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).

Highlights

  • 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

  • Identification of Human PP4C-binding Proteins—tandem affinity purification (TAP) tagging and mass spectrometry, in which a protein complex of interest is purified in a two-step affinity enrichment process and its components are identified by the fragment ion spectra of selected tryptic peptides, have proven to be invaluable tools in the characterization of protein complexes [16, 21,22,23]

  • Using an iterative TAP tagging/mass spectrometry approach, we report that the mammalian PP4 catalytic subunit is a component of several different mutually exclusive subcomplexes: one containing PP4R1, another containing alpha4 and the TRiC/CCT complex, and a third (PP4cs) containing PP4R2 and a novel protein that we termed PP4R3

Read more

Summary

Introduction

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. Mammalian PP4 Module Interaction Network—To confirm the observed novel interactions and to better understand the supramolecular architecture of PP4C-containing complexes in mammals, we cloned the human alpha4, PP4R1 (PPP4R1), PP4R2 (PPP4R2), and PP4R3␣ (KIAA2010) ORFs into TAP tag vectors, established HEK293 cell lines stably expressing these tagged proteins, and repeated the purification and mass spectrometric identification process for each of these proteins and their interacting partners (Fig. 1, A and D, and Table I).

Results
Conclusion

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call

Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.