A reinvestigation of the multisite phosphorylation of the transcription factor c-Jun

Abstract

We have used phospho-speci®c antibodies to re-examine the multisite phosphorylation of c-Jun in murine RAW macrophages and embryonic ®broblasts. Our results indicate that JNK isoforms are required and suf®cient for the phosphorylation of Thr91 and Thr93, as well as the phosphorylation of Ser63 and Ser73, in response to LPS or anisomycin in macrophages and TNFa or anisomycin in ®broblasts. However, the phorbol ester (TPA) and EGF-induced phosphorylation of Ser63 and Ser73 is mediated by ERK1/ERK2, as well as JNK1/JNK2, in ®broblasts from wild-type mice and by ERK1/ERK2 alone in ®broblasts from JNK-de®cient mice. The phosphorylation of Thr239 is catalysed by GSK3 and the phosphorylation of Ser243 by an as yet unidenti®ed protein kinase. The inhibition of GSK3 is not required for the dephosphorylation of Thr239 in response to LPS, and nor is the phosphorylation of Thr91 and Thr93 required for the TPA-or EGF-induced dephosphorylation of Thr239 in ®broblasts. The agonistinduced dephosphorylation of Thr239 may involve a conformational change that exposes Thr239 to dephosphorylation and/or the activation of a Thr239 phosphatase.

Identification of a novel phosphorylation site in c-jun directly targeted in vitro by protein kinase D

“…Previous studies determined MAPK sites Ser63, Ser73, Thr91, and Thr93 in the c-jun Nterminus [1,3]. Although it is well accepted that phosphorylation of these sites in response to stimuli regulates the activity and protein stability of c-jun, these events do not exclude the possibility of additional modulatory inputs.…”

Proteomic identification of C/EBP-DBD multiprotein complex: JNK1 activates stem cell regulator C/EBPα by inhibiting its ubiquitination

“…After stringent washing, interaction between JNK1 and GST-DBD was observed (Figure 2a; lane 5). We next examined whether JNK1 interacts with full-length C/EBPa and performed GST pull down assay using GST-DBD, GST-C/EBPa and GST alone from 25 ng/ml anisomycin-induced (U937 radioimmunoprecipitation assay (RIPA*) and uninduced (U937 RIPA) cell lysates; anisomycin is a potent activator of JNK which acts on upstream activators of JNK (Bogoyevitch et al, 1995;Morton et al, 2003). Immunoblot against JNK1 showed direct interaction of JNK1 (Figure 2b; lanes 2, 6 and 8 with fast migrating lanes) and phospho-JNK1 (pJNK) (Figure 2b; lanes 1, 5 and 7 with slow migrating bands as compared to respective uninduced conditions) with GST-DBD and GST-C/EBPa.…”

c-Jun activation is required for 4-hydroxytamoxifen-induced cell death in breast cancer cells

“…JNK induces c-Jun transactivation by phosphorylating the c-Jun N-terminal domain at serines 63/73 and threonines 91/93 (Bannister et al, 1995;Papavassiliou et al, 1995;Morton et al, 2003;Weiss et al, 2003;Nateri et al, 2005;Vinciguerra et al, 2008). By forming stable homodimeric or heterodimeric complexes with Fos or ATF family members, c-Jun constitutes the inducible activator protein-1 (AP-1) transcription factor.…”