BAD Ser¹²⁸ is not phosphorylated by c-Jun NH₂- terminal kinase for promoting apoptosis

The phosphorylation and regulation of the proapoptotic Bcl-2 family protein BAD by c-Jun NH₂-terminal kinase (JNK) is controversial. JNK can suppress interleukin-3 withdrawal-induced apoptosis via phosphorylation of BAD at Thr²⁰¹. However, it has also been reported that JNK promotes apoptosis through phosphorylation of BAD at Ser¹²⁸. Here, we report that JNK is not a BAD Ser¹²⁸ kinase. JNK phosphorylates murine BAD (mBAD), but not human BAD (hBAD), in which Ser⁹¹ is equivalent to Ser¹²⁸ in mBAD. In contrast, Cdc2, which phosphorylates Ser ¹²⁸, phosphorylates both mBAD and hBAD. Replacement of Ser ¹²⁸ by alanine has no effects on BAD phosphorylation by JNK in vitro and in vivo. Two-dimensional phosphopeptide mapping in combination with phosphoamino acid analysis reveals that JNK does not phosphorylate BAD at Ser¹²⁸. Elimination of Ser¹²⁸ phosphorylation has no effects on the proapoptotic activity of BAD in apoptosis induced by UV via JNK or growth factor withdrawal. Thus, our results show that Ser¹²⁸ is not phosphorylated by JNK for promoting cell death.