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The Journal of Immunology, 2001, 167: 6210-6216.
Copyright © 2001 by The American Association of Immunologists

2B4 (CD244)-Mediated Activation of Cytotoxicity and IFN-{gamma} Release in Human NK Cells Involves Distinct Pathways1

Samuel S. Chuang, Pappanaicken R. Kumaresan and Porunelloor A. Mathew2

Department of Molecular Biology and Immunology and Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 76107


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
2B4 (CD244), a member of the CD2 subset of the Ig superfamily receptors, is expressed on all human NK cells, a subpopulation of T cells, basophils and monocytes. 2B4 activates NK cell mediated cytotoxicity, induces secretion of IFN-{gamma} and matrix metalloproteinases, and NK cell invasiveness. Although there has been several molecules shown to interact with 2B4, the signaling mechanism of 2B4-mediated activation of NK cells is still unknown. In this study, we found cross-linking of 2B4 on YT cells, a human NK cell line, results in the increased DNA binding activity of activator protein-1 (AP-1), an important regulator of nuclear gene expression in leukocytes. We investigated the possible role of various signaling molecules that may be involved in the activation of lytic function of YT cells via 2B4. Treatment of YT cells with various specific inhibitors indicate that 2B4-stimulation of YT cells in spontaneous and Ab-dependent cytotoxicity is Ras/Raf dependent and involves multiple MAPK signaling pathways (ERK1/2 and p38). However, only inhibitors of transcription and p38 inhibited 2B4-mediated IFN-{gamma} release indicating distinct pathways are involved in cytotoxicity and cytokine release. In this study we also show that 2B4 constitutively associates with the linker for activation of T cells (LAT) and that 2B4 may mediate NK cell activation via a LAT-dependent signaling pathway. These results indicate that 2B4-mediated activation of NK cells involves complex interactions involving LAT, Ras, Raf, ERK and p38 and that cytolytic function and cytokine production may be regulated by distinct pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Natural killer cells recognize and kill virally infected cells, parasites and certain tumor cells (1, 2, 3). NK cells play an important role by their ability to target MHC class I negative cells that escape recognition by cytotoxic T cells (4, 5). In addition to cytolytic activity, NK cells also produce several regulatory cytokines including IFN-{gamma}, TGF{beta}, IL-1{beta}, IL-10 and GM-CSF as well as matrix metalloproteinases (MMPs) (6, 7, 8, 9). The mechanisms that control NK cell activation and cytotoxicity are believed to be determined by a delicate balance between stimulatory and inhibitory signals received from surface receptors (10, 11, 12). NK cells also mediate the rejection of MHC mismatched bone marrow stem cells (13). Engagement of cytolytic activity can be inhibited by MHC class I molecules on target cells interacting with MHC class I receptors expressed on the surface of NK cells. NK cell inhibitory receptors belong to the Ig and lectin gene superfamilies. One common feature of the killer cell inhibitory receptors is the presence of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic domains (3, 10). The ITIM motifs become phosphorylated upon receptor binding which results in the recruitment of protein SH2 domain bearing tyrosine phosphatase (SHP)3 1 and/or SHP-2. These phosphatases can dephosphorylate several substrates within the activating signaling cascade or a distal activating receptor and inhibit NK cell activation (14, 15).

Although there has been great progress in understanding the inhibitory mechanisms that regulate NK cell function, our knowledge of the activating signaling pathways is slowly emerging. Several surface molecules have been identified that can activate natural cytotoxicity. Activating receptors such as 2B4, CD2, CD16, KIR3DS1-5, NKp30, NKp44, and NKp46 are members of the Ig superfamily (16, 17, 18). These molecules bear high homology on their extracellular domains while lacking the immunoreceptor tyrosine-based inhibitory motifs on their cytoplasmic tails. There are also members of the lectin gene superfamily that can transduce an activating signal. Members of the lectin superfamily, such as NKR-P1A and NKR-P1C, NKG2 family, and the LY49 family can form homo- and heterodimer complexes that can be inhibitory or stimulatory. Although many of the activating surface molecules have been identified, information on the signaling cascade from the cell surface to within is fragmentary.

2B4 was originally identified on mouse NK cells and the subset of T cells that mediate non-MHC-restricted killing (18, 19, 20, 21). It is a novel member of the CD2 subfamily which includes signaling lymphocyte-activation molecule, CD48, CD58, CD84, CS1, and Ly9, and is expressed on all NK cells, a subset of CD8+ T cells, basophils, and monophils (22, 23, 24). The cytoplasmic domain of 2B4 contains novel tyrosine motifs (TxYxxV/I) that associate with signaling adaptor molecule, signaling lymphocyte-activation molecule-associated protein (SAP) whose defect forms the basis for X-linked lymphoproliferative syndrome (XLP) (25, 26, 27, 28). It is thought that NK cells in XLP patients are defective in their activation through 2B4 signaling (29, 30, 31). In addition to modulating cytolytic function, 2B4 activation of NK cells induces cytokine production as well as invasiveness (9, 19, 32). CD48 was recently identified as the high-affinity counterreceptor of 2B4 in both mice and humans (33, 34). CD48–2B4 interactions are physiologically important since they enhance the lytic function of human NK cells (23). It has been reported that 2B4 may function as a coreceptor in human NK cell activation (35).

In this paper, we investigated whether the AP-1 pathway is activated in human NK cells upon 2B4 stimulation. AP-1 is an important regulator of nuclear gene expression in leukocytes, having been found responsive to a wide range of stimuli and regulating a large number of genes (36, 37). We observed that AP-1 DNA-binding activity increased in response to 2B4 stimulation, in particular AP-1 complexes containing JunB. To dissect the mechanisms involved in 2B4-mediated cytotoxicity, we set out to examine whether 2B4 stimulation of YT cells utilized the Ras-dependent mitogen activated protein kinase (MAPK) pathway in natural cytotoxicity and Ab-dependant cellular cytotoxicity (ADCC). Our results show that 2B4-induced NK cell cytotoxicity is dependent on the extracellular signal-regulated kinase (ERK) and p38 MAPK pathways and can be inhibited by inhibitors of Ras and Raf. Examination of 2B4-stimulated release of IFN-{gamma} revealed the role of the p38 MAPK but not the ERK1/2 pathway in IFN-{gamma} production. Immunoprecipitations revealed the constitutive association of linker for activation of T cells (LAT) with 2B4.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines, Abs, and chemicals

YT (human NK cell line), K562 (human erythroleukemia cell line), and P815 (mouse lymphoma cell line) were cultured in culture medium (RPMI 1640 supplemented with 10% FBS (HyClone, Logan, UT), 2 mM glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, 10 mM HEPES, and 10 mM nonessential amino acids). Cells were maintained at 37°C in a humidified 5% CO2/95% air incubator. Cell culture reagents were obtained from Life Technologies (Gaithersburg, MD) unless otherwise noted. C1.7 Ab, which recognizes human 2B4 (25), was purchased from Coulter (Orlando, FL). Mouse Abs against c-Jun (catalog no. SC-822X), JunB (catalog no. SC-8051X), anti-phospho-p38 (catalog no. SC-7973), anti-phospho-ERK (catalog no. SC-7383), polyclonal goat Abs anti-p38 (catalog no. SC-535), and anti-ERK (catalog no. SC-94) and protein A/G PLUS-agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-LAT rabbit polyclonal Ab was purchased from Upstate Biotechnology (Lake Placid, NY). All enzymes were purchased from New England Biolabs (Beverly, MA) unless otherwise stated. Poly(dI-dC) was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). All custom-synthesized oligonucleotides used in this study were supplied by Integrated DNA Technologies (Coralville, IA). All inhibitors used in this study were purchased from Calbiochem (San Diego, CA).

Nuclear extraction and EMSA

Nuclear extracts were isolated from YT cells and NK-92 cells and YT cells incubated with mAb C1.7 (200 ng/ml concentration) (38) and protein DNA-binding reactions were conducted in EMSAs (39). One million YT cells were incubated with C1.7 mAb for 2, 4, 6, and 8 h in 12-well plates at 37°C in a humidified 5% CO2/95% air incubator. After the stimulation period, the cells were collected, washed in ice-cold PBS, and lysed as described elsewhwere (38). A typical binding reaction mixture contained 2 µg of nuclear protein, 1 µg of poly(dI-dC), and radiolabeled oligonucleotide (20,000 cpm, ~0.2 ng) in 10 µl of reaction volume. The double-stranded radiolabeled oligonucleotide probe codes for the human 2B4 (h2B4) promoter sequence lies (-111 to -89) relative to the start of transcription and contains a functional AP-1 site (40). The mixture was incubated on ice for 30 min and then electrophoresed through a 4% polyacrylamide gel under nondenaturing conditions in 0.25x Tris-borate-EDTA at 200 V for 70 min. The gel was dried and then exposed to film. The bands were visualized by autoradiography and quantified using the image quantitation program AlphaEase (Alpha Innotech, San Leandro, CA).

51Cr release cytotoxicity assay

K562 cells and P815 cells, where indicated, were used as target cells and labeled by incubating 1 x 106 cells with 2 MBq of Na251CrO4 (NEN Research Products, Boston, MA) for 90 min at 37°C under 5% CO2 in air. The target cells were then washed three times in culture medium. Ten thousand labeled target cells (100 µl) were incubated with the effector YT cell suspension (100 µl), with and without mAb C1.7 (200 ng/ml). Effector YT cells were resuspended and added at 1, 2, 5, 10, and 20 times the number of labeled target cells. After incubation for 4 h at 37°C under 5% CO2 in air, the cells were pelleted at 250 x g for 5 min, 100 µl of the supernatants was removed, and their radioactivity was measured. The percentage of specific lysis was calculated by the following equation: (a - b/c - b) x 100, where a is the radioactivity of the supernatant of target cells mixed with effector cells, b is that in the supernatant of target cells incubated alone, and c is that in the supernatant after lysis of target cells with 1% Nonidet P-40. All data points in each graph represent the average of four independent trials with similar results. Determination of statistical significance was determined on each data point representing 2B4-mediated cytotoxicity assays performed with inhibitor-treated effector cells or target cells compared with assays conducted with nontreated effector and target cells with Student’s t test. Data groups were considered significantly different when p < 0.05.

Inhibitor treatment of cells

In assays using inhibitors, both effector YT cells and target K562 and P815 cells were subjected to treatment in culture medium as indicated. Cells were incubated in actinomycin D (20 µg/ml) for 2 h to inhibit RNA polymerase. To inhibit p38, cells were incubated in SB203580 at 10 and 50 µM concentrations for 1 h. Cells were incubated in PD089059 (100 µM) for 1 h to inhibit MAPK kinase 1 (MEK1). Cells were incubated in FTI-277 at 7.5 and 30 µM for 16 h to inhibit H-Ras processing. All incubations with inhibitors were conducted at 37°C under 5% CO2 in air. At the end of the incubation period, the cells were used in 51Cr release cytotoxicity assays.

Immunoblot analysis

YT cells were incubated (1 x 107/100 µl; 37°C) for the indicated times with the different stimuli. These were C1.7 mAb (10 µg/ml) and PMA (50 ng/ml). After stimulation, the cells were lysed with 900 µl of lysis buffer (1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 10 mM HEPES (pH 7.5), 0.15 M NaCl, 10% glycerol, 1 mM PMSF, 1 mM Na3VO4, 50 mM NaF, 1 mM EDTA, and 10 µg/ml each of aprotinin and leupeptin). Forty micrograms of protein lysate was analyzed in 8% SDS-PAGE (reducing conditions). Western blots were performed according to the manufacturer’s chemiluminescence detection system instructions (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Western blots were hybridized with anti-phospho-p38 and anti-phospho-ERK mAbs to detect phosphorylated forms of p38 and ERK, respectively. The Western blots were then stripped and reprobed with anti-p38 and anti-ERK Abs to detect total amounts of p38 and ERK, respectively.

IFN-{gamma} release assay

Inhibitor-treated or untreated YT cells (500,000) were stimulated or unstimulated with C1.7 mAb (400 ng/ml) in flat-bottom 24-well plates for 1 h at 37°C under 5% CO2 in air. Target K562 cells (50,000) were then added. After incubation for 16 h at 37°C under 5% CO2 in air, 100 µl of cell-free supernatant was collected. IFN-{gamma} concentration was then quantitated with an ELISA kit according to the manufacturer’s instructions (CLB, Amsterdam, The Netherlands). Each condition was tested in at least four independent trials.

Immunoprecipitations

Approximately 1 x 108 YT cells were either left untreated or treated with 1 µg/ml C1.7 mAb for 2 h at 37°C. Cells were then lysed with 1% digitonin, 10 mM Tris (pH 7.4), 150 mM NaCl, 100 µg/ml PMSF, and protease inhibitor mixture (Sigma-Aldrich, St. Louis, MO) for 1 h on ice. The lysates were precleared with the addition of control mouse IgG Ab (Upstate Biotechnology) for 3 h followed by protein G/A plus agarose and rotated for 3 h at 4°C. The precleared supernatant were used to immunoprecipitate 2B4 with C1.7 mAb (5 µg/ml) overnight with slow shaking followed by protein G/A plus agarose mix for 3 h at 4°C. Isotype control IgG (22b5) was used as negative control immunoprecipitations. Immunoprecipitates were separated by 10% SDS-PAGE and detected as described in Immunoblot analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
2B4 stimulation induces AP-1 activation

Our studies on the transcriptional regulation of the mouse and human 2B4 gene have revealed the presence of functional AP-1 sites in both promoters (40, 41). The AP-1 site that resides -106 to -100 relative to the start of transcription is necessary for human 2B4 gene expression (40). Recently, it has been found that engagement of NK cell cytotoxicity regulates AP-1 expression (42). To determine whether 2B4 stimulation of YT cells, a human NK cell line, results in activation of AP-1, we isolated nuclear protein after 2B4 stimulation. We then performed EMSAs with double-stranded radiolabeled probes coding for the promoter sequence (-111 to -89) of the human 2B4 promoter that contains a functional AP-1 site (Fig. 1Go). Examination of the levels of AP-1 DNA-binding activity through densitometric analysis revealed that AP-1 DNA-binding activity increased in response to 2B4 cell stimulation, reaching maximal stimulation 6 h after 2B4 stimulation of the cells (Fig. 1Go, B and C). Supershift analysis using Ab specific for c-Jun and JunB found AP-1 factors that contained either protein component increased (Fig. 1Go, D–F). However, the presence of the supershifted band of JunB increased proportionately as shown in Fig. 1GoB, indicating that 2B4 stimulation of NK cells results in selective activation of AP-1 heterodimers involving JunB.



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  		FIGURE 1. AP-1 DNA-binding activities are increased by 2B4 stimulation. A, An EMSA experiment was performed with a radiolabeled double-stranded oligonucleotide coding for the h2B4 promoter sequence spanning (-111 to -89) relative to the start of transcription. -ve lane is a binding reaction done in the absence of YT cell nuclear extract. +ve lane is a binding reaction done in the presence of 2 µg pf YT cell nuclear extract. Unlabeled competitor DNAs were included in the binding reactions containing YT nuclear extract at 100 and 200 molar excess, respectively. The competitor DNAs used were wild-type (wt) cold probe; AP-1, double-stranded oligonucleotides coding for AP-1 consensus binding site. B, YT cells were treated with C1.7 mAb for the indicated time intervals at 37°C. Thereafter, nuclear protein extracts were prepared from the treated cells and analyzed for AP-1 DNA-binding activity by EMSA. DNA-binding reactions were performed with 2 µg of nuclear protein extract and radiolabeled double-stranded oligonucleotides coding for the h2B4 promoter sequence spanning (-111 to -89) relative to the start of transcription as outlined in Materials and Methods. C, Graphical representation of AP-1 activation observed in B. Nuclear protein extracts incubated with anti-c-Jun Ab (D) and anti-JunB Ab (E). F, Graphical representation of c-Jun () and JunB () activation observed in C and D, respectively. Arrow, AP-1-DNA complex. Bracket, Ab supershifted DNA-protein complex. N.S. are nonspecific bands. Free represents unbound probe.

 
2B4-mediated cytotoxicity is transcription dependent

Previously, we and others have established that 2B4 stimulation of NK cell lines results in an increase in spontaneous cytotoxic and redirected ADCC (rADCC) activity (9, 22, 23, 35). AP-1 induction and other critical transcriptional events may be involved in 2B4 signaling in NK cells. To determine whether 2B4-mediated cytotoxicity is transcription dependent, 51Cr release cytotoxicity assays were performed using effector and target cells pretreated for 2 h with 20 µg/ml actinomycin D. Actinomycin D inhibits RNA polymerases by complexing with the DNA preventing transcription. Pretreatment of YT cells with actinomycin D also inhibited natural cytotoxicity against target K562 cells (data not shown). Pretreatment of YT effector cells resulted in significantly lower levels of 2B4-mediated cytotoxicity of K562 cells (p < 0.05) and rADCC (p < 0.02) against P815 cells than those mediated by control nontreated effector cells (Fig. 2Go). However, pretreatment of target cells with actinomycin D did not alter their susceptibility to NK cell cytotoxicity. Thus, these results show that critical transcriptional events are involved in 2B4-mediated cytotoxicity.



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  		FIGURE 2. Effect of actinomycin D on 2B4-stimulated YT cell cytotoxicity. YT and target K562 and P815 cells were pretreated for 2 h at 37°C with actinomycin D (20 µg/ml) as described in Materials and Methods. YT cells were incubated (1 h, 37°C) without ({blacksquare}) or with C1.7 mAb at 200 ng/ml concentrations ({square}). Pretreated effector cells (x) were then incubated (1 h, 37°C) with C1.7 mAb (200 ng/ml). Pretreated target cells ({diamondsuit}) were incubated with 2B4-stimulated effector cells. All data points in each graph represents the average of four independent trials with similar results.

 
Role of p38 and MEK1/ERK signaling in 2B4 stimulation of NK cells

Several transcription factors including AP-1, NF-{kappa}B, and Ets can be activated by the MAPK signaling pathways. We undertook the task of examining the roles of the p38 and MEK1/ERK signaling pathways in the 2B4 signal cascade. To accomplish this, we used the selective inhibitor of p38, SB203580, in pretreatments of YT cells and target K562 cells before their use in 51Cr release cytotoxicity assays. YT cells pretreated with increasing concentrations of SB203580 and then stimulated with anti-2B4 mAb resulted in progressively lower spontaneous cytotoxicity against K562 cells (p < 0.05; Fig. 3GoA). SB203580-pretreated NK cells’ ability for rADCC against P815 cells was also diminished. Pretreatment of either target cell failed to inhibit 2B4-stimulated cytotoxicity significantly. These results show that p38 plays a role in 2B4-stimulated YT cells in ADCC as well as spontaneous cytotoxicity.



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  		FIGURE 3. Effect of MAPK inhibitors in YT lysis of tumor cells. A, YT and target K562 and P815 cells were pretreated for 1 h at 37°C with 10 and 50 µM with SB203580. B, Cells were pretreated for 1 h at 37°C with 100 µM PD089059. YT cells were incubated (1 h, 37°C) without ({blacksquare}) or with C1.7 mAb at 200 ng/ml concentrations ({square}). Effector cells pretreated with low (x) or high (•) concentrations of inhibitors were then incubated (1 h, 37°C) with C1.7 mAb (200 ng/ml). Target cells pretreated with low ({diamondsuit}) or high ({blacktriangleup}) concentrations of inhibitors were then incubated with 2B4-stimulated effector cells All data points in each graph represent the average of four independent trials with similar results.

 
We next examined whether the MEK1/ERK signaling pathway is part of the signaling cascade that is activated by 2B4 ligation. We used the MEK1 inhibitor PD089059 to determine the importance of MEK1 during 2B4 activation of NK cells (43). PD089059 pretreatment of YT cells before their use in 51Cr release cytotoxicity assays resulted in a significant reduction in cytolytic activity from 2B4-stimulated YT cells against both K562 (p < 0.02) and P815 cells (p < 0.05 for 20:1, 10:1, and 5:1 E:T ratios; Fig. 3GoB). This indicates the that MEK1/ERK signaling pathway regulates 2B4 activation of the lytic function of NK cells. It appears that 2B4 signaling utilizes multiple MAPK pathways in activating cytolytic function. We have examined the effect of pretreatment with PD089059 and SB203580 on YT cells. We found that inhibition with either MEK1 or p38 decreased the natural cytotoxicity of YT cells against target K562 cells (data not shown). Previous studies have found that the MEK1/ERK pathway is required for NK cells to acquire lymphokine-activated killer activity. However, once the NK cells were engaged, treatment with the inhibitor PD089059 had little effect on NK activity (44). The MEK1/ERK signaling pathway can activate AP-1, members of the Ets-1 family, whereas activation of the p38 MAPK pathway leads to NF-{kappa}B activation (37, 45, 46, 47). Activation of these pathways can lead to important transcriptional events, such as AP-1 activation, that are essential for the engagement of NK cell cytotoxicity.

Signal transduction through the p38 and ERK MAPK pathways requires the phosphorylation of p38 and ERK, respectively. To determine whether 2B4 ligation results in the alteration of phosphorylation states of p38 and ERK, YT cells were stimulated with C1.7 mAb and then lysed and analyzed by Western blot. 2B4 engagement by anti-2B4 mAb resulted in tyrosine phosphorylation of both p38 and ERK (Fig. 4Go). However, PMA stimulation of YT cells resulted in phosphorylated forms of ERK only.



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  		FIGURE 4. 2B4 stimulation results in phosphorylation of ERK and p38. YT cells were stimulated with anti-2B4 mAb and PMA for the indicated times at 37°C. A, Cell lysates were then prepared and analyzed by Western blotting with anti-phospho-ERK (upper panel). The same membrane was stripped and reprobed with anti-ERK to check for total protein amounts (lower panel). B, Cell lysates were then prepared and analyzed by Western blotting with anti-phospho-p38 (upper panel). The same membrane was stripped and reprobed with anti-p38 to check for total protein amounts (lower panel).

 
2B4 stimulation of NK cells utilizes a Ras-dependent pathway

In general, the ERK pathway can be activated by signaling through Ras-Raf (36, 37). It has been recently reported that Ras becomes activated after target ligation or IL-2 induction (48). To determine whether 2B4 activates NK cell function in a Ras-dependent manner, we performed 51Cr release assays with FTI-277-treated cells. FTI-277 is a farnesyl transferase inhibitor that prevents H-Ras from localizing to the plasma membrane and becoming functionally active both in vivo and in vitro. This induces accumulation of nonfarnesylated cytoplasmic H-Ras, which bind Raf protein to form inactive Ras-Raf complexes (49). YT cells were treated for 16 h at 37°C with 7.5 and 30 µM FTI-277, known levels that inhibit Ras activation (48). Pretreatment of YT cells with FTI-277 markedly diminished 2B4-stimulated NK cell cytotoxicity against K562 and P815 target cells (Fig. 5Go). However, FTI-277 treatment of target cells had no effect on target lysis in the same assay (Fig. 5Go). These 51Cr release cytotoxicity assays were also conducted using ZM 336372, an inhibitor of Raf. ZM336372 treatment of YT effector cells also significantly inhibited 2B4-mediated cytolytic activity (data not shown). Thus, it appears Ras and Raf are essential for 2B4-stimulated NK cell cytotoxicity in YT cells.



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  		FIGURE 5. Effect of Ras inhibitor in YT lysis of tumor cells. YT and target K562 and P815 cells were pretreated for 1 h at 37°C with FTI-277 at 7.5 and 30 µM concentrations. YT cells were incubated (1 h, 37°C) without ({blacksquare}) or with C1.7 mAb at 200 ng/ml concentrations ({square}). Effectors cells pretreated with low (x) or high (•) concentrations of inhibitors were then incubated (1 h, 37°C) with C1.7 mAb (200 ng/ml). Target cells pretreated with low ({diamondsuit}) or high ({blacktriangleup}) concentrations of inhibitors were then incubated with 2B4-stimulated effector cells All data points in each graph represent the average of four independent trials with similar results.

 
IFN-{gamma} release is dependent on the p38 pathway

IFN-{gamma} production by NK cells is a major function of NK cells in response to NK cell-target cell contact (1). Our studies have revealed that binding of 2B4 by C1.7 mAb results in the increase in cytolytic activity of YT cells and the activation of Ras, Raf, and the ERK and p38 MAPK pathways. Using similar inhibitor treatment protocols, we investigated whether 2B4-induced IFN-{gamma} production was under the control of the same pathways. Pretreatment of YT cells with actinomycin D and SB203580 inhibited IFN-{gamma} production (Fig. 6Go), whereas inhibitors of Ras, Raf, and the ERK MAPK pathway did not. The induction of IFN-{gamma} production required the activity of p38 as shown by the ability of SB203580 to completely inhibit 2B4-induced cytokine production.



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  		FIGURE 6. p38 activation controls 2B4-induced IFN-{gamma} production in YT cells. YT cells (5 x 105) were incubated with medium only or cell-permeable inhibitors as described in Materials and Methods. The untreated and treated cells were then stimulated with anti-2B4 mAb for 1 h at 37°C under 5% CO2 in air. Target K562 cells (5 x 104) were then added. After incubation for 16 h at 37°C under 5% CO2 in air, cell-free supernatants were collected. IFN-{gamma} concentration was quantitated by ELISA.

 
2B4 is constitutively associated with LAT

Both 2B4 and LAT are localized in the glycolipid-enriched microdomains (GEM) fractions of the cell membrane (50). To determine whether LAT associates with 2B4 in NK cells, 2B4 was immunoprecipitated from the YT cells, which was either unstimulated or stimulated with anti-2B4 mAb. Immunoprecipitates were analyzed by Western blot using anti-2B4 mAb and subsequently after stripping the blot was reprobed with anti-LAT polyclonal Ab. As shown in Fig. 7Go, LAT is associated with 2B4 consitutively. Densitometric analysis indicates that the level of association between 2B4 and LAT remains unchanged after 2B4 stimulation.



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  		FIGURE 7. 2B4 is constitutively associated with LAT. YT cells were lysed and immunoprecipitated with anti-2B4 mAb. Immunoprecipitates were analyzed by Western blotting with anti-2B4 mAb. The same membrane was stripped and reprobed with anti-LAT Ab. Lane 1, cells stimulated with anti-2B4 mAb (200 ng/ml) for 2 h at 37°C before lyses; lane 2, immunoprecipitation with unstimulated YT cells; lane 3, YT cells lysed and immunoprecipitated with a control IgG; and lane 4, YT cells lysed and immunoprecipitated with protein A plus G only. The migration positions of 2B4 and LAT are indicated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Transcription factor AP-1 has been found to be an important regulator of gene expression in leukocytes involving T and B cell activation, Ig production, cytokine expression, cell differentiation, and development (36). In NK cells, AP-1 factors have been implicated in cell activation, cytolytic activity, and cytokine production (42, 44, 51). Our previous studies in understanding the transcription of the 2B4 gene in both mouse and humans has revealed that AP-1 plays an important role in the regulation of expression of 2B4 (40, 41). We have examined the effect of 2B4 stimulation of YT cells on AP-1 DNA-binding activity and found that AP-1 DNA-binding activity increased significantly. Supershift EMSAs revealed that increases in AP-1 complexes containing JunB correlated strongly with increases in AP-1 DNA-binding activity. These results are consistent with other findings that show an increase in JunB mRNA and the increase in JunB-containing AP-1 complexes during natural cytotoxicity (42).

MAPK signaling pathways activates AP-1 and other transcription factors including NF-{kappa}B and Ets proteins. IL-2 activation of human NK cells results in an increase in ERK activity but not p38 activity, and IL-2-activated NK functions are dependent on the ERK pathway (44). FcR-triggered TNF-{alpha} secretion and integrin-mediated IFN-{gamma} production also are dependent on the ERK pathway (52, 53). In our study, YT cytotoxicity in spontaneous and rADCC against target K562 and P815 cells was increased by 2B4 ligation with Ag-specific mAb. Pretreatment of YT cells with various cell-permeable specific inhibitors revealed the activation of the Ras/Raf/ERK and p38 MAPK pathways during 2B4-mediated signaling. Previously, it has been found that p38 and MEK1/ERK signaling are involved in direct tumor cell lysis by NK cells while the c-Jun N-terminal kinase MAPK pathway does not play a significant role (44, 48, 52). The p38 MAPK pathway’s role in other NK cell functions include Fc{gamma}RIIIA-induced granule exocytosis and target cell-induced IFN-{gamma} mRNA accumulation (52). Although some studies have shown that activation of natural cytotoxicity can be Ras independent, IL-2 activation of the MAPK pathway was completely dependent on intact Ras (44, 48). It appears that the role of these proteins in natural cytotoxicity is complex and dependent on the activation state of the NK cells.

2B4 stimulation of NK cells may utilize distinct but overlapping pathways from those engaged by other receptors and these pathways regulate different aspects of NK cell function. Ras inhibitor, FTI-277, could not interfere with perforin and granzyme B polarization toward the contact point with a target cell (48). 2B4 stimulation of NK cells revealed the activation of the ERK and p38 pathway as well as an integral role for Ras ( Figs. 3–5GoGoGo). However, pretreatment of YT cells with an inhibitor of Ras did not inhibit 2B4/target cell-stimulated production of IFN-{gamma} (Fig. 6Go). Alternatively, treatment of YT cells using actinomycin D and the p38 inhibitor SB203580 did inhibit IFN-{gamma} production. Additionally, SB202190, a p38 inhibitor, inhibited accumulation of target (K562)-induced IFN-{gamma} mRNA in NK cells (52). This suggests that 2B4-mediated IFN-{gamma} production is Ras/Raf independent, unlike {beta}1 integrin- and IL-2-activated secretion of IFN-{gamma} (44, 53).

Our study demonstrated that 2B4 associates constitutively with LAT (Fig. 7Go). Bottino et al. (54) have also shown that 2B4 is constitutively associated with LAT and Ab ligation of 2B4 leads to phosphorylation of 2B4 and LAT. Furthermore, phosporylated LAT recruits other signaling molecules, phospholipase C (PLC) {gamma} and Grb2 (54). The role of PLC{gamma}1 or PLC{gamma}2 in natural cytotoxicity has not been clearly defined. Both are found in NK cells and become tyrosine phosphorylated upon Fc{gamma}R activation (55). In other signaling pathways, LAT is required for FcR-dependent phosphorylation of PLC{gamma}. LAT has been found to associate with Grb2, Gads, and PLC{gamma}1 in T cell Ag receptor-mediated signaling (56, 57). In T cells, the activation pathways through LAT may be determined by the signaling molecules that it associates with (58, 59, 60). The Ras pathway is blocked in LAT-deficient cells, resulting in inhibition of MAPK activation and transcriptional activity of AP-1 (61, 62). On the other hand, overexpression of LAT in NK cells leads to increased ADCC and spontaneous cytotoxicity (56). LAT has emerged as a major transmembrane adaptor protein in T cell activation after TCR engagement by Ag-MHC (58, 59, 60, 63). TCR engagement results in the phosphorylation of LAT which then associates with other adaptor proteins including PLC{gamma}, Grb-2, Grap, phosphatidylinositol 3-kinase, and Gads which can then form multimeric signaling complexes by the recruitment of other adaptor proteins including Vav, SLP-76 Sos, Shc, Pyk-2, and Graf (58, 59, 60). Many of these adaptor proteins are also expressed in NK cells and have been found to play various roles in the signaling cascade in natural cytotoxicity and ADCC. Grb2-Sos complex associates with LAT and activates the Ras-dependent MEK/ERK pathway (36, 43, 64). Many other kinases have also been implicated in NK cell activation including phosphatidylinositol 3-kinase (65), Syk (66), and Pyk2 (67, 68, 69). Pyk2 is recruited from the cytoplasm and associates with paxillin and microtubule organizing center and transported to the GEM fraction where it associates with Grb2, Shc, and possibly Vav (53, 67, 68, 70). Pyk2 can activate the Ras/Raf kinase pathway as well as the Rac-1 pathway (71, 72). Overexpression of Pyk2 leads to the activation of p38 MAPK (73). Thus, it appears there are complex interactions that occur at the GEM where 2B4 signaling may be controlled by LAT and the adaptor molecules it associates with. It has been suggested that 2B4 functions as a coreceptor in human NK cell activation and requires coengagement of other activating receptors (35). It is possible that NK cell activation is dependent on the recruitment of key adaptor molecules to the GEM fractions before engagement of cell cytotoxicity can occur.

It has been found that NK inhibitory receptors can inhibit phosphorylation of 2B4, thus blocking NK cell activation (15). A possible mechanism may be dependent on SHP-1 recruitment by inhibitory killer Ig-related receptors that could dephosphorylate 2B4 directly or other downstream signaling molecules including LAT, SLP-76, Pyk2, and PLC{gamma} (15, 74, 75, 76). It has been shown that the 2B4 cytoplasmic tail can associate many adaptor molecules including SHP-2 and SAP (25). It has also been found that 2B4 can associate with SHP-2 in murine NK cells and SHP-1 in primary NK cell isolates (31, 77). Several studies indicate defective 2B4 signaling in NK cells from XLP patients (26, 27, 29, 30, 31). Mutations in SAP results in defective NK cell activation through 2B4, possibly due to loss of inhibition of SHP-1–2B4 interactions (25, 30). It is conceivable that SAP regulates 2B4 function by preventing SHP-1 from associating and dephosphorylating the 2B4 cytoplasmic tail as well as other adaptor molecules that are part of the signaling complex.

Activation of NK cells via h2B4 triggers many events that may be controlled through transcription including an increase in transcription of IFN-{gamma} and MMP-2 (9, 78). Although we have found 2B4 signaling results in the activation of both the ERK and p38 MAPK pathway, surprisingly, only inhibitors of the p38 pathway resulted in the inhibition of 2B4-mediated IFN-{gamma} secretion. This indicates that the MAPK pathways regulate different aspects of NK cell activation. Examination of the role of another NK cell surface receptor KIR2DL4 (CD158d) also supports the concept of functional distinction between cytotoxicity and cytokine production (79). KIR2DL4 induction of resting NK cells induced IFN-{gamma} production but not cytotoxicity. We have observed that the LLT-1 receptor (80), another NK cell molecule, induces IFN-{gamma} secretion while failing to increase cytolytic activity (H. K. Pham, S. S. Chuang, and P. A. Mathew, unpublished observation). Cumulatively, these data show that NK cell functions may be differentially controlled and stimulated as determined by the signals of individual receptors.

In summary, our study demonstrates that activation of NK cells through surface 2B4 is mediated via pathways that may involve adaptor molecules including LAT, Ras, and Raf, resulting in the activation of the p38 and MEK1/ERK MAPK pathways. This data along with other recent findings reveal the complexity of 2B4 signaling and its role in NK cell activation.


   Acknowledgments
 
We thank Kent S. Boles for insightful comments and review of this manuscript.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grants AI 38938 and CA 85753. This work was supported in part by a grant from the University of North Texas Health Science Center Institute for Cancer Research. We appreciate support of our work from Bank One through the University of North Texas Health Science Center Institute for Cancer Research. Back

2 Address correspondence and reprint requests to Dr. Porunelloor A. Mathew, Department of Molecular Biology and Immunology, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107-2699. E-mail address: pmathew{at}hsc.unt.edu Back

3 Abbreviations used in this paper: SHP, SH2 domain bearing tyrosine phosphatase; SAP, signaling lympocyte-activation molecule-associated protein; XLP, X-linked lymphoproliferative syndrome; MAPK, mitogen-activated protein kinase; ADCC, Ab-dependent cellular cytotoxicity; ERK, extracellular signal-regulated kinase; LAT, linker for activation of T cells; h2B4, human 2B4; MEK1, MAPK kinase 1; rADCC, redirected ADCC; GEM, glycolipid-enriched microdomains; MMP, metalloproteinase; PLC, phospholipase C. Back

Received for publication March 28, 2001. Accepted for publication September 26, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Trinchieri, G.. 1989. Biology of natural killer cells. Adv. Immunol. 47:187.[Medline]
  2. Scott, P., G. Trinchieri. 1995. The role of natural killer cells in host-parasite interactions. Curr. Opin. Immunol. 7:34.[Medline]
  3. Lanier, L. L.. 1998. NK cell receptors. Annu. Rev. Immunol. 16:359.[Medline]
  4. Lanier, L. L.. 1998. Follow the leader: NK cell receptors for classical and nonclassical MHC class I. Cell 92:705.[Medline]
  5. Moretta, L., E. Ciccone, A. Moretta, P. Hoglund, C. Ohlen, K. Karre. 1992. Allorecognition by NK cells: nonself or no self?. Immunol. Today 13:300.[Medline]
  6. Biron, C. A.. 1997. Activation and function of natural killer cell responses during viral infections. Curr. Opin. Immunol. 9:24.[Medline]
  7. Naume, B., T. Espevik. 1994. Immunoregulatory effects of cytokines on natural killer cells. Scand. J. Immunol. 40:128.[Medline]
  8. Kitson, R. P., P. M. Appasamy, U. Nannmark, P. Albertsson, M. K. Gabauer, R. H. Goldfarb. 1998. Matrix metalloproteinases produced by rat IL-2-activated NK cells. J. Immunol. 160:4248.[Abstract/Free Full Text]
  9. Chuang, S. S., M. H. Kim, L. A. Johnson, P. Albertsson, R. P. Kitson, U. Nannmark, R. H. Goldfarb, P. A. Mathew. 2000. 2B4 stimulation of YT cells induces natural killer cell cytolytic function and invasiveness. Immunology 100:378.[Medline]
  10. Tomasello, E., M. Blery, E. Vely, E. Vivier. 2000. Signaling pathways engaged by NK cell receptors: double concerto for activating receptors, inhibitory receptors and NK cells. Semin. Immunol. 12:139.[Medline]
  11. Moretta, A., R. Biassoni, C. Bottino, L. Moretta. 2000. Surface receptors delivering opposite signals regulate the function of human NK cells. Semin. Immunol. 12:129.[Medline]
  12. Long, E. O., S. Rajagopalan. 2000. HLA class I recognition by killer cell Ig-like receptors. Semin. Immunol. 12:101.[Medline]
  13. George, T., Y. Y. Yu, J. Liu, C. Davenport, S. Lemieux, E. Stoneman, P. A. Mathew, V. Kumar, M. Bennett. 1997. Allorecognition by murine natural killer cells: lysis of T-lymphoblasts and rejection of bone-marrow grafts. Immunol. Rev. 155:29.[Medline]
  14. Blery, M., L. Olcese, E. Vivier. 2000. Early signaling via inhibitory and activating NK receptors. Hum. Immunol. 61:51.[Medline]
  15. Watzl, C., C. C. Stebbins, E. O. Long. 2000. NK cell inhibitory receptors prevent tyrosine phosphorylation of the activation receptor 2B4 (CD244). J. Immunol. 165:3545.[Abstract/Free Full Text]
  16. Moretta, L., R. Biassoni, C. Bottino, M. C. Mingari, A. Moretta. 2000. Human NK-cell receptors. Immunol. Today 21:420.[Medline]
  17. Colonna, M., H. Nakajima, M. Cella. 1999. Inhibitory and activating receptors involved in immune surveillance by human NK and myeloid cells. J. Leukocyte Biol. 66:718.[Abstract]
  18. Mathew, P. A., B. A. Garni-Wagner, K. Land, A. Takashima, E. Stoneman, M. Bennett, V. Kumar. 1993. Cloning and characterization of the 2B4 gene encoding a molecule associated with non-MHC-restricted killing mediated by activated natural killer cells and T cells. J. Immunol. 151:5328.[Abstract]
  19. Garni-Wagner, B. A., A. Purohit, P. A. Mathew, M. Bennett, V. Kumar. 1993. A novel function-associated molecule related to non-MHC-restricted cytotoxicity mediated by activated natural killer cells and T cells. J. Immunol. 151:60.[Abstract]
  20. Schuhmachers, G., K. Ariizumi, P. A. Mathew, M. Bennett, V. Kumar, A. Takashima. 1995. Activation of murine epidermal gamma delta T cells through surface 2B4. Eur. J. Immunol. 25:1117.[Medline]
  21. Schuhmachers, G., K. Ariizumi, P. A. Mathew, M. Bennett, V. Kumar, A. Takashima. 1995. 2B4, a new member of the immunoglobulin gene superfamily, is expressed on murine dendritic epidermal T cells and plays a functional role in their killing of skin tumors. J. Invest. Dermatol. 105:592.[Medline]
  22. Boles, K. S., H. Nakajima, M. Colonna, S. S. Chuang, S. E. Stepp, M. Bennett, V. Kumar, P. A. Mathew. 1999. Molecular characterization of a novel human natural killer cell receptor homologous to mouse 2B4. Tissue Antigens 54:27.[Medline]
  23. Nakajima, H., M. Cella, H. Langen, A. Friedlein, M. Colonna. 1999. Activating interactions in human NK cell recognition: the role of 2B4-CD48. Eur. J. Immunol. 29:1676.[Medline]
  24. Boles, K. S., P. A. Mathew. 2001. Molecular cloning of CS1, a novel human natural killer cell receptor belonging to the CD2 subset of the immunoglobulin superfamily. Immunogenetics 52:302.[Medline]
  25. Tangye, S. G., S. Lazetic, E. Woollatt, G. R. Sutherland, L. L. Lanier, J. H. Phillips. 1999. Cutting edge: human 2B4, an activating NK cell receptor, recruits the protein tyrosine phosphatase SHP-2 and the adaptor signaling protein SAP. J. Immunol. 162:6981.[Abstract/Free Full Text]
  26. Benoit, L., X. Wang, H. F. Pabst, J. Dutz, R. Tan. 2000. Defective NK cell activation in X-linked lymphoproliferative disease. J. Immunol. 165:3549.[Abstract/Free Full Text]
  27. Sayos, J., K. B. Nguyen, C. Wu, S. E. Stepp, D. Howie, J. D. Schatzle, V. Kumar, C. A. Biron, C. Terhorst. 2000. Potential pathways for regulation of NK and T cell responses: differential X-linked lymphoproliferative syndrome gene product SAP interactions with SLAM and 2B4. Int. Immunol. 12:1749.[Abstract/Free Full Text]
  28. Nakajima, H., M. Colonna. 2000. 2B4: an NK cell activating receptor with unique specificity and signal transduction mechanism. Hum. Immunol. 61:39.[Medline]
  29. Tangye, S. G., J. H. Phillips, L. L. Lanier, K. E. Nichols. 2000. Functional requirement for SAP in 2B4-mediated activation of human natural killer cells as revealed by the X-linked lymphoproliferative syndrome. J. Immunol. 165:2932.[Abstract/Free Full Text]
  30. Nakajima, H., M. Cella, A. Bouchon, H. L. Grierson, J. Lewis, C. S. Duckett, J. I. Cohen, M. Colonna. 2000. Patients with X-linked lymphoproliferative disease have a defect in 2B4 receptor-mediated NK cell cytotoxicity. Eur. J. Immunol. 30:3309.[Medline]
  31. Parolini, S., C. Bottino, M. Falco, R. Augugliaro, S. Giliani, R. Franceschini, H. D. Ochs, H. Wolf, J. Y. Bonnefoy, R. Biassoni, et al 2000. X-linked lymphoproliferative disease: 2B4 molecules displaying inhibitory rather than activating function are responsible for the inability of natural killer cells to kill epstein-barr virus-infected cells. J. Exp. Med. 192:337.[Abstract/Free Full Text]
  32. Valiante, N. M., G. Trinchieri. 1993. Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes. J. Exp. Med. 178:1397.[Abstract/Free Full Text]
  33. Latchman, Y., P. F. McKay, H. Reiser. 1998. Identification of the 2B4 molecule as a counter-receptor for CD48. J. Immunol. 161:5809.[Abstract/Free Full Text]
  34. Brown, M. H., K. Boles, P. A. van der Merwe, V. Kumar, P. A. Mathew, A. N. Barclay. 1998. 2B4, the natural killer and T cell immunoglobulin superfamily surface protein, is a ligand for CD48. J. Exp. Med. 188:2083.[Abstract/Free Full Text]
  35. Sivori, S., S. Parolini, M. Falco, E. Marcenaro, R. Biassoni, C. Bottino, L. Moretta, A. Moretta. 2000. 2B4 functions as a co-receptor in human NK cell activation. Eur. J. Immunol. 30:787.[Medline]
  36. Foletta, V. C., D. H. Segal, D. R. Cohen. 1998. Transcriptional regulation in the immune system: all roads lead to AP-1. J. Leukocyte Biol. 63:139.[Abstract]
  37. Wisdom, R.. 1999. AP-1: one switch for many signals. Exp. Cell Res. 253:180.[Medline]
  38. Schreiber, E., P. Matthias, M. M. Muller, W. Schaffner. 1989. Rapid detection of octamer binding proteins with "mini-extracts," prepared from a small number of cells. Nucleic Acids Res. 17:6419.[Free Full Text]
  39. Chuang, S. S., D. Banerjee, H. K. Das. 1999. Purified apolipoprotein B gene regulatory factor-3 is DNA topoisomerase I. Eur J Biochem 263:773.[Medline]
  40. Chuang, S. S., H. K. Pham, P. R. Kumaresan, P. A. Mathew. 2001. A prominent role for activator protein-1 in the transcription of the human 2B4 (CD244) gene in natural killer cells. J. Immunol. 166:6188.[Abstract/Free Full Text]
  41. Chuang, S. S., Y. Lee, S. E. Stepp, P. R. Kumaresan, P. A. Mathew. 1999. Molecular cloning and characterization of the promoter region of murine natural killer cell receptor 2B4. Biochim. Biophys. Acta 1447:244.[Medline]
  42. Bernard, K., A. Cambiaggi, S. Guia, F. Bertucci, S. Granjeaud, R. Tagett, C. N'Guyen, B. R. Jordan, E. Vivier. 1999. Engagement of natural cytotoxicity programs regulates AP-1 expression in the NKL human NK cell line. J. Immunol. 162:4062.[Abstract/Free Full Text]
  43. English, J., G. Pearson, J. Wilsbacher, J. Swantek, M. Karandikar, S. Xu, M. H. Cobb. 1999. New insights into the control of MAP kinase pathways. Exp. Cell Res. 253:255.[Medline]
  44. Yu, T. K., E. G. Caudell, C. Smid, E. A. Grimm. 2000. IL-2 activation of NK cells: involvement of MKK1/2/ERK but not p38 kinase pathway. J. Immunol. 164:6244.[Abstract/Free Full Text]
  45. Wasylyk, B., J. Hagman, A. Gutierrez-Hartmann. 1998. Ets transcription factors: nuclear effectors of the Ras-MAP-kinase signaling pathway. Trends Biochem. Sci. 23:213.[Medline]
  46. Su, B., M. Karin. 1996. Mitogen-activated protein kinase cascades and regulation of gene expression. Curr. Opin. Immunol. 8:402.[Medline]
  47. Schulze-Osthoff, K., D. Ferrari, K. Riehemann, S. Wesselborg. 1997. Regulation of NF-{kappa}B activation by MAP kinase cascades. Immunobiology 198:35.[Medline]
  48. Wei, S., D. L. Gilvary, B. C. Corliss, S. Sebti, J. Sun, D. B. Straus, P. J. Leibson, J. A. Trapani, A. D. Hamilton, M. J. Weber, J. Y. Djeu. 2000. Direct tumor lysis by NK cells uses a Ras-independent mitogen-activated protein kinase signal pathway. J. Immunol. 165:3811.[Abstract/Free Full Text]
  49. Lerner, E. C., Y. Qian, M. A. Blaskovich, R. D. Fossum, A. Vogt, J. Sun, A. D. Cox, C. J. Der, A. D. Hamilton, S. M. Sebti. 1995. Ras CAAX peptidomimetic FTI-277 selectively blocks oncogenic Ras signaling by inducing cytoplasmic accumulation of inactive Ras-Raf complexes. J. Biol. Chem. 270:26802.[Abstract/Free Full Text]
  50. Klem, J., M. Bennett, V. Kumar, and J. Schatzle. 2000. the NK receptor, 2B4, is constitutively localized in glycolipid-enriched microdomains and associates with LAT. FASEB J. 14:90.36 (Abstr.).
  51. Trotta, R., K. A. Puorro, M. Paroli, L. Azzoni, B. Abebe, L. C. Eisenlohr, B. Perussia. 1998. Dependence of both spontaneous and antibody-dependent, granule exocytosis-mediated NK cell cytotoxicity on extracellular signal- regulated kinases. J. Immunol. 161:6648.[Abstract/Free Full Text]
  52. Trotta, R., K. Fettucciari, L. Azzoni, B. Abebe, K. A. Puorro, L. C. Eisenlohr, B. Perussia. 2000. Differential role of p38 and c-Jun N-terminal kinase 1 mitogen- activated protein kinases in NK cell cytotoxicity. J. Immunol. 165:1782.[Abstract/Free Full Text]
  53. Mainiero, F., A. Gismondi, A. Soriani, M. Cippitelli, G. Palmieri, J. Jacobelli, M. Piccoli, L. Frati, A. Santoni. 1998. Integrin-mediated ras-extracellular regulated kinase (ERK) signaling regulates interferon gamma production in human natural killer cells. J. Exp. Med. 188:1267.[Abstract/Free Full Text]
  54. Bottino, C., R. Augugliaro, R. Castriconi, M. Nanni, R. Biassoni, L. Moretta, A. Moretta. 2000. Analysis of the molecular mechanism involved in 2B4-mediated NK cell activation: evidence that human 2B4 is physically and functionally associated with the linker for activation of T cells. Eur. J. Immunol. 30:3718.[Medline]
  55. Ting, A. T., L. M. Karnitz, R. A. Schoon, R. T. Abraham, P. J. Leibson. 1992. Fc{gamma} receptor activation induces the tyrosine phosphorylation of both phospholipase C (PLC)-{gamma}1 and PLC-{gamma}2 in natural killer cells. J. Exp. Med. 176:1751.[Abstract/Free Full Text]
  56. Jevremovic, D., D. D. Billadeau, R. A. Schoon, C. J. Dick, B. J. Irvin, W. Zhang, L. E. Samelson, R. T. Abraham, P. J. Leibson. 1999. Cutting edge: a role for the adaptor protein LAT in human NK cell- mediated cytotoxicity. J. Immunol. 162:2453.[Abstract/Free Full Text]
  57. Zhang, W., R. P. Trible, M. Zhu, S. K. Liu, C. J. McGlade, L. E. Samelson. 2000. Association of Grb2, Gads, and phospholipase C-{gamma}1 with phosphorylated LAT tyrosine residues. Effect of LAT tyrosine mutations on T cell angigen receptor-mediated signaling. J. Biol. Chem. 275:23355.[Abstract/Free Full Text]
  58. Tomlinson, M. G., J. Lin, A. Weiss. 2000. Lymphocytes with a complex: adapter proteins in antigen receptor signaling. Immunol. Today 21:584.[Medline]
  59. Zhang, W., L. E. Samelson. 2000. The role of membrane-associated adaptors in T cell receptor signalling. Semin. Immunol. 12:35.[Medline]
  60. Schraven, B., A. Marie-Cardine, C. Hubener, E. Bruyns, I. Ding. 1999. Integration of receptor-mediated signals in T cells by transmembrane adaptor proteins. Immunol. Today 20:431.[Medline]
  61. Finco, T. S., T. Kadlecek, W. Zhang, L. E. Samelson, A. Weiss. 1998. LAT is required for TCR-mediated activation of PLC{gamma}1 and the Ras pathway. Immunity 9:617.[Medline]
  62. Zhang, W., B. J. Irvin, R. P. Trible, R. T. Abraham, L. E. Samelson. 1999. Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient Jurkat cell line. Int. Immunol. 11:943.[Abstract/Free Full Text]
  63. Zhang, W., C. L. Sommers, D. N. Burshtyn, C. C. Stebbins, J. B. DeJarnette, R. P. Trible, A. Grinberg, H. C. Tsay, H. M. Jacobs, C. M. Kessler, et al 1999. Essential role of LAT in T cell development. Immunity 10:323.[Medline]
  64. Galandrini, R., G. Palmieri, M. Piccoli, L. Frati, A. Santoni. 1996. CD16-mediated p21ras activation is associated with Shc and p36 tyrosine phosphorylation and their binding with Grb2 in human natural killer cells. J. Exp. Med. 183:179.[Abstract/Free Full Text]
  65. Bonnema, J. D., L. M. Karnitz, R. A. Schoon, R. T. Abraham, P. J. Leibson. 1994. Fc receptor stimulation of phosphatidylinositol 3-kinase in natural killer cells is associated with protein kinase C-independent granule release and cell-mediated cytotoxicity. J. Exp. Med. 180:1427.[Abstract/Free Full Text]
  66. Brumbaugh, K. M., B. A. Binstadt, D. D. Billadeau, R. A. Schoon, C. J. Dick, R. M. Ten, P. J. Leibson. 1997. Functional role for Syk tyrosine kinase in natural killer cell-mediated natural cytotoxicity. J. Exp. Med. 186:1965.[Abstract/Free Full Text]
  67. Gismondi, A., J. Jacobelli, F. Mainiero, R. Paolini, M. Piccoli, L. Frati, A. Santoni. 2000. Cutting edge: functional role for proline-rich tyrosine kinase 2 in NK cell-mediated natural cytotoxicity. J. Immunol. 164:2272.[Abstract/Free Full Text]
  68. Sancho, D., M. Nieto, M. Llano, J. L. Rodriguez-Fernandez, R. Tejedor, S. Avraham, C. Cabanas, M. Lopez-Botet, F. Sanchez-Madrid. 2000. The tyrosine kinase PYK-2/RAFTK regulates natural killer (NK) cell cytotoxic response, and is translocated and activated upon specific target cell recognition and killing. J. Cell Biol. 149:1249.[Abstract/Free Full Text]
  69. Nieto, M., J. L. Rodriguez-Fernandez, F. Navarro, D. Sancho, J. M. Frade, M. Mellado, A. C. Martinez, C. Cabanas, F. Sanchez-Madrid. 1999. Signaling through CD43 induces natural killer cell activation, chemokine release, and PYK-2 activation. Blood 94:2767.[Abstract/Free Full Text]
  70. Gismondi, A., L. Bisogno, F. Mainiero, G. Palmieri, M. Piccoli, L. Frati, A. Santoni. 1997. Proline-rich tyrosine kinase-2 activation by beta 1 integrin fibronectin receptor cross-linking and association with paxillin in human natural killer cells. J. Immunol. 159:4729.[Abstract]
  71. Blaukat, A., I. Ivankovic-Dikic, E. Gronroos, F. Dolfi, G. Tokiwa, K. Vuori, I. Dikic. 1999. Adaptor proteins Grb2 and Crk couple Pyk2 with activation of specific mitogen-activated protein kinase cascades. J. Biol. Chem. 274:14893.[Abstract/Free Full Text]
  72. Dikic, I., G. Tokiwa, S. Lev, S. A. Courtneidge, J. Schlessinger. 1996. A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation. Nature 383:547.[Medline]
  73. Pandey, P., S. Avraham, S. Kumar, A. Nakazawa, A. Place, L. Ghanem, A. Rana, V. Kumar, P. K. Majumder, H. Avraham, R. J. Davis, S. Kharbanda. 1999. Activation of p38 mitogen-activated protein kinase by PYK2/related adhesion focal tyrosine kinase-dependent mechanism. J. Biol. Chem. 274:10140.[Abstract/Free Full Text]
  74. Kumar, S., S. Avraham, A. Bharti, J. Goyal, P. Pandey, S. Kharbanda. 1999. Negative regulation of PYK2/related adhesion focal tyrosine kinase signal transduction by hematopoietic tyrosine phosphatase SHPTP1. J. Biol. Chem. 274:30657.[Abstract/Free Full Text]
  75. Binstadt, B. A., D. D. Billadeau, D. Jevremovic, B. L. Williams, N. Fang, T. Yi, G. A. Koretzky, R. T. Abraham, P. J. Leibson. 1998. SLP-76 is a direct substrate of SHP-1 recruited to killer cell inhibitory receptors. J. Biol. Chem. 273:27518.[Abstract/Free Full Text]
  76. Valiante, N. M., J. H. Phillips, L. L. Lanier, P. Parham. 1996. Killer cell inhibitory receptor recognition of human leukocyte antigen (HLA) class I blocks formation of a pp36/PLC-{gamma} signaling complex in human natural killer (NK) cells. J. Exp. Med. 184:2243.[Abstract/Free Full Text]
  77. Schatzle, J. D., S. Sheu, S. E. Stepp, P. A. Mathew, M. Bennett, V. Kumar. 1999. Characterization of inhibitory and stimulatory forms of the murine natural killer cell receptor 2B4. Proc. Natl. Acad. Sci. USA 96:3870.[Abstract/Free Full Text]
  78. Johnson, L. A., R. H. Goldfarb, P. A. Mathew. 2000. Regulation of IFN-{gamma} production following 2B4 activation in human NK cells. In Vivo 14:625.[Medline]
  79. Rajagopalan, S., J. Fu, E. O. Long. 2001. Cutting Edge: Induction of IFN-{gamma} production, but not cytotoxicity, by the killer cell Ig-like receptor KIR2DL4 (CD 158d) in resting NK cells. J. Immunol. 167:1877.[Abstract/Free Full Text]
  80. Boles, K. S., R. Barten, P. R. Kumaresan, J. Trowsdale, P. A. Mathew. 1999. Cloning of a new lectin-like receptor expressed on human NK cells. Immunogenetics 50:1.[Medline]



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S. Malarkannan, J. Regunathan, H. Chu, S. Kutlesa, Y. Chen, H. Zeng, R. Wen, and D. Wang
Bcl10 Plays a Divergent Role in NK Cell-Mediated Cytotoxicity and Cytokine Generation
J. Immunol., September 15, 2007; 179(6): 3752 - 3762.
[Abstract] [Full Text] [PDF]

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J. C. D. Wiseman, L. L. Ma, K. J. Marr, G. J. Jones, and C. H. Mody
Perforin-Dependent Cryptococcal Microbicidal Activity in NK Cells Requires PI3K-Dependent ERK1/2 Signaling
J. Immunol., May 15, 2007; 178(10): 6456 - 6464.
[Abstract] [Full Text] [PDF]

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P. Eissmann and C. Watzl
Molecular Analysis of NTB-A Signaling: A Role for EAT-2 in NTB-A-Mediated Activation of Human NK Cells.
J. Immunol., September 1, 2006; 177(5): 3170 - 3177.
[Abstract] [Full Text] [PDF]

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H. Sasanuma, A. Tatsuno, S. Hidano, K. Ohshima, Y. Matsuzaki, K. Hayashi, C. A. Lowell, D. Kitamura, and R. Goitsuka
Dual function for the adaptor MIST in IFN-{gamma} production by NK and CD4+NKT cells regulated by the Src kinase Fgr
Blood, May 1, 2006; 107(9): 3647 - 3655.
[Abstract] [Full Text] [PDF]