Microcystin-LR stabilizes c-myc protein by inhibiting protein phosphatase 2A in HEK293 cells
Introduction
Toxic cyanobacterial blooms have become a worldwide environmental concern. Microcystins (MCs) are produced by cyanobacteria, which are harmful to aquatic organisms and mammals including humans (Azevedo et al., 2002, Carmichael et al., 2001). In 1996, 60 dialysis patients in Caruaru of Brazil were died due to the acute exposure to MCs in water used for dialysis (Pouria et al., 1998). Epidemiological studies have indicated that MCs might be responsible for the increased incidence of liver cancer in certain areas of China (Ueno et al., 1996, Yu, 1989). So far, more than 80 structural analogs of MCs have been identified, of which microcystin-LR (MC-LR) is the most studied and the most toxic (Hoeger et al., 2005). MC-LR is characterized with regard to hepatotoxicity, nephrotoxicity, neurotoxicity, reproductive toxicity and carcinogenicity (Chen et al., 2013, Feurstein et al., 2011, Li et al., 2012a, Suput, 2011, Wang et al., 2010a, Zhao et al., 2012). The International Agency for Research on Cancer (IARC) has classified MC-LR as a possible human carcinogen (Cogliano et al., 2008, Grosse et al., 2006). In recent years, MC-LR has attracted increasing attention because of its tumor-promoting capacity.
The c-myc gene, a proto-oncogene, encodes a transcription factor involved in the control of cell proliferation, differentiation and apoptosis (Cole, 1986, Dang., 1999, Prendergast, 1999). Overexpression of c-myc plays a significant role in cancer development (Nesbit et al., 1999, Rapp et al., 2009). Because of its potent effects on cell fate, cells have evolved sophisticated methods to ensure proper expression of c-myc. The expression of c-myc is regulated at multiple levels: transcription, post-transcription and post-translation (Jones and Cole, 1987, Kelly et al., 1983, Sears et al., 1999). The half-life of c-myc is very short in cells due to proteasomal degradation. Previous study has indicated that the phosphorylation of threonine 58 and serine 62, two phosphorylation sites in the N-terminus of c-myc, can regulate c-myc protein stability (Sears et al., 2000). Threonine 58 phosphorylation destabilizes c-myc, while serine 62 phosphorylation stabilizes c-myc. The well-known regulatory cycle of c-myc, from synthesis to degradation, includes: (1) new c-myc protein synthesis; (2) serine 62 of c-myc is phosphorylated by extracellular receptor kinase (ERK); (3) threonine 58 is phosphorylated by glycogen synthase kinase (GSK-3β); (4) Pin1 prolyl isomerase recognizes the threonine 58 and serine 62 phosphorylated c-myc and catalyzes c-myc protein conformational change; (5) serine 62 is then dephosphorylated by protein phosphatase 2A (PP2A); (6) c-myc protein is degraded by the ubiquitin/proteasome system (Sears, 2004).
The most studied and accepted mechanism of MC-LR toxicity is the inhibition of protein phosphatase 1 and 2A (PP1 and PP2A) by directly binding to the catalytic subunits of these enzymes (Mackintosh et al., 1995, Xing et al., 2006). PP2A is a major serine/threonine phosphatase in cells and is involved in regulating proliferation, growth, differentiation, and apoptosis (Janssens and Goris, 2001). Unlike c-myc, PP2A is generally regarded as a tumor suppressor (Janssens et al., 2005). Several studies have shown that PP2A might be able to affect the phosphorylation status of threonine 58 and serine 62 of c-myc in the direct or indirect pathways (Arnold and Sears, 2008, Dias et al., 2010, Henriksson et al., 1993, Lutterbach and Hann, 1994, Pulverer et al., 1994, Takumi et al., 2010). So, theoretically, given its potent effect on PP2A activity, MC-LR might be able to regulate c-myc protein stability. In addition, previous studies have shown that MC-LR could induce c-myc expression at the transcriptional level (Li et al., 2009, Takumi et al., 2010). Therefore, MC-LR might induce c-myc expression at both transcriptional and post-translational levels. However, the exact connection and molecular mechanisms between MC-LR toxicity and c-myc response remains unclear.
In the present study, we used HEK293 cells because kidney is an important target organ of MC-LR and the endogenous c-myc protein level in HEK293 cells is low (Supplementary Fig. 1). To clarify the exact connection between MC-LR toxicity and c-myc response, two stable c-myc protein point mutants (c-mycT58A and c-mycS62A) and three promoter mutants (P1 mut1, P1 mut2, P2 mut) were employed. The results suggested that MC-LR up-regulates the c-myc protein level mainly via the MC-LR/PP2A/serine 62 pathway.
Section snippets
Cell culture and reagents
HEK293 cells were obtained from the American Type Culture Collection and were maintained in Dulbecco's modified Eagle's medium (Hyclone, UT) supplemented with 10% (v/v) fetal bovine serum (Hyclone, UT) at 37 °C in a humidified atmosphere with 5% CO2. Cells were counted using an automated cell counter (TC10, Bio-Rad, CA). MC-LR (purity ≥ 95%) was extracted and purified from the freeze-dried surface blooms collected from Lake Dianchi, Yunnan, China, following the method described previously (Wang et
Effect of MC-LR on c-myc protein stability
To determine whether MC-LR could affect the stability of c-myc protein, HEK293 cells were transfected with exogenous wild-type c-myc expression vector and then treated with MC-LR. The expression of exogenous c-myc was verified by Western blot. As shown in Supplementary Fig. 1, the exogenous c-myc was clearly indicated by the c-myc antibody. While the endogenous c-myc protein level could hardly be detected, being much lower than the exogenous c-myc. Western blot assay was performed to assess the
Discussion
MC-LR has attracted increasing interest because of its tumor-promoting activity in hepatocytes of animals and humans. IARC has classified MC-LR as a possible human carcinogen (Cogliano et al., 2008). In this study, we showed that MC-LR increase c-myc protein stability by altering the phosphorylation status of serine 62. Furthermore, we identified that the TATA box of P1 promoter might play a critical role in MC-LR-induced up-regulation of c-myc promoter activity. These results suggested that
Conclusions
In the present study, we showed that MC-LR increased c-myc protein level mainly by altering the phosphorylation status of serine 62, which was mediated by the inhibition of the activity of PP2A. Moreover, we demonstrated that the TATA box for P1 of c-myc might play an important role in MC-LR-induced up-regulation of c-myc mRNA, which may also contribute to MC-LR-induced up-regulation of c-myc protein level. And we also found that MC-LR may have a relatively weak negative effect on c-myc protein
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
The authors thank Dr. Xiaoyang Wan for helping to revise the manuscript. This work was supported by the National Natural Science Foundation of China (31322013) and the State Key Laboratory of Freshwater Ecology and Biotechnology (2011FBZ07).
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These authors contributed equally to this work.