Ly-49D Signal Transduction 16-Kda Tyrosine Phosphoprotein Required

Cutting Edge: Characterization of an Associated 16-kDa Tyrosine Phosphoprotein Required for Ly-49D Signal Transduction

This information is current as Llewellyn H. Mason, Jami Willette-Brown, Stephen K. Anderson, of October 1, 2021. Pierre Gosselin, Elizabeth W. Shores, Paul E. Love, John R. Ortaldo and Daniel W. McVicar J Immunol 1998; 160:4148-4152; ; http://www.jimmunol.org/content/160/9/4148 Downloaded from

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Cutting Edge: Characterization of an Associated 16-kDa Tyrosine Phosphoprotein Required for Ly-49D Signal Transduction1

Llewellyn H. Mason,2* Jami Willette-Brown,* Stephen K. Anderson,† Pierre Gosselin,* Elizabeth W. Shores,‡ Paul E. Love,§ John R. Ortaldo,* and Daniel W. McVicar* Downloaded from

ITIMs3 in their cytoplasmic domains that are phosphorylated on Ly-49D is an activating receptor on NK cells that does not become stimulation, leading to the recruitment of SHP-1 phosphatase and tyrosine phosphorylated upon activation. This report demon- attenuation of intracellular signals (4, 5). A similar system exists in strates that immunoprecipitation of Ly-49D, following pervana- human NK cells, where signaling by the p58 receptor family also date treatment or speciﬁc Ab cross-linking, coprecipitates a 16-

uses SHP-1 phosphatase to inhibit NK cell activation (6). How- http://www.jimmunol.org/ kDa tyrosine-phosphorylated protein (pp16). Immunoblotting ever, receptors exist in both systems that can activate NK cells. In experiments and data from TCR-␨/Fc⑀RI␥ double knockout mice the human, p50 receptors have truncated cytoplasmic domains that confirm that pp16 is not TCR-␨, TCR-␩,orFc⑀RI␥. Association lack ITIMs (7) and have been shown to mediate reverse Ab-de- of pp16 with Ly-49D involves a transmembrane arginine since 2ϩ pendent cellular cytotoxicity and mobilize intracellular Ca in the R54L) abolishes association with pp16 mutation to leucine (Ly-49D presence of specific mAb (8). Olcese et al. have recently demon- in transfected P815 cells. In addition, Ly-49DR54L transfectants strated that p50 receptors are associated with a multimeric com- .fail to mediate Ca2؉ mobilization following Ab cross-linking plex of proteins that when appropriately stimulated become phos- Therefore, signaling through Ly-49D on NK cells depends on as- phorylated (killer cell-activatory receptor-associated polypeptides) sociation with a distinct tyrosine phosphoprotein (pp16) in a man- by guest on October 1, 2021 (9). These molecules exist as a 12–16 kDa group of polypeptides ner analogous to that of TCR and FcR. Expression of this novel that can be detected upon internal radiolabeling of NK cells and signaling peptide in both the NK and myeloid lineages indicates immunoprecipitation. Since the human killer cell-activatory recep- that pp16 is likely involved in the signal transduction cascade of tors contain a lysine in their transmembrane domain, they specu- additional receptor families. The Journal of Immunology, 1998, lated that this amino acid is important for the association of killer 160: 4148–4152. cell-activatory receptor with killer cell-activatory receptor-associated polypeptides (9). urine NK cells express Ly-49 receptors that can initi- Our laboratory has shown that murine Ly-49D, in the presence ate either inhibitory or activating signals to regulate of mAb 12A8, activates NK cells by inducing reverse Ab-depen- ϩ M lytic function. Ly-49A, Ly-49C, and Ly-49G2 have dent cellular cytotoxicity of FcR targets (10). Ly-49D contains a been shown to inhibit NK cell function on recognition of class I cytoplasmic domain that is comparable in length with other Ly-49 ligands on target cells (1–3). These inhibitory receptors contain receptors. We have recently demonstrated that Ly-49D is not phosphorylated following pervanadate stimulation, consistent with its *Laboratory of Experimental Immunology, Division of Basic Sciences, and †Intra- lack of an ITIM. However, stimulation of NK cells with pervana- mural Research Support Program, SAIC Frederick, National Cancer Institute-Fred- date and immunoprecipitation of Ly-49D did reveal associated ty- erick Cancer Reseach and Development Center, Frederick, MD 21702; ‡Division of Hematologic Products, Center for Biologics Evaluation and Research, Food and Drug rosine-phosphorylated proteins (pp16). These associated phospho- Administration, Washington, DC; and §Laboratory of Mammalian Genes and Devel- proteins exist as disulfide-linked dimers that are highly opment, National Institute of Child Health and Human Development, National Insti- phosphorylated following either pervanadate stimulation of NK tutes of Health, Bethesda, MD 20892 cells or specific receptor cross-linking. In this report, we charac- Received for publication January 15, 1998. Accepted for publication February 23, 1998. terize these phosphoproteins and provide data to prove that the The costs of publication of this article were defrayed in part by the payment of page arginine in the transmembrane domain of Ly-49D is critical for charges. This article must therefore be hereby marked advertisement in accordance pp16 association and subsequent signaling by this receptor. with 18 U.S.C. Section 1734 solely to indicate this fact. 1 The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. 2 Address correspondence and reprint requests to Dr. L. H. Mason, NCI-FCRDC 3 Abbreviations used in this paper: ITIM, immunoreceptor tyrosine-based inhibitory Building 560, Room 31-93, Frederick, MD 21702-1201. E-mail address: motif; pp16, 16-kDa tyrosine-phosphorylated protein; SA-HRP, streptavidin-horse- [email protected] radish peroxidase.

● The Journal of Immunology 4149

FIGURE 1. Immunoprecipitation and anti- phosphotyrosine blotting of Ly-49D receptor- associated phosphoproteins. A, IL-2-cultured C57BL/6 (B6) NK cells were surface biotinylated, lysed, and immunoprecipitated, and proteins were separated on 8 to 16% SDS-PAGE under nonreducing conditions. Proteins were transferred to Immobilon and blotted with SA- HRP. B, NK cells were treated with pervanadate (1 mM), lysed in Triton X-100, immunoprecipitated, separated on SDS-PAGE as above, and immunoblotted with biotinylated 4G10 followed by SA-HRP. C, NK cells treated as in B were separated on 16% SDS-PAGE under reducing conditions. Downloaded from

Materials and Methods lecular masses of 28 to 32 kDa were readily observed. Since mAb NK cell isolation 12A8 reacts with both Ly-49A and Ly-49D, we repeated these

experiments with a new mAb (4E5) that reacts with Ly-49D, but http://www.jimmunol.org/ Splenic NK cells were isolated from C57BL/6 (B6) mice4 and grown for 7 to 10 days in 1000 U/ml Cetus recombinant IL-2 as previously not Ly-49A, Ly-49B, Ly-49C/I, Ly-49H, or Ly-49G2 (data not described (11). shown). Figure 1A shows a representative experiment in which pervanadate-treated B6 NK cells were surface biotinylated, immu- Antibodies noprecipitated, separated on SDS-PAGE without reduction, and The following mAb were used: 4D11 (Ly-49G2 (3)); 12A8 (Ly-49A/D blotted with SA-HRP. Immunoprecipitation with mAb 4D11 (Ly- (10)); 4E5 (Ly-49D; manuscript in preparation); and RM2-1 (CD2 (12)). 49G2) and mAb 12A8 (Ly-49A&D) resulted in 90- to 100-kDa Antisera to Fc⑀RI␥ and TCR-␨ were kindly provided by Dr. J. P. Kinet and Dr. A. Weissman, respectively. proteins, consistent with the reported molecular masses for these receptors. Immunoprecipitation with mAb 4E5 yields a band of 95

Stimulation, immunoprecipitation, electrophoresis, and blotting to 100 kDa, consistent with that reported for Ly-49D. Furthermore, by guest on October 1, 2021 Cell stimulation, immunoprecipitation, and immunoblotting were per- transfection of Cos-7 cells with Ly-49D cDNA, and immunopre- formed as previously described (5). Pervanadate stimulation of cells uti- cipitation with both mAb12A8 and 4E5 detected proteins of sim- lized 1 mM pervanadate for 15 min at 37°C. ilar size (data not shown). These data confirmed the ability of both Site-directed mutagenesis and transfection mAb 12A8 and 4E5 to immunoprecipitate Ly-49D. In Figure 1B, a parallel blot with anti-phosphotyrosine blotting of immunopre- A substitution mutant was generated within the Ly-49D transmembrane domain in which the arginine at position 54 was mutated to a leucine cipitates from pervanadate-treated NK cells demonstrates that both (Ly-49DR54L). Mutation of the Ly-49D cDNA was performed with the mAb 12A8 and 4E5 coimmunoprecipitated Ly-49D-associated Transformer Site-Directed Mutagenesis Kit (Clontech, Palo Alto, CA) ac- phosphoproteins of ϳ28 to 32 kDa under nonreducing conditions. cording to the manufacturer’s instructions. The mutant construct was con- ControlimmunoprecipitationsincludedmAb4D11thatdetectsphos- firmed by sequencing. P815 cells were electroporated as previously phorylated Ly-49G2 and mAb 12A8 that detects phosphorylated described (5). Ly-49A with which it cross-reacts. Ly-49D, which is not phos- Calcium flux phorylated, is not seen after immunoprecipitation with mAb 4E5 2ϩ 2ϩ Analysis of the changes in intracellular Ca concentration ([Ca ]i) was and anti-phosphotyrosine blotting. Figure 1C depicts the Ly-49D- conducted using a DeltaScan fluorometer (Photon Technologies, Princeton, associated phosphoproteins under reducing conditions at an appar- NJ) and the calcium-sensitive fluorochrome Indo-1 as previously described ent molecular mass of ϳ16 kDa. Therefore, immunoprecipitation (13). The cells were stimulated with primary mAb followed 25 s later by of Ly-49D, with either of two mAb reacting with different receptor rabbit anti-rat Ab. Data are expressed as the ratio of bound dye to free dye, 2ϩ epitopes, detects a strongly associated tyrosine phosphoprotein that a direct indication of [Ca ]i. exists as a ϳ32-kDa dimer in its fully phosphorylated form. Results and Discussion To compare the kinetics between pervanadate- and receptor- Previous work from our laboratory has demonstrated that phos- mediated induction of pp16 phosphorylation, a time course exper- phorylation of the tyrosine within the ITIMs of Ly-49A, Ly-49C, iment was performed. After stimulation for various lengths of and Ly-49G2 occurs following pervanadate stimulation of NK time, aliquots of cells were lysed, immunoprecipitated, separated cells (5). Ly-49D, however, does not contain any tyrosines and is on SDS-PAGE under nonreducing conditions, and blotted with therefore not phosphorylated. Although anti-phosphotyrosine blot- anti-phosphotyrosine. Figure 2A shows a time course experiment ting of mAb 12A8 immunoprecipitates of NK cells did not reveal using pervanadate to stimulate B6 NK cells. These experiments phosphorylation of Ly-49D, associated phosphoproteins with mo- suggest that at least four phosphorylated species of pp16 are observed within 2 min after pervanadate stimulation. From 4 to 8 min after stimulation, the maximally phosphorylated form is predom- 4 Animal care was provided in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication inant. More importantly, phosphorylation of pp16 can be induced No. 86-23, 1985). through cross-linking of Ly-49D as seen in Figure 2B. In these 4150 CUTTING EDGE

FIGURE 2. Kinetics of pp16 phosphorylation by pervanadate stimulation and Ly-49D receptor cross-linking. A, IL-2-cultured B6 NK cells (3 ϫ 106/lane) were stimulated with pervanadate for the indicated time periods followed by lysis, immunoprecipitation with mAb 4E5/protein G-agarose, and treated as in Figure 1B for blotting with anti-phosphotyrosine. B,5ϫ 106 NK cells in 100 ␮l of HBSS were placed on ice for 5 min, after which 5 ␮g of mAb 4E5 were added and allowed to react for 5 min. A secondary rabbit anti-rat antiserum (RART) (10 ␮g) was added to each tube, and the tubes were placed in a 37°C water bath for the indicated times. The NK cells were immedi- ately lysed by the addition of 1 ml of ice-cold lysis buffer for 30 min. Lysates were cleared, immunoprecipitated by the addition of mAb 4E5/protein G-agarose, and processed as above. Downloaded from experiments, phosphorylation of the dimeric form of pp16 oc- nized by antisera to TCR-␨ or Fc⑀RI␥. These results suggest that curs, within 30 s, and is maximal by 2 min after cross-linking. pp16 is a novel phosphoprotein that may function as a signal trans- Phosphorylation of pp16 is not mediated by the Ly-49D-speciﬁc duction molecule in murine NK cells.

mAb 4E5 alone or by rabbit anti-rat antisera. Only cross-linking To confirm the immunologic data indicating that pp16 was nei- http://www.jimmunol.org/ by a secondary anti-rat Ab mediates efficient pp16 phosphory- ther TCR-␨ nor Fc⑀RI␥, spleens were obtained from mice that lation. possessed null mutations of both the TCR-␨ and Fc⑀RI␥ loci (dou- Phosphoproteins in the range of 14 to 28 kDa are reminiscent of ble knockouts). Splenic adherent lymphokine-activated killer cells proteins involved in TCR and FcR signaling. Therefore, immuno- were prepared and after 7 days of culture in IL-2, were analyzed precipitation experiments were performed on B6 NK cells to de- for expression of Ly-49s by flow cytometry analysis. IL-2-cultured termine whether pp16 might be TCR-␨ or Fc⑀RI␥, both of which NK cells from the double knockout mice appeared to express a are found in NK cells (13). Figure 3A depicts such an experiment normal Ly-49 receptor phenotype (data not shown). We next per- in which pervanadate-treated NK cells were lysed, immunopre- formed immunoprecipitation experiments and anti-phospho- cipitated with antisera to TCR-␨ or Fc⑀RI␥, and blotted with anti- tyrosine immunoblotting to determine whether pp16 was associ- by guest on October 1, 2021 phosphotyrosine. This experiment suggests that pp16 is not the ated with Ly-49D in the mutant mice. Figure 3B demonstrates that same as the phosphorylated forms of TCR-␨ or Fc⑀RI␥. While pp16 is definitely present in these double knockout mice, because immunoprecipitation with antisera to Fc⑀RI␥ yielded 2 phosphor- both mAb 12A8 and 4E5 immunoprecipitate pp16. These results ylated bands of ϳ14 and ϳ18 kDa, antisera to TCR-␨ yielded a clearly illustrate that pp16 is not a product of the TCR-␨ or Fc⑀RI␥ single phosphorylated protein of ϳ21 kDa, consistent with the loci and may therefore represent a novel signaling molecule. reported sizes of these phosphorylated receptors. However, both Comparison of the structure of Ly-49D with other Ly-49 family mAb 12A8 and 4E5 immunoprecipitated a 16-kDa phosphoprotein members revealed two significant differences: the lack of an ap- that migrated to a different position than phosphoproteins recog- propriate ITIM; and an arginine residue within the transmembrane

FIGURE 3. pp16 is not TCR-␨ or Fc⑀RI␥. A,9ϫ 106 B6 NK cells were stimulated with pervanadate, lysed, immunoprecipitated as indicated, and blotted with anti-phosphotyrosine after separation on 16% SDS-PAGE under reducing conditions. Control immuno- precipitations are RM2–1 (CD-2/lane 1), 4D11 (Ly-49G2/lane 2), anti- Fc⑀RI␥ Ab alone (no lysate/lane 3), and anti-TCR-␨ Ab alone (no lysate/lane 8). B,4ϫ 106 B6 NK cells from TCR-␨ and Fc⑀RI␥ double knockout (K.O.) mice were treated with pervanadate lysed and blotted for tyrosine- phosphor- ylated proteins after separation on 8 to 16% SDS-PAGE under nonreducing conditions. Controls consisted of RM2–1 (CD-2, lane 1) and 4D11 (Ly-49G2, lane 2). The Journal of Immunology 4151

FIGURE 4. A, Mutation of the Ly-49D transmembrane arginine abolishes pp16 association. The Ly-49D transmembrane region was mutated by a single substi- Downloaded from tution at amino acid 54 from an arginine to a leucine (Ly-49DR54L) using site-directed mutagenesis. Both wild-type (WT) Ly-49D and Ly-49DR54L were stably transfected into P815 cells by electroporation and se- lection in G418. Cells were stimulated with pervanadate for 15 min, lysates were immunoprecipitated with the designated mAb, and proteins were separated by http://www.jimmunol.org/ electrophoresis under reducing conditions and immunoblotted with anti-phosphotyrosine as described previously. B, Mutation of the arginine residue of the Ly-49D transmembrane domain ablates calcium mobilization in P815. Parental P815 or P815 stably expressing wild-type Ly-49D or Ly-49DR54L were stimulated with either anti-Ly-49D (4E5) or anti-Ly-49G mAb (4D11) as indicated (arrow 1). Primary Ab was cross-

linked with rabbit anti-rat Ab after 25 s (arrow 2). by guest on October 1, 2021 2ϩ [Ca ]i was monitored over time and is presented as the ratio of dye bound to Ca2ϩ (emission at 402 nm) over dye free of Ca2ϩ (emission at 486 nm) in arbitrary units.

domain. We have demonstrated previously that tyrosine phos- and generated stable transfectants of the mutant and wild-type Ly- phorylation of Ly-49D does not occur following pervanadate 49D in the P815 mastocytoma cell line, which was previously stimulation, most likely due to its lack of an ITIM. However, the found to express pp16 (data not shown). Figure 4A presents the presence of a charged residue in the Ly-49D transmembrane re- data obtained after pervanadate stimulation of transfected P815 gion suggested that this residue might mediate the association of cells followed by lysis, immunoprecipitation with mAb 12A8 or this receptor with pp16 in a manner analogous to the association of 4E5, and anti-phosphotyrosine blotting. The data demonstrate that TCR-␨ and Fc⑀RI␥ with their receptors. Therefore, we constructed although both forms of the receptor are expressed (data not a point mutation changing arginine-54 to leucine (Ly-49DR54L) shown), immunoprecipitation of Ly-49D coimmunoprecipitates 4152 CUTTING EDGE pp16 only in cells transfected with wild-type Ly-49D and not with References Ly-49DR54L. Moreover, Western blotting with a rabbit antiserum 1. Karlhofer, F. M., R. K. Ribaudo, and W. M. Yokoyama. 1992. MHC class I raised against pp16 detects pp16 in wild-type Ly-49D immuno- alloantigen specificity of Ly-49ϩ IL-2-activated natural killer cells. Nature 358: precipitates but not immunoprecipitates of Ly-49DR54L (data not 66. shown). These data demonstrate that physical association of pp16 2. Yu, Y. Y., T. George, J. R. Dorfman, J. Roland, V. Kumar, and M. Bennett. 1996. The role of Ly49A and 5E6 (Ly49C) molecules in hybrid resistance mediated by with Ly-49D is mediated by the arginine within the transmem- murine natural killer cells against normal T cell blasts. Immunity 4:67. brane domain of Ly-49D. 3. Mason, L. H., J. R. Ortaldo, H. A. Young, V. Kumar, M. Bennett, and Having established that Ly-49D is capable of physically inter- S. K. Anderson. 1995. Cloning and functional characteristics of murine large granular lymphocyte-1: a member of the Ly-49 gene family (Ly-49G2). J. Exp. acting with pp16 in P815, we asked whether this ectopically ex- Med. 182:293. pressed receptor was able to couple to the P815 signal transduction 4. Nakamura, M. C., E. C. Niemi, M. J. Fisher, L. D. Shultz, W. E. Seaman, and mechanism. Figure 4B demonstrates that stimulation of parental J. C. Ryan. 1997. Mouse Ly-49A interrupts early signaling events in natural killer cell cytotoxicity and functionally associates with the SHP-1 tyrosine phosphatase. P815 with cross-linked mAb 4E5 had no effect on the intracellular J. Exp. Med. 185:673. 2ϩ calcium concentration ([Ca ]i). In contrast, cross-linking mAb 5. Mason, L. H., P. Gosselin, S. K. Anderson, W. E. Fogler, J. R. Ortaldo, and 4E5 on P815 cells stably expressing wild-type Ly-49D showed a D. W. McVicar. 1997. Differential tyrosine phosphorylation of inhibitory vs. 2ϩ activating Ly-49 receptor proteins and their recruitment of SHP-1 phosphatase. substantial increase in [Ca ]i consistent with coupling of Ly-49D J. Immunol. 159:4187. to the P815 signaling apparatus. This response was specific in that 6. Burshtyn, D. N., A. M. Scharenberg, N. Wagtmann, S. Rajagopalan, K. Berrada, treatment of Ly-49D transfectants with mAb recognizing Ly-49G T. Yi, J. P. Kinet, and E. O. Long. 1996. Recruitment of tyrosine phosphatase (4D11) showed no increase in calcium levels. Next we used this HCP by the killer cell inhibitory receptor. Immunity 4:77. 7. Biassoni, R., C. Cantoni, M. Falco, S. Verdiani, C. Bottino, M. Vitale, R. Conte, model to test the importance of pp16 in Ly-49D signaling by cross- A. Poggi, A. Moretta, and L. Moretta. 1996. The human leukocyte antigen Downloaded from linking Ly-49DR54L, a receptor incapable of physically interacting (HLA)-C-specific “activatory” or “inhibitory” natural killer cell receptors display R54L highly homologous extracellular domains but differ in their transmembrane and with pp16. Cross-linking of Ly-49D in two independent intracytoplasmic portions. J. Exp. Med. 183:645. clones failed to induce calcium mobilization in P815, even though 8. Moretta, A., S. Sivori, M. Vitale, D. Pende, L. Morelli, R. Augugliaro, C. Bottino, these cells express higher levels of surface Ly-49D than their wild- and L. Moretta. 1995. Existence of both inhibitory (p58) and activatory (p50) receptors for HLA-C molecules in human natural killer cells. J. Exp. Med. 182: type counterparts (data not shown). 875.

Together our data suggest the absolute requirement for pp16 9. Olcese, L., A. Cambiaggi, G. Semenzato, C. Bottino, A. Moretta, and E. Vivier. http://www.jimmunol.org/ association in Ly-49D signal transduction. These ﬁndings suggest 1997. Human killer cell activatory receptors for MHC class I molecules are in- cluded in a multimeric complex expressed by natural killer cells. J. Immunol. that analogous to other multichain immune receptors such as the 158:5083. TCR, B cell receptor, and FcR, Ly-49D lacks intrinsic signaling 10. Mason, L. H., S. K. Anderson, H. R. Smith, W. M. Yokoyama, properties and instead forms multimeric complexes with low m.w., R. Winkler-Pickett, and J. R. Ortaldo. 1996. The Ly-49D receptor activates mu- tyrosine-phosphorylated polypeptides. Within other receptor sys- rine natural killer cells. J. Exp. Med. 184:2119. 11. Mason, L., S. L. Giardina, T. Hecht, J. Ortaldo, and B. J. Mathieson. 1988. tems, these low m.w. proteins serve as tyrosine kinase substrates LGL-1: a non-polymorphic antigen expressed on a major population of mouse that recruit additional src homology 2 (SH2)-containing kinases natural killer cells. J. Immunol. 140:4403. such as members of the Syk/Zap70 family (14, 15). Preliminary 12. Yagita, H., T. Nakamura, H. Karasuyama, and K. Okumura. 1989. Monoclonal antibodies speciﬁc for murine CD2 reveal its presence on B as well as T cells. data using anti-pp16 antisera directly demonstrates expression of Proc. Natl. Acad. Sci. USA 86:645. by guest on October 1, 2021 pp16 not only in NK cells but also in cells of the myeloid lineage. 13. O’Shea, J. J., D. W. McVicar, D. B. Kuhns, and J. R. Ortaldo. 1992. A role for This leads to the speculation that pp16 may serve as the Syk/Zap70 protein tyrosine kinase activity in natural cytotoxicity as well as antibody-depen- dent cellular cytotoxicity: effects of herbimycin A. J. Immunol. 148:2497. docking site within receptor complexes other than Ly-49D. Can- 14. Isakov, N., R. L. Wang, and L. E. Samelson. 1994. The role of tyrosine kinases didate receptors include the recently described positive signaling and phosphotyrosine-containing recognition motifs in regulation of the T cell- receptors of the ILT/MIR (16, 17) and PIRA families (18, 19). antigen receptor-mediated signal transduction pathway. J. Leukoc. Biol. 55:265. 15. Weiss, A., and D. R. Littman. 1997. Signal transduction by lymphocyte antigen Recently, Lanier et al. (20) cloned an ITAM-containing polypep- receptors. Cell 76:263. tide, DAP12, that associates with noninhibitory isoforms of human 16. Wagtmann, N., S. Rojo, E. Eichler, H. Mohrenweiser, and E. O. Long. 1997. A killer cell-inhibitory receptor molecules and appears to be involved new human gene complex encodes the killer cell inhibitory receptors and a re- in NK cell activation. We are currently examining the possibility lated family of monocyte/macrophage receptors. Curr. Biol. 7:615. 17. Samaridis, J., and M. Colonna. 1997. Cloning of novel immunoglobulin super- that the Ly-49D-associated pp16 represents the murine DAP12. family receptors expressed on human myeloid and lymphoid cells: structural evidence for new stimulatory and inhibitory pathways. Eur. J. Immunol. 27:660. Acknowledgments 18. Hayami, K. 1997. Molecular cloning of a novel murine cell-surface glycoprotein homologous to killer cell inhibitory receptors. J. Biol. Chem. 272:7320. We thank Dr. Deborah N. Burshtyn for critical reading of the manuscript, 19. Kubagawa, H., P. D. Burrows, and M. D. Cooper. 1997. A novel pair of immu- Dr. Kazuo Maruyama for the PMKit vector, and Ms. Joyce Vincent for noglobulin-like receptors expressed by B cells and myeloid cells. Proc. Natl. Acad. Sci. USA 94:5261. manuscript preparation. In addition, we thank Dr. Jean-Pierre Kinet for ⑀ ␥ 20. Lanier, L. L., B. C. Corliss, J. Wu, C. Leons, and J. H. Phillips. 1998. Immuno- rabbit antisera to Fc RI and Dr. Allan Weissman for rabbit antisera to the receptor DAP12 bearing a tyrosine-based activation motif is involved in activat- ␨-chain of the TCR. ing NK cells. Nature 391:703.