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

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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 References This article cites 20 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/160/9/4148.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 1, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. c 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-z/FceRIg double knockout mice the human, p50 receptors have truncated cytoplasmic domains that confirm that pp16 is not TCR-z, TCR-h,orFceRIg. Association lack ITIMs (7) and have been shown to mediate reverse Ab-de- of pp16 with Ly-49D involves a transmembrane arginine since 21 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 Ca21 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-associ- ated 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- 1 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 phos- phorylated 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. Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 c 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 biotinyl- ated, lysed, and immunoprecipitated, and pro- teins 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 pervana- date (1 mM), lysed in Triton X-100, immuno- precipitated, separated on SDS-PAGE as above, and immunoblotted with biotinylated 4G10 fol- lowed by SA-HRP. C, NK cells treated as in B were separated on 16% SDS-PAGE under re- ducing 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 FceRIg and TCR-z 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
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