title: Protein phosphatase 6 regulatory subunits composed of ankyrin repeat domains.
abstract: Protein phosphatase 6 (PP6) is an essential Ser/Thr phosphatase conserved among eukaryotes. The Saccharomyces cerevisiae homologue of PP6 called Sit4 depends on association with SAPS domain subunits. This study used a human SAPS domain subunit FLAG-PP6R1 to identify endogenous interacting proteins. Mass spectrometry identified coprecipitating proteins as PP6 catalytic subunit and three ankyrin repeat proteins (Ankrd28, Ankrd44, and Ankrd52). These proteins have extensive sequence identity to one another but segregate into separate branches on a phylogenetic tree for vertebrate species, suggesting individual biological functions. Tagged Ankrd28 coprecipitated with PP6, not with PP2A or PP4, and with SAPS domain subunits PP6R1 and PP6R3. Tagged PP6 coprecipitated endogenous SAPS domain subunits and Ankrd28. The C-terminal region of PP6R1 was sufficient to coprecipitate Ankrd28, but not PP6, demonstrating that PP6R1 acts as a scaffold with separate regions for binding to PP6 and to Ankrd28. Endogenous PP6 holoenzymes with PP6R1 and PP6R3 subunits were resolved by DEAE chromatography and eluted together with Ankrd28 at Mr > 440 kDa from Superose 12. Knockdown of PP6R1 or Ankrd28, but not PP6R3, produced equivalent enhancement of IkappaBepsilon degradation in response to TNFalpha. The results suggest that PP6 functions as a heterotrimer, composed of the PP6 catalytic subunit bound to a SAPS domain scaffold subunit that associates with Ankrd28. We propose that the SAPS and ankyrin repeat regulatory subunits determine the function and specificity of PP6.
authors.affiliation: Center for Cell Signaling and Department of Microbiology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
authors.forename: Bjarki
authors.initials: B
authors.lastname: Stefansson
authors.forename: Takashi
authors.initials: T
authors.lastname: Ohama
authors.forename: Abbi E
authors.initials: AE
authors.lastname: Daugherty
authors.forename: David L
authors.initials: DL
authors.lastname: Brautigan
mesh_terms: D000595:Amino Acid Sequence; D017089:Ankyrin Repeat; D002478:Cells, Cultured; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000235:genetics; D010802:Phylogeny; D034741:RNA, Small Interfering / Q000235:genetics; D016415:Sequence Alignment
chemical_list: D034741:RNA, Small Interfering; C000621177:ANKRD52 protein, human; D010749:Phosphoprotein Phosphatases; C106079:protein phosphatase 6