XB-ART-238J Biol Chem August 11, 2006; 281 (32): 22624-34.
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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.
PubMed ID: 16769727
Article link: J Biol Chem
Species referenced: Xenopus laevis
Genes referenced: ppp4c ppp6c ppp6r1 ppp6r2 ppp6r3 ptpa
Article Images: [+] show captions
|FIGURE 1. Protein sequences of SAPS-related proteins and sequence alignment. Sequences for PP6R1 (KIAA1115, accession number BC002799) , PP6R2 (KIAA0685, accession number BC000976) , and PP6R3 (C11orf23, accession number DQ111954) were aligned using the Clustal method (see “Experimental Procedures”), and identical residues in all three proteins are printed in reverse. The SAPS domain (according to Pfam assignment) is underlined with a thick black line. PP6R3 appears to have multiple alternatively spliced forms (Q3KR35, Q5H9R7, Q96MB2, Q9HCL4, Q68CR3, Q9H2K5, Q9H2K6, OBS#8642489 Incyte Human cDNA from Open Biosystems). These spliced forms produce differences in the N-terminal half of the SAPS domain, whereas the C-terminal half appears the same. The schematic below the sequences shows the relative size and position of the SAPS domains in the PP6R1, PP6R2, and PP6R3 proteins. The broken line between the N terminus and the SAPS domain in PP6R3 denotes the existence of multiple spliced versions of PP6R3.|
|FIGURE 2. Phylogenetic tree of SAPS-related proteins. A parsimony consensus (unrooted) tree was prepared as described under “Experimental Procedures.” Accession numbers of sequences used to construct the phylogenetic tree are included in the figure except PP6R1 H. sapiens BAA83067.3, PP6R1 M. musculus Q7TSI3, PP6R1 X. laevis Q6PCI0, PP6R2 H. sapiens Q7Z5G5, PP6R2 M. musculus Q8R3Q2, PP6R2 X. laevis Q6GNW5, PP6R3 H. sapiens Q9H2K6, PP6R3 M. musculus Q922D4, and PP6R3 X. laevis Q6NRF1. Sc, S. cerevisiae; Sp, S. pombe; Ce, C. elegans; Dm, D. melanogaster; Os, Oryza sativa; At, Arabidopsis thaliana; XI, X. laevis; Mm, M. musculus; Hs, H. sapiens.|