Methodology article

Displacement affinity chromatography of protein phosphatase one (PP1) complexes

Greg BG Moorhead¹ , Laura Trinkle-Mulcahy² , Mhairi Nimick¹ , Veerle De Wever¹ , David G Campbell³ , Robert Gourlay³ , Yun Wah Lam² and Angus I Lamond²

¹Department of Biological Sciences, University of Calgary, 2500 University Dr. N.W. Calgary, AB T2N 1N4, Canada

²Wellcome Trust Biocentre, MSI/WTB Complex, University of Dundee, Dundee, DD1 5EH, UK

³MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee, Scotland DD1 5EH, UK

author email corresponding author email

BMC Biochemistry 2008, 9:28doi:10.1186/1471-2091-9-28

Published:	10 November 2008

Abstract

Background

Protein phosphatase one (PP1) is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PP1 is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif.

Results

We demonstrate that a peptide based on the RVXF/W motif can effectively displace PP1 bound proteins from PP1 retained on the phosphatase affinity matrix microcystin-Sepharose. Subsequent co-immunoprecipitation experiments confirmed that each identified binding protein was either a direct PP1 interactor or was in a complex that contains PP1. Our results have linked PP1 to numerous new nuclear functions and proteins, including Ki-67, Rif-1, topoisomerase IIα, several nuclear helicases, NUP153 and the TRRAP complex.

Conclusion

This modification of the microcystin-Sepharose technique offers an effective means of purifying novel PP1 regulatory subunits and associated proteins and provides a simple method to uncover a link between PP1 and additional cellular processes.