Swe1p blocks entry into mitosis through inhibitory phosphorylation of a conserved tyrosine residue, Y19, in the cyclin-dependent kinase Cdc28p ( Booher et al., 1993). The cell cycle arrest is enacted by Swe1p, the sole Wee1-family kinase in S. This in turn triggers cell cycle arrest in G2 through the morphogenesis checkpoint ( Lew and Reed, 1995 McMillan et al., 1998). Bud formation requires a polarized actin cytoskeleton, so that during some stress responses bud formation is temporarily halted. Stress responses often involve transient depolarization of the actin cytoskeleton ( Chowdhury et al., 1992 Delley and Hall, 1999) in addition to changes in gene expression ( Gasch et al., 2000).
Yeast cells deploy a panoply of stress responses to adapt to changes in environmental conditions. The other region did not appear to affect interactions with known Swe1p regulators, suggesting that other as-yet-unknown regulators exist. One of these regions mediates interaction of Swe1p with Hsl7p, showing that the Swe1p-Hsl7p interaction is critical for Swe1p neck targeting and degradation.
Third, a screen for functional but nondegradable mutants of SWE1 identified two small regions of Swe1p that are key to its degradation. Second, cyclin/Cdc28p does not influence Swe1p neck targeting, but can directly phosphorylate Swe1p, suggesting that it acts downstream of neck targeting in the Swe1p degradation pathway. First, we show here that Met30p (and by implication SCF Met30) is not, in fact, required for Swe1p degradation. The basis for regulation of Swe1p degradation by the morphogenesis checkpoint remains unclear, and in order to elucidate that regulation we have dissected the Swe1p degradation pathway in more detail, yielding several novel findings. In addition, Swe1p degradation is stimulated by its substrate, cyclin/Cdc28p, and Swe1p is thought to be a target of the ubiquitin ligase SCF Met30 acting with the ubiquitin-conjugating enzyme Cdc34p. Swe1p degradation involves the relocalization of Swe1p from the nucleus to the mother-bud neck, and neck targeting requires the Swe1p-interacting protein Hsl7p. The Swe1p stabilization promotes cell cycle arrest through Swe1p-mediated inhibitory phosphorylation of Cdc28p until the cells can recover from the perturbation and resume bud formation. However, Swe1p degradation is halted by the morphogenesis checkpoint, which responds to insults that perturb bud formation. While SAM's mechanism is NF-κB-dependent, MTA has both NF-κB-dependent and -independent mechanisms.Swe1p, the sole Wee1-family kinase in Saccharomyces cerevisiae, is synthesized during late G1 and is then degraded as cells proceed through the cell cycle. In conclusion, SAM and MTA down-regulate BHMT expression in HepG2 cells in part by inducing NF-κB, which acts as a repressor for the human BHMT gene. Indeed, MTA treatment resulted in increased expression ER stress markers. Lower BHMT expression can impair homocysteine metabolism, which can induce ER (endoplasmic reticulum) stress. The NF-κB binding site at −301 is responsible, at least in part, for this effect.
Overexpression of p50 and p65 decreased BHMT promoter activity, while blocking NF-κB activation increased BHMT expression and promoter activity, and prevented SAM but not MTA's ability to inhibit BHMT expression. SAM and MTA treatment increased NF-κB nuclear binding and NF-κB-driven luciferase activities, and increased nuclear binding activity of multiple histone deacetylase co-repressors to the NF-κB sites. Maximal suppression was observed with the BHMT promoter construct −347/+33, which contains a number of NF-κB (nuclear factor κB) binding sites. Both SAM and MTA treatment of HepG2 cells resulted in a dose- and time-dependent decrease in BHMT mRNA levels, which paralleled their effects on the BHMT promoter activity. To facilitate these studies, we cloned the 2.7 kb 5′-flanking region of the human BHMT gene (GenBank® accession number AY325901). The present study examined regulation of the human BHMT gene by SAM and its metabolite, MTA (5′-methylthioadenosine). Transcriptional regulation of the human BHMT is also unknown. SAM ( S-adenosylmethionine) inhibits BHMT activity, but whether SAM modulates BHMT gene expression is unknown. Access content during the Covid-19 pandemicīHMT (betaine–homocysteine methyltransferase) remethylates homocysteine to form methionine.
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