Protein phosphatase 6 subunit with conserved Sit4-associated protein domain targets IkappaBepsilon.
Protein Ser/Thr phosphatases compose a PPP family that includes type-2 PP2A, PP4, and PP6, each with essential functions. The human PP6 gene rescues sit4(ts) mutants of Saccharomyces cerevisiae, and Sit4 phosphatase function depends on multiple Sit4-associated protein (SAP) subunits. We report here finding a SAPS sequence domain encoded in only a single gene each in Schizosaccharomyces pombe, Caenorhabditis elegans, and Drosophila but in three distinct open reading frames in Xenopus, Mus musculus, and Homo sapiens. The SAPS proteins are more divergent in sequence than PP6. Northern hybridization showed differential distribution of the human SAPS-related mRNA in multiple human tissues, named as PP6R1, PP6R2, and PP6R3. Antibodies were generated, distribution of endogenous PP6, PP6R1, PP6R2, and PP6R3 proteins was examined by immunoblotting, and the abundance of mRNA and protein in various tissues did not match. FLAG-tagged PP6R1 and PP6R2 expressed in HEK293 cells co-precipitated endogenous PP6, but not PP2A or PP4, showing specificity for recognition of phosphatases. The SAPS domain of PP6R1 alone was sufficient for association with PP6, and this predicts that conserved sequence motifs in the SAPS domain accounts for the specificity. FLAG-PP6R1 and FLAG-PP6R2 co-precipitated HA-IkappaBepsilon. Knockdown of PP6 or PP6R1 but not PP6R3 with siRNA significantly enhanced degradation of endogenous IkappaBepsilon in response to tumor necrosis factor-alpha. The results show SAPS domain subunits recruit substrates such as IkappaBepsilon as one way to determine specific functions for PP6.
Stefansson
Bjarki
B
Center for Cell Signaling, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
Brautigan
David L
DL
D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans; D002460:Cell Line; D004331:Drosophila melanogaster; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology*; D010802:Phylogeny; D011485:Protein Binding; D054648:Protein Phosphatase 2; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014018:Tissue Distribution; D014982:Xenopus laevis
D020672:I-kappa B Proteins; C105627:NFKBIE protein, human; D011518:Proto-Oncogene Proteins; D029701:Saccharomyces cerevisiae Proteins; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; C104343:SIT4 protein, S cerevisiae; C106079:protein phosphatase 6