Ampk Alpha Subunit Ssp2 And Glycogen Synthase Kinases Gsk3/Gsk31 Are Involved In Regulation Of Sterol Regulatory Element-Binding Protein (Srebp) Activity In Fission Yeast

Sterol regulatory element-binding protein (SREBP), a highly conserved family of membrane-bound transcription factors, is an essential regulator for cellular cholesterol and lipid homeostasis in mammalian cells. Sre1, the homolog of SREBP in the fission yeast Schizosaccharomyces pombe (S. pombe), regulates genes involved in the transcriptional responses to low sterol as well as low oxygen. Previous study reported that casein kinase 1 family member Hhp2 phosphorylated the Sre1 N-terminal transcriptional factor domain (Sre1N) and accelerated Sre1N degradation, and other kinases might exist for regulating the Sre1 function. To gain insight into the mechanisms underlying the Sre1 activity and to identify additional kinases involved in regulation of Sre1 function, we developed a luciferase reporter system to monitor the Sre1 activity through its binding site called SRE2 in living yeast cells. Here we showed that both ergosterol biosynthesis inhibitors and hypoxia-mimic CoCl2 caused a dose-dependent increase in the Sre1 transcription activity, concurrently, these induced transcription activities were almost abolished in Delta sre1 cells. Surprisingly, either AMPK alpha Subunit Ssp2 deletion or Glycogen Synthase Kinases Gsk3/Gsk31 double deletion significantly suppressed ergosterol biosynthesis inhibitors- or CoCl2-induced Sre1 activity. Notably, the Delta ssp2 Delta gsk3 Delta gsk31 mutant showed further decreased Sre1 activity when compared with their single or double deletion. Consistently, the Delta ssp2 Delta gsk3 Delta gsk31 mutant showed more marked temperature sensitivity than any of their single or double deletion. Moreover, the fluorescence of GFP-Sre1N localized at the nucleus in wild-type cells, but significantly weaker nuclear fluorescence of GFP-Sre1N was observed in Delta ssp2, Delta gsk3 Delta gsk31, Delta ssp2 Delta gsk3, Delta ssp2 Delta gsk31 or Delta ssp2 Delta gsk3 Delta gsk31 cells. On the other hand, the immunoblot showed a dramatic decrease in GST-Sre1N levels in the Delta gsk3 Delta gsk31 or the Delta ssp2 Delta gsk3 Delta gsk31 cells but not in the Delta ssp2 cells. Altogether, our findings suggest that Gsk3/Gsk31 may regulate Sre1N degradation, while Ssp2 may regulate not only the degradation of Sre1N but also its translocation to the nucleus.