Figure 5. Phosphorylation of JNK by PKC contributes to UV-induced apoptosis.

Endogenous JNK levels were reduced by siRNA. SW1 cells were transfected with pRS or pRS-JNK (see methods) and stable lines resistant to puromycin were established. Expression of JNK was assessed by immunoblot. (B) Expression of JNK mutants in SW1 cells whose endogenous JNK levels were decreased by siRNA. SW1 cells described in A were transfected with FLAG-tagged JNK-WT, JNK-S129A and JNK-APF (an inactive form of JNK) and stable lines resistant to both puromycin and hygromycin were established. Expression of the exogenous proteins was assessed by immunoblot with FLAG antibody. (C) JNK-S129A is less active than JNK-WT. Cells described in B were treated with Go6976 for 30min followed by UV treatment for an additional 30min. Cells were lysed and exogenous JNK immunoprecipitated with FLAG antibodies. The activity of JNK in the immunoprecipitates was assessed by an immunokinase reaction using c-Jun as a substrate. (D) Cells expressing JNK-S129A are more resistant to UV-induced apoptosis. Cells described in B were UV-irradiated (40J/m²) and quantification of apoptotic cells was determined at the indicated time points. (E) The PKC inhibitor Go6976 inhibits both the early and late phases of JNK activation by UV. SW1 cells expressing both JNK siRNA and FLAG-JNK-WT were treated with Go6976 for 30min followed by UV treatment (40J/m²). Protein extracts were obtained at the indicated time points following UV treatment. JNK activity was assessed by an immunokinase reaction using c-Jun as a substrate. Similar results were obtained in three separate experiments. One asterisk indicates p ≤ 0.05 and two asterisk indicate p ≤ 0.01.