Mapping of protein phosphatase-6 association with its SAPS domain regulatory subunit using a model of helical repeats.
BACKGROUND
Helical repeat motifs are common among regulatory subunits for type-1 and type-2A protein Ser/Thr phosphatases. Yeast Sit4 is a distinctive type-2A phosphatase that has dedicated regulatory subunits named Sit4-Associated Proteins (SAPS). These subunits are conserved, and three human SAPS-related proteins are known to associate with PP6 phosphatase, the Sit4 human homologue.


RESULTS
Here we show that endogenous SAPS subunit PP6R3 co-precipitates half of PP6 in cell extracts, and the SAPS region of PP6R3 is sufficient for binding PP6. The SAPS domain of recombinant GST-PP6R3 is relatively resistant to trypsin despite having many K and R residues, and the purified SAPS domain (residues 1-513) has a circular dichroic spectrum indicative of mostly alpha helical structure. We used sequence alignments and 3D-jury methods to develop alternative models for the SAPS domain, based on available structures of other helical repeat proteins. The models were used to select sites for charge-reversal substitutions in the SAPS domain of PP6R3 that were tested by co-precipitation of endogenous PP6c with FLAG-tagged PP6R3 from mammalian cells. Mutations that reduced binding with PP6 suggest that SAPS adopts a helical repeat similar to the structure of p115 golgin, but distinct from the PP2A-A subunit. These mutations did not cause perturbations in overall PP6R3 conformation, evidenced by no change in kinetics or preferential cleavage by chymotrypsin.


CONCLUSIONS
The conserved SAPS domain in PP6R3 forms helical repeats similar to those in golgin p115 and negatively charged residues in interhelical loops are used to associate specifically with PP6. The results advance understanding of how distinctive helical repeat subunits uniquely distribute and differentially regulate closely related Ser/Thr phosphatases.
Guergnon
Julien
J
Center for Cell Signalling, University of Virginia School of Medicine, Charlottesville, VA 22908 USA.
Derewenda
Urszula
U
Edelson
Jessica R
JR
Brautigan
David L
DL
D020816:Amino Acid Motifs / Q000235:genetics*; D052005:Bone Morphogenetic Protein Receptors, Type I; D045744:Cell Line, Tumor; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D054648:Protein Phosphatase 2 / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics*; D016415:Sequence Alignment; D017384:Sequence Deletion; D019076:Transgenes / Q000235:genetics
D052005:Bone Morphogenetic Protein Receptors, Type I; C517189:PPP2CA protein, human; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2