Hydroxyurea sensitivity reveals a role for ISC1 in the regulation of G2/M.
Saccharomyces cerevisiae cells lacking ISC1 (inositol phosphosphingolipase C) exhibit sensitivity to genotoxic agents such as methyl methanesulfonate and hydroxyurea (HU). Cell cycle analysis by flow cytometry revealed a G(2)/M block in isc1Delta cells when treated with methyl methanesulfonate or HU. Further investigation revealed that the levels of Cdc28 phosphorylated on Tyr-19, which plays an essential role in the regulation of the G(2)/M checkpoint, were higher in synchronized and asynchronous cells lacking ISC1 in response to HU. Use of a Cdc28-Y19F mutant protected isc1Delta from the G(2)/M block. In wild type cells, HU induced a loss of the Swe1p kinase, the enzyme that phosphorylates Cdc28-Tyr-19, correlating with resumption of the cell cycle. In the isc1Delta cells, however, the levels of Swe1p remained at sustained high levels in response to HU. Significantly, deletion of SWE1 in an isc1Delta background overcame the G(2)/M block in response to HU. The double isc1Delta/swe1Delta mutant also overcame the growth defect on HU. Taken together, these findings implicate Isc1p as an upstream regulator of Swe1p levels and stability and Cdc28-Tyr-19 phosphorylation, in effect signaling recovery from the effects of genotoxic stress and allowing G(2)/M progression.
Matmati
Nabil
N
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29403, USA.
Kitagaki
Hiroshi
H
Montefusco
David
D
Mohanty
Bidyut K
BK
Hannun
Yusuf A
YA
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017489:CDC28 Protein Kinase, S cerevisiae / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects* / Q000502:physiology; D004249:DNA Damage / Q000187:drug effects / Q000502:physiology; D016195:G2 Phase / Q000187:drug effects* / Q000502:physiology; D055786:Gene Knockout Techniques; D006918:Hydroxyurea / Q000494:pharmacology*; D008698:Mesylates / Q000494:pharmacology; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D008698:Mesylates; D029701:Saccharomyces cerevisiae Proteins; C045880:methanesulfonic acid; C099685:SWE1 protein, S cerevisiae; D011505:Protein-Tyrosine Kinases; D017489:CDC28 Protein Kinase, S cerevisiae; C419391:ISC1 protein, S cerevisiae; D010738:Type C Phospholipases; D006918:Hydroxyurea