Identification of Protein Phosphatase-1-binding Proteins by Microcystin-Biotin Affinity Chromatography

doi:10.1074/jbc.271.45.28478

Identification of Protein Phosphatase-1-binding Proteins by Microcystin-Biotin Affinity Chromatography*

From the Department of Pharmacology, and Markey Center for Cell Signaling, University of Virginia, Charlottesville, Virginia 22908

^‡ To whom correspondence should be addressed:
Timothy A. J. Haystead, Ph.D., Department of Pharmacology, Box 448, University of Virginia Health Sciences Center, Charlottesville, VA 22908
.

Abstract

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.

Footnotes

↵* This work was supported in part by the Markey Center at the University of Virginia and by National Institutes of Health Grant PO1 HL19242-20. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PP

protein phosphatase

MC

microcystin LR

EDT

ethanedithiol

TCEP

Tris-(2-carboxyethyl)phosphine hydrochloride

HPLC

high performance liquid chromatography

PAGE

polyacrylamide gel electrophoresis

AKAPS

A kinase anchoring protein.
↵² J. Scott, personal communication.
- Received February 12, 1996.
- Revision received August 22, 1996.