Volume 9 Issue 8 - July 17, 2009
PP2B-mediated dephosphorylation of c-Jun C terminus regulates phorbol ester-induced c-Jun/Sp1 interaction in A431 cells.
Ben-Kuen Chen1,*, Chi-Chen Huang1,*, Wei-Chiao Chang1, Yun-Ju Chen1, Ushio Kikkawa2, Ken-ichi Nakahama3, Ikuo Morita3 and Wen-Chang Chang1,‡

1Department of Pharmacology, College of Medicine, and Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University, Tainan 701, Taiwan
2Biosignal Research Center, Kobe University, Kobe 657-8501, Japan
3Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo, Japan

Molecular Biology of the Cell, 2007, 18, 1118-1127

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The proto-oncogene c-Jun is one of the components of AP1, a transcription factor complex believed to mediate a variety of cellular processes including growth, proliferation, transformation, and apoptosis (Shaulian & Karin, 2002). For example, in tumorigenic processes, cellular transformation and malignancy induced by Ha-Ras require c-Jun transactivation activity (Smeal et al., 1991). Additionally, c-Jun is also involved in angiogenesis, invasion and metastasis to control tumorigenesis (Jochum et al., 2001). Thus, aberrant expression and increased transactivation activity of c-Jun are observed in a wide spectrum of human cancers (Eferl & Wagner, 2003). Since c-Jun plays an important role in the regulation of tumorigenesis, to understand the mechanisms involved in the regulation of c-Jun activation is a pivotal issue. The transcriptional activity of c-Jun is dependent on protein phosphorylated on Ser-63, Ser-73, Thr-91 and/or Thr-93 within the N-terminal domain of the molecule by extracellular signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) (Smeal et al., 1991; Adler et al., 1992; Treisman, 1996). In contrast to the activation of c-Jun by N-terminal phosphorylation, the phosphorylation of c-Jun on three C-terminal residues close to the DNA binding domain has been reported to inhibit DNA binding (Hunter and Karin, 1992). In addition, many genes are regulated by c-Jun through the binding between the C-terminal domain of c-Jun and transcription factors Sp1, ATF, smad, NFAT, Ets, and STAT (Chinenov and Kerppola, 2001). In spite of this wealth of knowledge regarding the regulatory mechanisms underpinning c-Jun, the regulation and correlation of dephosphorylation of c-Jun C-terminus and its interaction with transcription factors, like as Sp1, are not completely clarified.

In order to clarify the mechanisms involved in the regulation of c-Jun/Sp1 interaction, which may reveal how c-Jun/Sp1-regulated genes are controlled by such complex, we devoted to find out phosphatases to be responsible for the dephosphorylation of c-Jun C-terminus and to promote protein interaction. We found that PP2B played a functional role in enhancing the interaction between c-Jun and Sp1 ensuing the transcriptional activation of 12(S)-lipoxygenase gene upon the PMA treatment. In addition, we also found that PP2B siRNA inhibited c-Jun/Sp1 interaction but had no effect on the PMA-induced expression of c-Jun and nuclear distribution of c-Jun. Although, the role of PP2B in regulating the transcription and nuclear localization through dephosphorylation of downstream targets such as the PP2B-dependent cytoplasmic subunits of the NFATc transcription complex has been documented (Shibasaki et al., 1996; Rao et al., 1997), our results suggest that the regulation of PMA-induced gene expression mediated by PP2B was not due to an enhancement in the nuclear transport of c-Jun and Sp1, but might be due to the regulation of c-Jun/Sp1 interaction. 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.

In this study, we provided several new findings as follows. The first, a large group of transcription factors including the bZIP, NFAT, Ets, Smad and bHLH families can activate or repress the transcription in conjunction with c-Jun C-terminus by binding to regulatory elements adjacent to AP1 sites (Chinenov and Kerppola, 2001). However, it is not clear exactly how proteins interact with each other. In the present study, we found that dephosphorylation at the Ser-243 of c-Jun was required for its interaction with Sp1. These results reveal that PP2B-mediated dephosphorylation of c-Jun C-terminus may play a role in the regulation of interaction between c-Jun and their coactivators as well as Sp1, Smad, NFAT etc. The second, PP2B has relatively narrower substrate specificity including transcription factors, e.g. NFAT (Luo et al., 1996a), the transcription factor Elk-1 (Tian and Karin, 1999), and the heat shock protein, hsp25 (Gaestel, 1992). We found that transcription factor c-Jun could be a new target of PP2B. These results indicate that PP2B may participate in tumorigenesis through the regulation of c-Jun interaction with cooperators. Finally, although the mutation of Ser-243 prevents c-Jun from being earmarked for destruction by the Fbw7 ubiquitin ligase complex (Wei et al., 2005), it is not clear which phosphatase could regulate dephosphorylation of c-Jun at Ser-243 to enhance its stability until our finding was reported. Thus, PP2B-regulated dephosphorylation of c-Jun C-terminus may thus not only participate in the regulation of c-Jun/Sp1 interaction but also play a role in the stabilization of c-Jun.

In conclusion, as shown in Fig. 1, we found that dephosphorylation of c-Jun C-terminus regulated by PP2B was required for the c-Jun/Sp1 interaction in gene regulation. Our present findings strongly support the notion that the up-regulation of gene expression, like as p21WAF1/CIP1 may also be mediated through PP2B by regulating the interaction of c-Jun with Sp1. It is apparent that PP2B also participates in regulating other genes in addition to 12(S)-lipoxygenase through the c-Jun/Sp1 interaction. Indeed, we found that PP2B regulated the EGF-induced promoter activity of keratin 16 gene (Wang Y. N. and Chang, W. C., unpublished results) and PMA-induced cPLA2α expression. An increasing number of genes, e.g. 12(S)-lipoxygenase, keratin 16, human cytosolic phospholipase A2 (cPLA2), neuronal nicotinic acetylcholine receptor β4 (nAChRβ4), p21WAF1/CIP1, human vimentin and lamin A/C regulated by the c-Jun/Sp1 interaction are identified (Kardassis et al., 1999; Chen and Chang, 2000; Blaine et al., 2001; Melnikova and Gardner, 2001; Wang and Chang, 2003; Wu et al., 2003; Okumura et al., 2004). Since genes regulated by the c-Jun/Sp1 interaction play important functions in cells, further elucidation of the mechanisms underlying the c-Jun/Sp1 interaction could provide important clues to how genes are regulated in general.
Fig. 1. PP2B dephosphorylates C-terminus of c-Jun to enhance c-Jun/Sp1 interaction and promote gene expression.

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