Downstream of tyrosine kinase 1 and 2 play opposing roles in CD200 receptor signaling

Abstract

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 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.

Fig. 1

Phosphorylation of Dok2 precedes phosphorylation of Dok1 in CD200R signaling. U937 cells expressing wild-type (Wt) or truncated (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. Immunoprecipitates were blotted with anti-phosphotyrosine mAb. Membranes were then washed extensively and re-probed with specific antibodies against the indicated proteins. Results are representative of three independent experiments.

Fig. 2

Crk and CrkL interact with Dok1 in CD200R signaling. U937 cells expressing wild-type (Wt) or truncated (Tr) CD200R were incubated for 10 min at 37 °C in the presence of CD200-COMP. Cells were then lysed and Dok2 (A) or Dok1 (B) was immunoprecipitated from lysates. Immunoprecipitates were blotted with specific antibodies against Crk or CrkL. After extensive washing, membranes were re-probed with antibodies against Dok2 or Dok1 to control for loading. Results are representative of three or more independent experiments.

Fig. 3

Nck is recruited to phosphorylated Dok2 in CD200R signaling. U937 cells expressing wild-type or truncated CD200R were incubated for 10 min at 37 °C in the presence of CD200-COMP. Cells were then lysed and Dok2 and Dok1 were immunoprecipitated from lysates. Immunoprecipitates were blotted with mAb against Nck. After extensive washing, membranes were re-probed with antibodies against Dok2 or Dok1 to control for loading. Results are representative of three independent experiments.

Fig. 4

Nck binds directly to Dok2. Phosphorylated peptides corresponding to sequences around Tyr362 of Dok1 or Tyr345 of Dok2 were immobilized on a BIAcore™ chip and recombinant human Nck SH2 domain was passed over flow cells at different concentrations at 37 °C. Equilibrium binding at each concentration of Nck binding to the phosphopeptides is shown. The hyperbolas represent best fits used for affinity calculation of equilibrium dissociation constants. Results are representative of two or more independent experiments.

Fig. 5

Knockdown of CrkL increases phosphorylation of CD200R signaling complexes. PLCγ (A), Crk (B), CrkL (C) and Nck (D) expression was knocked down by shRNA in U937 cells expressing wild-type (Wt) or truncated (Tr) CD200R. Cells were lysed after incubation for 10 min at 37 °C in the presence of CD200-COMP. Dok2 (E) or Dok1 (F) was immunoprecipitated from the lysates and precipitates blotted with anti-phosphotyrosine mAb to determine the phosphorylation state of Doks and co-precipitated proteins. After extensive washing, membranes were re-probed for RasGAP and Dok proteins. Results are representative of three independent experiments conducted using separately established knockdown cell lines.

Fig. 6

Dok1 inhibits Dok2-mediated CD200R signaling. (A) Dok1 expression was knocked down by shRNA in U937 cells expressing wild-type (Wt) or truncated (Tr) CD200R. (B) Cells were lysed after incubation for 10 min at 37 °C in the presence of CD200-COMP. Dok2 was immunoprecipitated from the lysates and precipitates blotted with anti-phosphotyrosine mAb to determine the phosphorylation state of Dok2 and co-precipitated proteins. After extensive washing, membranes were re-probed for RasGAP and Dok2. Results are representative of three independent experiments. Results are representative of three independent experiments conducted using separately established knockdown cell lines.

Fig. 7

Model for CD200R signaling. Dok2 binds to the third phosphotyrosine in the cytoplasmic tail of CD200R via its PTB domain. Subsequent phosphorylation of Dok2 results in the recruitment of RasGAP, Nck and Dok1. RasGAP hydrolyses RasGTP into the inactive form RasGDP, thereby inhibiting PI3K and Erk. Dok1 interacts constitutively with Crk and recruits CrkL upon tyrosine phosphorylation. CrkL subsequently inhibits phoshorylation of Dok2 by an unknown mechanism, resulting in inhibition of RasGAP activation.