Figure 5. Detection of S129 Phosphorylation In Vivo by Phosphopeptide Mapping and S129-Specific Phosphoantibodies.

(A and B) Mapping the site of PKC phosphorylation of JNK in vivo. Tryptic phosphopeptide mapping of JNK forms was carried out with JNK^wt, JNK^APF, or JNK^S129A transfected into 293 HEK cells that were incubated with [³²P]orthophosphate followed by UV irradiation ([A] and [B]) or anisomycin treatment ([B]). Phosphorylated proteins were IP with anti-Flag Ab followed by their separation on SDS-PAGE, and corresponding bands were cut and subjected to in-gel digestion with trypsin. The tryptic digests were separated with thin-layer electrophoresis (horizontal dimension) followed by ascending chromatography (vertical dimension) and visualized by phosphorimager. The phosphopeptides X, Y, Z, and V are indicated. Note that running conditions differ among (A) and (B).

(C) JNK is phosphorylated in S129 after UV irradiation. HEK293 T cells were transfected with JNK^wt (JNK2) (1 μg). Cells were serum starved and treated with UV as described in Figure 2B. Protein extracts (800 μg) were subjected to IP with anti-Flag Ab and blotted with the p-JNK^S129 antibody. The antibody used to develop the membrane in lane 3 was preincubated with the phosphopeptide used for immunization. The membrane was reprobed with rabbit anti-Flag Ab to reveal quantities of Flag-JNK in the immunoprecipitates (lower).

(D) Antibodies to phosphorylated S129 do not recognize JNK^S129A. HEK293 T cells were transfected with JNK^wt or JNK^S129A (1 μg). The experiment was performed as indicated in (C).

(E) Phosphoantibodies to S129 identify JNK phosphorylation upon exposure to PKC-dependent stimuli.

HEK293 T cells were subjected to the indicated treatment (see Experimental Procedures for details), and proteins were prepared 30 min later. Where indicated, cells were transfected with HA-CA-PKCβII (2 μg). Protein extracts (1 mg) were subjected to IP with mouse anti-JNK1 antibodies and blotted by using the p-JNK^S129 antibody. The membrane was reprobed with rabbit anti-JNK antibodies to reveal quantities of JNK in the immunoprecipitates (lower).