Inhibition of the NKG2D activation pathway is mediated through the reduced translocation of NF-κB into the nuclei of NK cells. (A) Diagram depicting the hypothesized interactions between activating NKG2D and inhibitory Ly49 pathways. Binding of H60 with NKG2D receptor leads to phosphorylation of DAP10 or DAP12, resulting in recruitment of protein tyrosine kinases (PTKs) belonging to the SFK family. The subsequent activation of PTKs results in the phosphorylation of downstream signaling proteins, eventually leading to nuclear translocation of NF-κB. Phosphorylation of ITIM sequences at the cytoplasmic domain of Ly49 molecules recruits and activates phosphatases, which in turn dephosphorylate substrates that are part of the NKG2D activation pathway. This results in the impaired translocation of NF-κB into the nuclei. (B) Gel-shift analysis was performed using nuclear extracts from fresh NK cells incubated with indicated target cells at a 40:1 E/T ratio for 2 hours at 37°C. Supershift analysis was performed on nuclear extracts from NK cells incubated with ST63 in the presence of 32P-labelled oligonucleotide and a mAb specific for NF-κB subunit, p50. Data presented are representative of 2 independent experiments.
