Displacement affinity chromatography of protein phosphatase one (PP1) complexes.
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 IIalpha, several nuclear helicases, NUP153 and the TRRAP complex.


CONCLUSIONS
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.
Moorhead
Greg B G
GB
Department of Biological Sciences, University of Calgary, 2500 University Dr, NW Calgary, AB T2N 1N4, Canada. moorhead@ucalgary.ca
Trinkle-Mulcahy
Laura
L
Nimick
Mhairi
M
De Wever
Veerle
V
Campbell
David G
DG
Gourlay
Robert
R
Lam
Yun Wah
YW
Lamond
Angus I
AI
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods*; D006003:Glycogen / Q000302:isolation & purification; D006367:HeLa Cells; D006801:Humans; D052998:Microcystins / Q000737:chemistry / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism; D054645:Protein Phosphatase 1 / Q000737:chemistry / Q000302:isolation & purification*; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D012685:Sepharose / Q000737:chemistry
D052998:Microcystins; D021122:Protein Subunits; C078588:microcystin; D006003:Glycogen; D012685:Sepharose; D054645:Protein Phosphatase 1