The NF-κB activation in lymphotoxin β receptor signaling depends on the phosphorylation of p65 at serine 536

NF-kappaB-inducing kinase (NIK) has been shown to play an essential role in the NF-kappaB activation cascade elicited by lymphotoxin beta receptor (LTbetaR) signaling. However, the molecular mechanism of this pathway remains unclear. In this report we demonstrate that both NIK and IkappaB kinase alpha (IKKalpha) are involved in LTbetaR signaling and that the phosphorylation of the p65 subunit at serine 536 in its transactivation domain 1 (TA1) plays an essential role. We also found that NF-kappaB could be activated in the LTbetaR pathway without altering the level of the phosphorylation of IkappaB and nuclear localization of p65. By using a heterologous transactivation system in which Gal4-dependent reporter gene is activated by the Gal4 DNA-binding domain in fusion with various portions of p65, we found that TA1 serves as a direct target in the NIK-IKKalpha pathway. In addition, mutation studies have revealed. the essential role of Ser-536 within TA1 of p65 in transcriptional control mediated by NIK-IKKalpha. Furthermore, we found that Ser-536 was phosphorylated following the stimulation of LTbetaR, and this phosphorylation was inhibited by the kinase-dead dominant-negative mutant of either NIK or IKKalpha. These observations provide evidence for a crucial role of the NIK-IKKalpha cascade for NF-kappaB activation in LTbetaR signaling.