PSF is tyrosine-phosphorylated in NPM/ALK-expressing cells and is a substrate of ALK tyrosine kinase activity. (A) Anti-ALK1 and anti-PSF immunoprecipitates (IPs) from SUDHL-1, K562, BaF3-Par, BaF3-KD, and BaF3-NA cells were Western blotted with an antiphosphotyrosine (anti-pTyr) antibody. (B) Anti-ALK1 immunoprecipitates from lymph nodes derived from patient 1 with NPM/ALK⁺ ALCL and NPM/ALK⁻ spleen tissue were Western blotted using the anti-pTyr antibody. (C) A radioactive in vitro kinase assay was performed with purified recombinant 6xHisALK kinase domain (rALK) and purified full-length 6xHis-PSF. Protein purity was checked by coomassie staining (Ci). Kinase assay was performed in the presence of rALK kinase domain alone (0.4 μg), PSF alone (0.25 μg), or rALK and PSF together. Samples were analyzed by SDS-PAGE and visualized by autoradiography (Cii). Bands corresponding to PSF and ALK are indicated by arrows. (D) Time courses of the phosphorylation of PSF peptides (400 μM) by GST-ALK. The peptide sequences are as follows: Tyr251, RGGRQHHPPYHQQHHQGP; Tyr293, PGEKTYTQRCRLFVGNLPADIT; Tyr470, EKLAQKNPMYQKERETPTR; Tyr488-490, TFEYEYSQRWKSLDEMEKQQR; Tyr527, EMEDAYHEHQANLLRQDLMRRQ; Tyr597-602, REESYSRMGYMDPRERDMR; Tyr624, MNMGDPYGSGGQKFPPLGGG; and Tyr698, GRGREEYEGPNKKPRF (target tyrosine in bold). (E) Immunoprecipitation with anti-HA (lanes 2,3) and anti-ALK1 (lanes 4,5) monoclonal antibodies of lysate derived from 293T cells transiently transfected with wild-type or mutant Y293F HA-PSF together with NPM/ALK. Immunoprecipitates were resolved by SDS-PAGE, and Western blotting with anti-pTyr and anti-HA antibodies was performed. Lane 1 shows lysate from nontransfected 293T cells.