ABSTRACT
Using a combination of tandem affinity purification tagging and mass spectrometry, we characterized a novel, evolutionarily conserved protein phosphatase 4 (PP4)-containing complex (PP4cs, protein phosphatase 4, cisplatin-sensitive complex) that plays a critical role in the eukaryotic DNA damage response. PP4cs is comprised of the catalytic subunit PP4C; a known regulatory subunit, PP4R2; and a novel protein that we termed PP4R3. The Saccharomyces cerevisiae PP4R3 ortholog Psy2 was identified previously in a screen for sensitivity to the DNA-damaging agent and anticancer drug cisplatin. We demonstrated that deletion of any of the PP4cs complex orthologs in S. cerevisiae elicited cisplatin hypersensitivity. Furthermore human PP4R3 complemented the yeast psy2 deletion, and Drosophila melanogaster lacking functional PP4R3 (flfl) exhibited cisplatin hypersensitivity, suggesting a highly conserved role for PP4cs in DNA damage repair. Finally we found that PP4R3 may target PP4cs to the DNA damage repair machinery at least in part via an interaction with Rad53 (CHK2).
Subject(s)
Cisplatin/pharmacology , Conserved Sequence , Evolution, Molecular , Phosphoprotein Phosphatases/metabolism , Cell Line , DNA Damage , Drug Hypersensitivity , Humans , Models, Biological , Multiprotein Complexes/chemistry , Multiprotein Complexes/isolation & purification , Multiprotein Complexes/metabolism , Mutation/genetics , Phosphoprotein Phosphatases/chemistry , Phosphoprotein Phosphatases/genetics , Phosphoprotein Phosphatases/isolation & purification , Protein Binding , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/analysis , Saccharomyces cerevisiae Proteins/metabolism , Sequence Alignment , Two-Hybrid System TechniquesABSTRACT
The Phosphatidylinositol-3 kinase/Protein Kinase B (PI3K/PKB) signaling pathway controls growth, metabolism, and lifespan in animals, and deregulation of its activity is associated with diabetes and cancer in humans. Here, we describe Susi, a coiled-coil domain protein that acts as a negative regulator of insulin signaling in Drosophila. Whereas loss of Susi function increases body size, overexpression of Susi reduces growth. We provide genetic evidence that Susi negatively regulates dPI3K activity. Susi directly binds to dP60, the regulatory subunit of dPI3K. Since Susi has no overt similarity to known inhibitors of PI3K/PKB signaling, it defines a novel mechanism by which this signaling cascade is kept in check. The fact that Susi is expressed in a circadian rhythm, with highest levels during the night, suggests that Susi attenuates insulin signaling during the fasting period.
