Microcystin-LR stabilizes c-myc protein by inhibiting protein phosphatase 2A in HEK293 cells

Highlights

• MC-LR stabilizes c-myc protein, which is mainly by the PP2A/serine 62 pathway.

• The TATA box for P1 is involved in the MC-LR-induced up-regulation of c-myc mRNA.

• MC-LR has triple effects on c-myc protein.

• C-myc response to MC-LR toxicity is mainly by the PP2A/serine 62 pathway.

Abstract

Microcystin-LR is the most toxic and the most frequently encountered toxin produced by the cyanobacteria in the contaminated aquatic environment. Previous studies have demonstrated that Microcystin-LR is a potential carcinogen for animals and humans, and the International Agency for Research on Cancer has classified Microcystin-LR as a possible human carcinogen. However, the precise molecular mechanisms of Microcystin-LR-induced carcinogenesis remain a mystery. C-myc is a proto-oncogene, abnormal expression of which contributes to the tumor development. Although several studies have demonstrated that Microcystin-LR could induce c-myc expression at the transcriptional level, the exact connection between Microcystin-LR toxicity and c-myc response remains unclear. In this study, we showed that the c-myc protein increased in HEK293 cells after exposure to Microcystin-LR. Coexpression of protein phosphatase 2A and two stable c-myc protein point mutants (either c-myc^T58A or c-myc^S62A) showed that Microcystin-LR increased c-myc protein level mainly through inhibiting protein phosphatase 2A activity which altered the phosphorylation status of serine 62 on c-myc. In addition, we also showed that Microcystin-LR could increase c-myc promoter activity as revealed by luciferase reporter assay. And the TATA box for P1 promoter of c-myc might be involved. Our results suggested that Microcystin-LR can stimulate c-myc transcription and stabilize c-myc protein, which might contribute to hepatic tumorigenesis in animals and humans.

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-myc^T58A and c-myc^S62A) 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.