PP2B-mediated Dephosphorylation of c-Jun C Terminus Regulates Phorbol Ester-induced c-Jun/Sp1 Interaction in A431 Cells

Abstract

The c-Jun/Sp1 interaction is essential for growth factor- and phorbol 12-myristate 13-acetate (PMA)-induced genes expression, including human 12(S)-lipoxygenase, keratin 16, cytosolic phospholipase A2, p21(WAF1/CIP1), and neuronal nicotinic acetylcholine receptor beta4. Here, we examined the mechanism underlying the PMA-induced regulation on the interaction between c-Jun and Sp1. We found that treatment of cells with PMA induced a dephosphorylation at the C terminus of c-Jun at Ser-243 and a concomitant inhibition of PP2B by using PP2B small interfering RNA, resulting in reduction of PMA-induced gene expression as well as the c-Jun/Sp1 interaction. The c-Jun mutant TAM-67-3A, which contains three substitute alanines at Thr-231, Ser-243, and Ser-249 compared with TAM-67, binds more efficaciously with Sp1 and is about twice as efficacious as TAM-67 in inhibiting the PMA-induced activation of the 12(S)-lipoxygenase promoter. Importantly, PP2B not only dephosphorylates the c-Jun at Ser-243 but also interacts with c-Jun in PMA-treated cells. PMA stimulates the association of the PP2B/c-Jun/Sp1 complex with the promoter. These findings indicate the dephosphorylation of c-Jun C terminus is required for the c-Jun/Sp1 interaction and reveal that PP2B plays an important role in regulating c-Jun/Sp1 interaction in PMA-induced gene expression.

Calcineurin-mediated dephosphorylation of c-Jun Ser-243 is required for c-Jun protein stability and cell transformation

“…Overexpression of active CaN (AI domain-deletion of CnA) decreases c-Jun phosphorylation at Ser-243 (Chen et al, 2007). Consistent with our previous result, inhibition of endogenous CaN with siRNA led to increased Ser-243 phosphorylation of c-Jun (Figure 1a), which further verified that CaN targets c-Jun at Ser-243.…”

“…Comparing the binding activities of c-Jun with CnA-DAI and CnA-DAI-CaM, the latter did not bind to c-Jun, which demonstrates the critical role of CaM-binding domain in CaN/c-Jun interaction CaN-mediated Ser-243 dephosphorylation of c-Jun stabilizes the c-Jun protein stability Previously, it has been reported that phosphorylation of c-Jun at Thr-239 and Ser-243 targets c-Jun for polyubiquitination and degradation, and the v-Jun point mutation on Ser-243 allows c-Jun to escape GSK3-dependent recognition and destruction by the E3 ligase (Wei et al, 2005). Additionally, we identified the phosphatase that dephosphorylates Ser-243 of c-Jun is CaN (Chen et al, 2007). We next determined whether CaN participates in regulating c-Jun protein stability through Ser-243 dephosphorylation of c-Jun.…”

“…Consistent with our previous result, inhibition of endogenous CaN with siRNA led to increased Ser-243 phosphorylation of c-Jun (Figure 1a), which further verified that CaN targets c-Jun at Ser-243. Using immunoprecipitation and GST-pull down assays, CaN interacts with the phosphorylated c-Jun C-terminus in vitro (Chen et al, 2007). Based on these observations, we determined whether the interaction between these two proteins occurred in the living cell by coexpressing CFPtagged wild-type CnA with YFP-tagged c-Jun.…”

“…Calmodulin-binding domain of CnA is required for binding to c-Jun Phosphorylated C-terminus of c-Jun has been identified as the interactive domain for CnA (Chen et al, 2007), but the specific domain within CnA involved in this interaction has not been described. To pursue this further, expression vectors bearing various CFP-tagged deletion mutants of CnA ( Figure 2a) combined with YFP-tagged c-Jun were cotransfected into cells and analysed by FRET assay.…”

“…It is well accepted that UV radiation exposure induces activation of Jun N-terminal kinase, which leads to N-terminus phosphorylation of c-Jun and enhances c-Jun transactivation activity (Karin, 1995). On the other hand, the phosphorylation of c-Jun C-terminus by GSK3 or CKII decreases the activities of DNA-binding and Sp1 protein interaction (Boyle et al, 1991;Lin et al, 1992;Chen et al, 2007). Recently, we identified that CaN is involved in the dephosphorylation of c-Jun C-terminus at Ser-243, and this modification increases the interaction between c-Jun and Sp1, which results in the enhancement of c-Jun-regulated gene transcription (Chen et al, 2007).…”