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 fission yeast (Schizosaccharomyces pombe) a novel high molecular mass protein was identified with serine/threonine phosphatase activity (referred to as PP-R) that was resistant to a panel of characteristic inhibitors of protein phosphatases. Purification of the native sds21 catalytic isoform of protein phosphatase-1 (PP-1) from an S. pombe knockout strain lacking dis2 (deltadis2) resulted predominantly in identification of PP-R. To test the hypothesis that the catalytic activity of PP-R comprised sds21, a parallel purification was performed of PP-1 activity from an S. pombe knockout strain lacking sds21 (deltasds21). Both deltasds21 and deltadis2 strains exhibited similar protein phosphatase activity profiles as determined by DEAE-sepharose, Mono-Q and Superdex gel filtration chromatography. However, the peak of protein phosphatase activity from deltasds21 S. pombe that co-migrated with PP-R from deltadis2 S. pombe exhibited the sensitivity to a panel of inhibitors that was characteristic of a type-1 protein phosphatase. These data suggest that the catalytic subunit of PP-R comprises sds21 and that the resistance to inhibitors may originate from structural differences between dis2 and sds21 isoforms. A key structural feature present in sds21, but lacking in dis2, is a classical phosphorylation consensus sequence surrounding serine-145 of sds21. The previous hypothesis was that PP-1 activity among several lower eukaryotes may be regulated directly by cAMP-dependent protein kinase (PKA) phosphorylation. However, this study demonstrated that recombinant sds21 is not a target for PKA in vitro. The constrained configuration of the putative PKA site on the PP-1 holoenzyme may restrict its ability to be targeted by PKA.
Dawson
J F
JF
Department of Biochemistry, University of Alberta, Edmonton, Canada. jf_dawson@hotmail.com
Holmes
C F
CF
D000595:Amino Acid Sequence; D002457:Cell Extracts; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D012568:Schizosaccharomyces / Q000737:chemistry / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins
D002457:Cell Extracts; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D007527:Isoenzymes; D029702:Schizosaccharomyces pombe Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C404792:SDS21 protein, S pombe