Rapid Identification of Protein Phosphatase 1-binding Proteins by Mixed Peptide Sequencing and Data Base Searching
CHARACTERIZATION OF A NOVEL HOLOENZYMIC FORM OF PROTEIN PHOSPHATASE 1*
- From the Departments of Pharmacology, Markey Center for Cell Signaling and Biochemistry, University of Virginia, Charlottesville, Virginia 22908
Abstract
Microcystin-affinity chromatography was used to purify 15 protein phosphatase 1 (PP1)-binding proteins from the myofibrillar fraction of rabbit skeletal muscle. To reduce the time and amount of material required to identify these proteins, proteome analysis by mixed peptide sequencing was developed. Proteins are resolved by SDS-polyacrylamide gel electrophoresis, electroblotted to polyvinylidene fluoride membrane, and stained. Bands are sliced from the membrane, cleaved briefly with CnBr, and applied without further purification to an automated Edman sequencer. The mixed peptide sequences generated are sorted and matched against the GenBank using two new programs, FASTF and TFASTF. This technology offers a simple alternative to mass spectrometry for the subpicomolar identification of proteins in polyacrylamide gels. Using this technology, all 15 proteins recovered in PP-1C affinity chromatography were sequenced. One of the proteins, PP-1bp55, was homologous to human myosin phosphatase, MYPT2. A second, PP-1bp80, identified in the EST data bases, contained a putative PP-1C binding site and a nucleotide binding motif. Further affinity purification over ATP-Sepharose isolated PP-1bp80 in a quaternary complex with PP-1C and two other proteins, PP-1bp29 and human p20. Recombinant PP-1bp80 also bound PP-1C and suppressed its activity toward a variety of substrates, suggesting that the protein is a novel regulatory subunit of PP-1.
Footnotes
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↵* This work was supported by Grants HL19242 and DK52378A (to T. A. J. H.) and LM04969 (to W. R. P.) from the National Institutes of Health.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.
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↵‡ To whom correspondence should be addressed: Dept. of Pharmacology, Box 448, University of Virginia Health Sciences Center, Charlottesville, VA 22908.
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↵2 W. R. Pearson and T. A. J. Haystead, manuscript in preparation.
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↵3 The FASTF and TFASTF programs can be obtained via FTP (ftp://ftp.virginia.edu/pub/fasta) or by contacting the authors.
- Abbreviations:
- PP
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protein phosphatase
- MBP
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myelin basic protein
- PBS
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phosphate-buffered saline
- GST
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glutathione S-transferase
- PTH
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phenylthiohydantoin
- PAGE
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polyacrylamide gel electrophoresis
- PVM
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polyvinylidene fluoride membrane.
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- Received June 10, 1998.
- Revision received July 13, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
