Identification of sds21 in fission yeast in an inhibitor-resistant high molecular mass protein phosphatase-1 complex

While characterizing the type-1 protein phosphatases sds21 and dis2 in fiss ion yeast (Schizosaccharomyces pombe) a novel high molecular mass protein w as identified with serine/threonine phosphatase activity (referred to as PP -R) that was resistant to a panel of characteristic inhibitors of protein p hosphatases. Purification of the native sds21 catalytic isoform of protein phosphatase-1 (PP-1) from an S. pombe knockout strain lacking dis2 (Delta d is2) resulted predominantly in identification of PP-R. To test the hypothes is that the catalytic activity of PP-R comprised sds21, a parallel purifica tion was performed of PP-1 activity from an S. pombe knockout strain lackin g sds21 (Delta sds21). Both Delta sds21 and Delta dis2 strains exhibited si milar protein phosphatase activity profiles as determined by DEAE-sepharose , Mono-Q and Superdex gel filtration chromatography. However, the peak of p rotein phosphatase activity from Delta sds21 S. pombe that co-migrated with PP-R from Delta dis2 S. pombe exhibited the sensitivity to a panel of inhi bitors that was characteristic of a type-1 protein phosphatase. These data suggest that the catalytic subunit of PP-R comprises sds21 and that the res istance to inhibitors may originate from structural differences between dis 2 and sds21 isoforms. A key structural feature present in sds21, but lackin g in dis2, is a classical phosphorylation consensus sequence surrounding se rine-145 of sds21. The previous hypothesis was that PP-1 activity among sev eral lower eukaryotes may be regulated directly by cAMP-dependent protein k inase (PKA) phosphorylation. However, this study demonstrated that recombin ant sds21 is not a target for PKA in vitro. The constrained configuration o f the putative PKA site on the PP-1 holoenzyme may restrict its ability to be targeted by PKA.