FIGURE 1.

Tyrosine phosphorylation of KAP1 induced by nuclear tyrosine kinases. A, COS-1 cells transfected with Lyn-HA or NLS-Lyn-HA were cultured for 24 h. Cells were fixed and stained with anti-HA antibody. The areas of nuclei are marked by dotted lines. DIC, differential interference-contrast. B, COS-1 cells transfected with the indicated plasmids were cultured for 24 h in the presence or absence of 10 μm PP2 during the last 12 h. Immunoblotting (Western blot; WB) was performed with the indicated antibodies. C, parental HeLa S3 or HeLa S3/NLS-Lyn cells treated with or without 0.5 mm sodium orthovanadate (Na₃VO₄) were cultured for 1.5 h. Immunoblotting was performed with the indicated antibodies. D, COS-1 cells transfected with the indicated plasmids were cultured for 24 h. Endogenous KAP1 was immunoprecipitated (IP) with anti-KAP1 antibody. Immunoblotting was performed with the indicated antibodies. E, COS-1 cells cotransfected with KAP1-wt plus the indicated plasmids were cultured for 24 h in the presence of 10 μm SU6656 or DMSO (dimethyl sulfoxide, solvent control) during the last 3 h. KAP1-wt was immunoprecipitated with anti-FLAG antibody. Immunoblotting was performed with the indicated antibodies. F, KAP1-wt immunoprecipitates were incubated with Lyn immunoprecipitates in reaction buffer. Samples were taken at the indicated times. Immunoblotting was performed for FLAG, Tyr(P), and Lyn. The levels of tyrosine phosphorylation at each incubation time (15, 30, and 60 min) relative to that of the control (0 min) are shown. G, KAP1-wt immunoprecipitates were incubated with Lyn or Lyn(KD) immunoprecipitates for 30 min in reaction buffer. Immunoblotting was performed with the indicated antibodies. H–L, COS-1 cells cotransfected with KAP1-wt plus the indicated plasmids were cultured for 24 h. KAP1-wt was immunoprecipitated with anti-FLAG antibody. Immunoblotting was performed with the indicated antibodies. Asterisks show degradation products.