but not by src, demonstrating a less restrictive specificity for src family PTKs in that experimental system(51) .

Since the role of syk family PTKs in FcR signaling in NK cells has not been well characterized, we examined whether this family of PTKs is involved. We demonstrated formally that both PTKs of the syk family, ZAP-70 and syk, were tyrosine-phosphorylated and activated after FcR stimulation. Subsequently, we tested to see if lck acts proximal to the syk family PTKs in FcR signaling. The observation that overexpression of lck enhances the FcR-induced tyrosine phosphorylation of both ZAP-70 and syk is consistent with the notion that activation of syk family PTKs is dependent on src family PTKs. These results support and extend the model proposed by Weiss (52) in which the sequential activation of TCR-associated PTKs (i.e. src family PTK proximally, syk family PTK distally) initiates T cell activation. These studies indicated that a src family PTK is required for the association of either ZAP-70 or syk with a CD8/ chimera(53, 54) . Similarly, a study conducted with avian B cells also demonstrates that the presence of a src family PTK is required for the proper activation of syk after BCR stimulation(55) . Furthermore, a study with chimeric receptors of FcR (CD16) linked to src and syk family PTKs shows that clustering of a combination of fyn or lck with ZAP-70 is sufficient to activate the cytolytic machinery in a cytotoxic T cell line(56) . Taken together, these studies reflect the tight linkage that is required between the src and syk families for proper PTK signaling after receptor activation in lymphoid cells.

Our finding that lck has a greater effect on the FcR-induced tyrosine phosphorylation of ZAP-70 than that of syk implies that the regulation of these two related PTKs may be different. Nonetheless, the presence of lck significantly enhances both phosphorylation events, suggesting that the optimal activation of both ZAP-70 and syk is dependent on src family PTK. This difference between ZAP-70 and syk which we observed in our experiments is consistent with that of recent reports. Indeed, it is becoming increasingly evident that antigen receptor-induced syk activation, but not ZAP-70 activation, can occur in the absence of a src family PTK. For instance, TCR stimulation of Jurkat T cells lacking lck can lead to the activation of syk, but not ZAP-70(57) , whereas BCR stimulation of B cells lacking detectable src family PTKs also results in syk activation(55) . In either case, although a src family PTK is not requisite for syk activation, the presence of a src family PTK greatly enhances receptor-induced syk activation(55, 57) . These results correlate with the earlier study showing that clustering of CD16/syk chimeras by itself is sufficient to trigger cytolysis, whereas CD16/ZAP-70 chimeras require coclustering with CD16/fyn chimeras to achieve the same effect(56) . Regardless of the difference between the two syk family members, it is clear from our data and those studies by Couture et al.(57) and Kurosaki et al.(55) that optimal activation of syk family PTKs after receptor stimulation is dependent on the presence of src family PTKs.

FcR cross-linking in NK cells leads to the activation of phosphoinositide-specific PLC and the subsequent generation of critical second messengers(26) . We and others have reported that this is due to the tyrosine phosphorylation of PLC- isoforms mediated by FcR-associated PTK(5, 6, 7) . However, the identity of the PTK responsible for directly phosphorylating PLC- has remained elusive. The use of the vaccinia virus expression system allowed us to test whether lck may be involved in coupling the FcR to PLC- phosphorylation. Our analysis clearly demonstrates that lck participates in the FcR-induced tyrosine phosphorylation of PLC-2. This demonstration leaves open the question as to how lck may be causing the tyrosine phosphorylation of PLC-2. Increased PLC-2 phosphorylation could be due to its direct phosphorylation by the elevated levels of lck, and this view is supported by the observation that PLC-1 can associate with lck in activated T cells(58) . Alternatively, if lck serves to activate ZAP-70 and syk, then the phosphorylation of PLC-2 may be mediated by these downstream PTKs. In line with this latter hypothesis, clustering of CD16/syk chimeras alone, or a combination of CD16/ZAP-70 and CD16/fyn chimeras, can induce the tyrosine phosphorylation of PLC-1(56) . Likewise, the targeted disruption of the syk locus in avian B cells, but not the src-related lyn, inhibits the BCR-induced tyrosine phosphorylation of PLC-2(51) . Regardless of the precise mechanism utilized, our results strongly suggest that the activation of lck can couple FcR ligation to the subsequent tyrosine phosphorylation and regulation of PLC-2.

In summary, this study suggests a regulatory role for lck in the FcR-initiated activation of both ZAP-70 and syk in NK cells. This interaction between src and syk family PTKs can have a potent influence on downstream signaling molecules as evidenced by the effect on PLC-2. The data presented here provide the foundation for examining the function of syk family PTKs in FcR signaling in NK cells. In addition, we can also begin to identify other critical signaling molecules that may be regulated by lck during NK cell activation. More broadly, these results provide further insights into the coordinated interaction between multichain immune recognition receptors and cytoplasmic PTKs.


FOOTNOTES

*
This research was supported by the Mayo Foundation and by National Institutes of Health Grants CA47752 and GM47286. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Immunology, Mayo Clinic and Foundation, Rochester, MN 55905. Tel.: 507-284-4563; Fax: 507-284-1637.

The abbreviations used are: NK, natural killer; FcR, low affinity IgG Fc receptors (FcR type III); PLC, phospholipase C; PTK, protein- tyrosine kinase; TCR, T cell antigen receptor; BCR, B cell antigen receptor; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; Tween 20, polyoxyethylene sorbitan monolaurate.


ACKNOWLEDGEMENTS

We thank Roger Perlmutter, Bart Sefton, and Sally Parsons for generous gifts of the src family cDNAs. We are also grateful to Hans Schreiber for providing the pSC11 vector, Augusto Ochoa for the antiserum, Lawrence E. Samelson for providing the cfb3 peptide, and to Theresa Lee for assistance with the preparation of this manuscript.


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