Downstream of Tyrosine Kinase 1 and 2 Play Opposing Roles in CD200 Receptor Signaling Robin Mihrshahi and Marion H. Brown This information is current as J Immunol published online 15 November 2010 of October 1, 2021. http://www.jimmunol.org/content/early/2010/11/14/jimmun ol.1002858 Downloaded from Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists http://www.jimmunol.org/ • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: by guest on October 1, 2021 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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 15, 2010, doi:10.4049/jimmunol.1002858 The Journal of Immunology Downstream of Tyrosine Kinase 1 and 2 Play Opposing Roles in CD200 Receptor Signaling Robin Mihrshahi and Marion H. Brown The CD200 receptor (CD200R) negatively regulates myeloid cells by interacting with its widely expressed ligand CD200. CD200R signals through a unique inhibitory pathway involving a direct interaction with the adaptor protein downstream of tyrosine kinase 2 (Dok2) and the subsequent recruitment and activation of Ras GTPase-activating protein (RasGAP). Ligand engagement of CD200R also results in tyrosine phosphorylation of Dok1, but this protein is not essential for inhibitory CD200R signaling in human myeloid cells. In this paper, we show that CD200R-induced phosphorylation of Dok2 precedes phosphorylation of Dok1, and that Dok2 and Dok1 recruit different downstream proteins. Compared with Dok2, Dok1 recruits substantially less RasGAP. In addition to binding RasGAP, Dok2 recruits the adaptor molecule Nck in response to ligand engagement of CD200R. CD200R-induced phosphorylation of Dok1 results in the recruitment of CT10 sarcoma oncogene cellular homologue-like (CrkL), whereas the closely related CT10 sarcoma oncogene cellular homologue interacts constitutively with Dok1. Knockdown of Dok1 or CrkL Downloaded from expression in U937 cells resulted in increased Dok2 phosphorylation and RasGAP recruitment to Dok2. These data are consistent with a model in which Dok1 negatively regulates Dok2-mediated CD200R signaling through the recruitment of CrkL. The Journal of Immunology, 2010, 185: 000–000. he CD200 receptor (CD200R) is a type 1 transmembrane phosphatases Src homology 2 domain-containing phosphatase 1 glycoprotein of the Ig superfamily present on most leu- (SHP1), SHP2, or the inositol phosphatase SHIP (20). The cyto- http://www.jimmunol.org/ T kocytes, especially cells of the myeloid lineage (1, 2). plasmic tail of CD200R contains three conserved tyrosines, of CD200R mediates inhibitory signaling in myeloid cells (3–5) and which the most membrane distal one is part of a phosphotyrosine- T cells (6) by interacting with its widely expressed ligand CD200 binding (PTB) domain recognition motif (NPxY) (21). Phos- through its N-terminal V-type domain (7). phorylation of this tyrosine motif is essential for inhibitory CD200 knockout mice have increased numbers of myeloid cells, CD200R signaling (22, 23) and binds directly to the PTB domain- are more susceptible to induction of autoimmune disorders, and die containing adaptor downstream of tyrosine kinase 2 (Dok2) of severe lung inflammation after influenza infection (8, 9). De- (22). Phosphorylation of CD200R-bound Dok2 results in the re- ficiency of CD200R likewise results in exaggerated inflammatory cruitment and activation of Ras GTPase-activating protein (Ras- responses to various stimuli (10). In vitro experiments show that GAP) and the subsequent inhibition of Ras-Erk signaling (22–24). by guest on October 1, 2021 ligation of CD200R causes inhibition of cellular activation in dif- CD200R ligation also causes phosphorylation of the closely re- ferent cells and tissues including human and mouse mast cells (3), lated Dok1 (22–24), but unlike Dok2, this protein is not essential macrophages (4, 5), MLRs (6, 11), and basophils (12). High levels for inhibitory CD200R signaling in human myeloid cells (22). of CD200 expression are a characteristic of various human can- We now provide evidence of a regulatory role for Dok1 in cers, and this is thought to facilitate evasion of immune recogni- CD200R signaling by analyzing the kinetics of phosphorylation tion by inhibiting the activation of CD200R-bearing leukocytes of Dok2 and Dok1, and characterizing differences in their inter- (6, 13–16). CD200 homologs are also expressed by a number of actions downstream of CD200R. Compared with Dok2, CD200R- viruses and have been shown to inhibit host responses against induced phosphorylation of Dok1 was delayed. RasGAP and the virally infected cells (5, 12, 17–19). adaptor protein Nck were preferentially associated with Dok2 and Unlike most other inhibitory receptors, CD200R does not the closely related adaptor proteins CT10 sarcoma oncogene cel- contain any ITIMs, which mediate cellular inhibition through the lular homologue (Crk) and Crk-like (CrkL) with Dok1. Knockdown phosphorylation-dependent recruitment of the protein tyrosine of either CrkL or Dok1 resulted in enhanced phosphorylation of Dok2 and increased recruitment and activation of RasGAP. These data fit a model in which Dok1 is recruited indirectly through Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, Dok2 in CD200R signaling and initiates a CrkL-dependent neg- United Kingdom ative feedback loop to regulate inhibition by CD200R. Received for publication August 23, 2010. Accepted for publication October 7, 2010. This work was supported by an Oxford University Clarendon Fund award (to R.M.) and by the U.K. Medical Research Council (G0601169 and G0400808). Materials and Methods Address correspondence and reprint requests to Dr. Marion H. Brown, Sir William Abs Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom. E-mail address: [email protected] Polyclonal rabbit anti-Crk (sc-289) and anti-Grb2 (sc-255), polyclonal goat anti-Dok2 (sc-8130), and monoclonal mouse anti-RasGAP (sc-63) Abs Abbreviations used in this paper: CD200R, CD200 receptor; COMP, cartilage olig- were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal mouse omeric matrix protein; Crk, CT10 sarcoma oncogene cellular homologue; CrkL, Crk- anti-CrkL and anti-phosphotyrosine (4G10) were from Millipore (Bedford, like; Ctr, control; Dok, downstream of tyrosine kinase; PLCg, phospholipase Cg; PTB, phosphotyrosine binding; RasGAP, Ras GTPase-activating protein; SH2, Src MA). Polyclonal rabbit anti-phospholipase Cg1 (anti-PLCg1) and peroxidase- homology 2; SHP, Src homology 2 domain-containing phosphatase; shRNA, short conjugated goat anti-rabbit Abs were from Cell Signaling Technology hairpin RNA; Tr, truncated; WT, wild-type. (Beverly, MA). Monoclonal anti-Nck was from BD Biosciences (San Jose, CA). Polyclonal rabbit anti-human Dok1 Ab (25) was a kind gift from Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 Dominique Davidson and Andre´ Veillette. Peroxidase-conjugated polyclonal www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002858 2 Dok1 AND Dok2 PLAY OPPOSING ROLES IN CD200R SIGNALING FIGURE 1. Phosphorylation of Dok2 precedes phosphorylation of Dok1 in CD200R signaling. U937 cells expressing WT (Wt) or Tr CD200R were incubated for 2.5, 5, or 10 min at 37˚C in the presence of CD200-COMP. Cells were then lysed, and Dok2 or Dok1 was immunoprecipitated from lysates. Immunopre- cipitates were blotted with anti-phosphotyrosine mAb. Membranes were then washed extensively and reprobed with specific Abs against the indicated proteins. Results are representative of three independent experiments. anti-mouse, anti-rabbit, and anti-goat Abs were from Sigma-Aldrich (St. nitrocellulose membranes in a Novex XCell II Blot Module and Western Louis, MO). blotted using the SNAP i.d. Protein Detection System (Millipore). Cell culture BIAcore analysis Downloaded from U937 cells expressing wild-type (WT) or signaling-deficient (cytoplasmic Surface plasmon resonance analyses using a BIAcore 3000 (BIAcore, tail truncated [Tr]) human CD200R have been described previously (22). Uppsala, Sweden) were conducted essentially as described previously (22, In brief, these cell lines were established by lentiviral transduction of 27). In brief, ∼4000 response units streptavidin was immobilized at 25˚C U937 cells with constructs containing either full-length human CD200R or on CM5 chips by amine coupling followed by immobilization of 50–150 a Tr version lacking the last 40 aa of its cytoplasmic tail. Cells were grown response units biotinylated peptides (Peptide Protein Research, Fareham, in RPMI 1640 supplemented with 5% heat-inactivated FCS, 1 mM sodium U.K.). Flow cells with streptavidin only were used as controls. Increasing pyruvate, nonessential amino acids, and 50 U/ml penicillin, 50 mg/ml concentrations of monomeric (fast protein liquid chromatography-purified), streptomycin (all PAA Laboratories, Teddington, U.K.). recombinant, soluble protein were then