Multiple protein kinases influence the redistribution of fission yeast Clp1/Cdc14 phosphatase upon genotoxic stress.
The Cdc14 phosphatase family antagonizes Cdk1 phosphorylation and is important for mitotic exit. To access their substrates, Cdc14 phosphatases are released from nucleolar sequestration during mitosis. Clp1/Flp1, the Schizosaccharomyces pombe Cdc14 orthologue, and Cdc14B, a mammalian orthologue, also exit the nucleolus during interphase upon DNA replication stress or damage, respectively, implicating Cdc14 phosphatases in the response to genotoxic insults. However, a mechanistic understanding of Cdc14 phosphatase nucleolar release under these conditions is incomplete. We show here that relocalization of Clp1 during genotoxic stress is governed by complex phosphoregulation. Specifically, the Rad3 checkpoint effector kinases Cds1 and/or Chk1, the cell wall integrity mitogen-activated protein kinase Pmk1, and the cell cycle kinase Cdk1 directly phosphorylate Clp1 to promote genotoxic stress-induced nucleoplasmic accumulation. However, Cds1 and/or Chk1 phosphorylate RxxS sites preferentially upon hydroxyurea treatment, whereas Pmk1 and Cdk1 preferentially phosphorylate Clp1 TP sites upon H(2)O(2) treatment. Abolishing both Clp1 RxxS and TP phosphosites eliminates any genotoxic stress-induced redistribution. Reciprocally, preventing dephosphorylation of Clp1 TP sites shifts the distribution of the enzyme to the nucleoplasm constitutively. This work advances our understanding of pathways influencing Clp1 localization and may provide insight into mechanisms controlling Cdc14B phosphatases in higher eukaryotes.
Howard Hughes Medical Institute and Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Matthew R
MR
Broadus
Kathleen L
KL
Gould
D018797:Cell Cycle Proteins / Q000378:metabolism*; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology; D004249:DNA Damage; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006918:Hydroxyurea / Q000494:pharmacology; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism*; D013312:Stress, Physiological / Q000187:drug effects
D018797:Cell Cycle Proteins; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide; D011494:Protein Kinases; C439269:Clp1 protein, S. pombe; D017027:Protein Tyrosine Phosphatases; D006918:Hydroxyurea