CLOCK is a substrate of SUMO and sumoylation of CLOCK upregulates the transcriptional activity of estrogen receptor-α

Abstract

Disruption of the circadian rhythm is now believed to associate with a number of hormone-related cancers, such as breast cancer, in which aberrant estrogen receptor-α (ERα) signaling is a major contributor. However, the molecular mechanisms underlying the function of core clock proteins in cancer are still largely undefined. In this study, we showed that circadian locomotor output cycles kaput (CLOCK), a key circadian protein, can interact with ERα. Furthermore, this interaction was enhanced by estrogen. We also showed that CLOCK can be sumoylated and sumoylation of CLOCK, which is also stimulated by estrogen, had two consequences: (1) it increased the transcriptional activity of CLOCK; and (2) it increased the CLOCK-modulated transcriptional activity of ERα, as shown by increased transcription of cyclin D1. Sumoylation of CLOCK occurred at two lysine residues, K67 and K851. The enhancement of ERα transcriptional activity exerted by wild-type but not mutant (2K/2R) CLOCK in response to estrogen indicated that sumoylation of CLOCK may have an important role in estrogen-dependent signaling. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay conducted with breast cancer cell lines (MCF-7 and T47D) demonstrated that sumoylation of CLOCK stimulated cell growth and increased the proportion of S phase cells in the cell cycle. The results of this study uncovered new insight into the connection between a major circadian protein and a major estrogen-dependent transcription factor, providing the basis for further research into the involvement of circadian proteins in breast cancer.

The intricate dance of post-translational modifications in the rhythm of life

“…SUMOylation at Lys259 induces ubiquitination of BMAL1 and increases transactivation and degradation 91,92 . In a similar manner, SUMOylation at Lys67 and Lys851 of human CLOCK increases the activity of CLOCK 93 , although it is unclear whether this modification destabilizes CLOCK. O-GlcNAcylation at Ser418 of BMAL1 and an unknown site of CLOCK stabilizes CLOCK and BMAL1 by inhibiting ubiquitination 94 (Fig.…”

SUMOylation of GPS2 protein regulates its transcription-suppressing function

“…COS-7, MCF-7, and T47D cells were used in our previous work (Venteclef et al ., 2010; Li et al ., 2013). COS-7 and MCF-7 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS; Hyclone, Beijing, China) and penicillin-streptomycin (100 U/ml penicillin and 0.1 mg/ml streptomycin).…”

“…GAL4-UAS-Luc reporter construct used in our previous study was purchased from Promega (E1370; Madison, WI; Li et al ., 2013; Yang et al ., 2013). It contains 9×GAL4 UAS and the DNA sequence of luciferase.…”

SUMOylation of PES1 upregulates its stability and function via inhibiting its ubiquitination

“…COS-7, MCF-7 and T47D cells have been used in our previous study [22, 23]. For the cell starvation assay, MCF-7 and T47D cells were cultured in medium containing 2% charcoal-stripped fetal bovine serum (Gibico) and without phenol red for several days, and then treated with or without E2 (10 nM) for several hours.…”

“…Total RNA was extracted as previously described [22]. The primers used for the RT-PCR analysis of cyclin D1 and GAPDH were as previously described [22]. C yclin D1 mRNA level was normalized with the GAPDH mRNA level.…”