Identification of protein phosphatase-1-binding proteins by microcystin-biotin affinity chromatography.
Biotinylated microcystin was used to affinity purify over avidin-Sepharose the entire cellular content of active forms of protein phosphatase (PP) 1 and 2A holoenzymes present in three subcellular fractions of skeletal muscle. Biotinylated microcystin displayed IC50 values in the nM range against PP-1C (1.58 +/- 0.6 nM S.E., n = 3), PP-2AC (0.63 +/- 0.2 nM S.E., n = 3) and SMPP-1M (5.9 +/- 1.3 S.E., n = 3). Subsequent anion-exchange chromatography and SDS-polyacrylamide gel electrophoresis of the microcystin-biotin eluates of the three fractions revealed a complex pattern of proteins associated with PP-1C and PP-2AC. Far Western analysis and the rebinding interaction with recombinant PP-1C distinguished proteins in the eluates that bound PP-1C from those that bound PP-2AC. In Far Western analysis, 29 distinct proteins were identified to bind PP-1C. Significantly, these same proteins, plus seven others, were also recovered from the isothiocyanate eluates from microcystin-Sepharose by a rebinding interaction with PP-1C-microcystin-biotin. The number of proteins and range of novel molecular masses (18-125 kDa) identified to interact with PP-1C by these two techniques cannot be accounted for by the previously characterized subunits of PP-1. Our findings further support the concept that PP-1C is regulated in vivo by multiple and distinct substrate-targeting subunits.
Department of Pharmacology, and Markey Center for Cell Signaling, University of Virginia, Charlottesville, Virginia 22908, USA.
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Qian
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Haystead
D000818:Animals; D001710:Biotin; D015153:Blotting, Western; D002352:Carrier Proteins / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008297:Male; D052998:Microcystins; D018482:Muscle, Skeletal / Q000201:enzymology; D010456:Peptides, Cyclic; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D054645:Protein Phosphatase 1; D051381:Rats; D017208:Rats, Wistar
D002352:Carrier Proteins; D052998:Microcystins; D010456:Peptides, Cyclic; D001710:Biotin; C078588:microcystin; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1