AMPKα Subunit Ssp2 and Glycogen Synthase Kinases Gsk3/Gsk31 are involved in regulation of sterol regulatory element-binding protein (SREBP) activity in fission yeast

Abstract

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 CoCl 2 caused a dose-dependent increase in the Sre1 transcription activity, concurrently, these induced transcription activities were almost abolished in Δsre1 cells. Surprisingly, either AMPKα Subunit Ssp2 deletion or Glycogen Synthase Kinases Gsk3/Gsk31 double deletion significantly suppressed ergosterol biosynthesis inhibitors-or CoCl 2-induced Sre1 activity. Notably, the Δssp2Δgsk3Δgsk31 mutant showed further decreased Sre1 activity when compared with their single or double deletion. Consistently, the Δssp2Δgsk3Δgsk31 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 Δssp2, Δgsk3Δgsk31, Δssp2Δgsk3, Δssp2Δgsk31 or Δssp2Δgsk3Δgsk31 cells. On the other hand, the immunoblot showed a dramatic decrease in GST-Sre1N levels in the Δgsk3Δgsk31 or the Δssp2Δgsk3Δgsk31 cells but not in the Δssp2 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.

Environmental control of Pub1 (NEDD4 family E3 ligase) inS. pombeis regulated by TORC2 and Gsk3

“…Fission yeast Gsk31 is an ortholog of Gsk3. Whilst Pub1 levels remained unaffected in the gsk31.D deletion strain, Pub1 protein levels were further reduced in gsk3.D gsk31.D double deletion when compared to gsk3.D (Figure 4A), indicating that the two Gsk3 kinases are capable of functional redundancy upon deletion (Miao et al, 2020;Qingyun et al, 2016). Although the growth rate of the gsk3.D gsk31.D double mutant on minimal EMM2 media is reduced (Figure 4B) it is sensitive to canavanine.…”

Environmental control of Pub1 (NEDD4 family E3 ligase) in Schizosaccharomyces pombe is regulated by TORC2 and Gsk3

“…Fission yeast Gsk31 is an ortholog of Gsk3. Whereas Pub1 levels remained unaffected in the gsk31.Δ deletion strain, Pub1 protein levels were further reduced in gsk3.Δ gsk31.Δ double deletion when compared with gsk3.Δ (Fig 4A ), indicating that the two Gsk3 kinases are capable of functional redundancy upon deletion (Qingyun et al, 2016;Miao et al, 2020). Although the growth rate of the gsk3.Δ gsk31.Δ double mutant on minimal EMM2 medium is reduced (Fig 4B) it is sensitive to canavanine.…”

Interplays of AMPK and TOR in Autophagy Regulation in Yeast

“…Furthermore, TORC2 appears to suppress autophagy by enhancing TORC1 activity. As glucose deprivation inhibits TORC2 activation while enhancing Gsk3/Gsk31 activity, these conditions may promote autophagy via reduced TORC1 activation [102,105,106] (Figure 2). loss of Ssp2 expression by inhibiting TORC1 activity via the Tsc1 and Tsc2 complex [104,105].…”