Identification of protein phosphatase-1-binding proteins by microcystin-biotin affinity chromatography.

Journal Article (Journal Article)

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.

Full Text

Duke Authors

Cited Authors

  • Campos, M; Fadden, P; Alms, G; Qian, Z; Haystead, TA

Published Date

  • November 8, 1996

Published In

Volume / Issue

  • 271 / 45

Start / End Page

  • 28478 - 28484

PubMed ID

  • 8910475

International Standard Serial Number (ISSN)

  • 0021-9258

Digital Object Identifier (DOI)

  • 10.1074/jbc.271.45.28478


  • eng

Conference Location

  • United States