title: Tandem affinity purification in transgenic mouse embryonic stem cells identifies DDOST as a novel PPP1CC2 interacting protein.
abstract: Members of the PP1 family of protein phosphatases achieve functional diversity through numerous and varied protein-protein interactions. In mammals, there are four PP1 isoforms, the ubiquitously expressed PPP1CA, PPP1CB, and PPP1CC1, and the testis specific splice isoform PPP1CC2. When the mouse Ppp1cc gene is deleted, the only phenotypic consequence is a failure of spermatogenesis in homozygous males. To elucidate the function of the Ppp1cc gene, we sought to identify novel protein-protein interactions. To this end, we have created SBP-3XFLAG-PPP1CC1 and SBP-3XFLAG-PPP1CC2 knock-in mouse embryonic stem cell lines using a gene-trap-based system. Tandem affinity purification using our knock-in cell lines identified 11 significant protein-protein interactions, including nine known PP1 interacting proteins and two additional proteins (ATP5C1 and DDOST). Reciprocal in vitro sedimentation assays confirmed the interaction between PPP1CC2 and DDOST that may have physiological implications in spermatogenesis. Immunolocalization studies revealed that DDOST localized to the nuclear envelope in dissociated spermatogenic cells and persists throughout spermatogenesis. The knock-in system described in this paper can be applied in creating tandem affinity-tagged knock-in embryonic stem cell lines with any gene for which a compatible gene-trap line is available.
authors.affiliation: Department of Cell & Systems Biology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Graham
authors.initials: G
authors.lastname: MacLeod
authors.forename: Susannah
authors.initials: S
authors.lastname: Varmuza
mesh_terms: D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods*; D017931:DNA Primers; D053595:Embryonic Stem Cells / Q000378:metabolism*; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D053719:Tandem Mass Spectrometry
chemical_list: D017931:DNA Primers; D010749:Phosphoprotein Phosphatases