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Displacement affinity chromatography of protein phosphatase one (PP1) complexes.

Moorhead GB, Trinkle-Mulcahy L, Nimick M, De Wever V, Campbell DG, Gourlay R, Lam YW, Lamond AI - BMC Biochem. (2008)

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, University of Calgary, 2500 University Dr, NW Calgary, AB T2N 1N4, Canada. moorhead@ucalgary.ca

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 IIalpha, 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.

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Related in: MedlinePlus

A peptide based on the PP1 interaction motif RVXF/W displaces PP1 regulatory subunits from the affinity matrix microcystin-Sepharose. Proteins extracted from isolated rat liver nuclei were incubated with the affinity (MC) or control (Con) matrix (Tris coupled). Proteins were displaced with the RPKRKRKNSRVTFSEDDEII peptide (a), while in (b), elution was performed with either RPKRKRKNSRVTFSEDDEII or GKKRVRWADLE prior to the 3 M NaSCN elution. The control matrix is only used in the panel (a) experiment while microcystin-Sepharose is used in all others. After concentration, samples were run on 10% SDS-PAGE, blotted to nitrocellulose and membranes were probed with anti-PR65 and PP1 antibodies. In panel (c) the membrane was probed with anti-ZAP, p99 or NIPP1 antibodies [31]. To test for the salt dependence of peptide displacement, columns were eluted with peptide plus or minus NaCl as indicated and samples blotted for NIPP1 (d). To determine optimal peptide concentration for displacement from the column, the protein loaded beads were divided into 3 equal parts and eluted with 0.1, 0.5 and 2 mM peptide (e). To test for the specificity of the peptide displacement, the beads were divided into 3 equal parts and eluted with either GKKRVRWADLE, the GKKRVRWADLE peptide with the key interacting residues changed to A (GKKRARAADLE), or a scrambled version of the GKKRVRWADLE peptide (KLRGEVAKDWR) and blotted for ZAP, p99 and NIPP1. Glycogen particles were isolated from rabbit skeletal muscle and PP1GM bound to the microcystin matrix followed by peptide elution first with the GKKRARAADLE peptide, then GKKRVRWADLE, followed by 3 M NaSCN.
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Figure 1: A peptide based on the PP1 interaction motif RVXF/W displaces PP1 regulatory subunits from the affinity matrix microcystin-Sepharose. Proteins extracted from isolated rat liver nuclei were incubated with the affinity (MC) or control (Con) matrix (Tris coupled). Proteins were displaced with the RPKRKRKNSRVTFSEDDEII peptide (a), while in (b), elution was performed with either RPKRKRKNSRVTFSEDDEII or GKKRVRWADLE prior to the 3 M NaSCN elution. The control matrix is only used in the panel (a) experiment while microcystin-Sepharose is used in all others. After concentration, samples were run on 10% SDS-PAGE, blotted to nitrocellulose and membranes were probed with anti-PR65 and PP1 antibodies. In panel (c) the membrane was probed with anti-ZAP, p99 or NIPP1 antibodies [31]. To test for the salt dependence of peptide displacement, columns were eluted with peptide plus or minus NaCl as indicated and samples blotted for NIPP1 (d). To determine optimal peptide concentration for displacement from the column, the protein loaded beads were divided into 3 equal parts and eluted with 0.1, 0.5 and 2 mM peptide (e). To test for the specificity of the peptide displacement, the beads were divided into 3 equal parts and eluted with either GKKRVRWADLE, the GKKRVRWADLE peptide with the key interacting residues changed to A (GKKRARAADLE), or a scrambled version of the GKKRVRWADLE peptide (KLRGEVAKDWR) and blotted for ZAP, p99 and NIPP1. Glycogen particles were isolated from rabbit skeletal muscle and PP1GM bound to the microcystin matrix followed by peptide elution first with the GKKRARAADLE peptide, then GKKRVRWADLE, followed by 3 M NaSCN.

Mentions: We initiated our PP1 peptide displacement study selecting RVXF/W containing peptides from the PP1 targeting subunits NIPP1 [25,30] and ZAP (ZAP3) [31,32]. They were synthesized and tested for their ability to displace PP1 binding proteins from complexes retained on the microcystin matrix. In brief, we isolated rat liver nuclei, extracted proteins and incubated extracts with Microcystin-Sepharose to bind the microcystin-sensitive protein phosphatases [31]. After extensive column washing, we incubated the matrix with the NIPP1 or ZAP peptides (RPKRKRKNSRVTFSEDDEII and GKKRVRWADLE, respectively) to selectively displace proteins retained on the matrix through PP1. This implies that PP1 itself and other microcystin-sensitive phosphatase complexes should be retained on the matrix and can subsequently be eluted with the chaotrophic agent sodium thiocyanate (NaSCN), which will also displace other bound proteins [9] including some that may be retained by binding non-specifically to the Sepharose bead. Blotting for PP1 and the PP2A regulatory subunit PR65 show this to be true (Figure 1a and 1b) with no retention of these phosphatase subunits on a control matrix coupled with Tris alone. The NIPP1 peptide (RPKRKRKNSRVTFSEDDEII) readily displaced PP1 regulatory subunits (data not shown), yet we chose to continue working with the ZAP peptide (GKKRVRWADLE, later also referred to as RVRW peptide) because it is small, readily soluble and an excellent match to the optimal PP1 binding peptide discovered through panning a random peptide library [24]. Indeed, the ZAP peptide RVRW motif and the additional C-terminal amino acids ADL and N-terminal basic amino acids (KK) were most frequently obtained in the random peptide library screen (Figure 2 in [24]). We thus predict that the ZAP sequence GKKRVRWADLE is the most suitable, high affinity peptide that will compete and displace most other proteins docked to PP1 through variations of the RVXF/W-motif. We acknowledge that even with an 'optimal binding peptide' there are certainly other PP1 interacting proteins on the matrix that may not be displaced because of completely novel PP1 interaction sites and/or additional interaction sites outside of the RVXF/W-motif [20] that could maintain the interaction with PP1 even if the RVXF/W site is displaced. Thus, the PP1 binding partners uncovered here still most likely only represent a sub-population of the total nuclear PP1 binding partners.


Displacement affinity chromatography of protein phosphatase one (PP1) complexes.

Moorhead GB, Trinkle-Mulcahy L, Nimick M, De Wever V, Campbell DG, Gourlay R, Lam YW, Lamond AI - BMC Biochem. (2008)

A peptide based on the PP1 interaction motif RVXF/W displaces PP1 regulatory subunits from the affinity matrix microcystin-Sepharose. Proteins extracted from isolated rat liver nuclei were incubated with the affinity (MC) or control (Con) matrix (Tris coupled). Proteins were displaced with the RPKRKRKNSRVTFSEDDEII peptide (a), while in (b), elution was performed with either RPKRKRKNSRVTFSEDDEII or GKKRVRWADLE prior to the 3 M NaSCN elution. The control matrix is only used in the panel (a) experiment while microcystin-Sepharose is used in all others. After concentration, samples were run on 10% SDS-PAGE, blotted to nitrocellulose and membranes were probed with anti-PR65 and PP1 antibodies. In panel (c) the membrane was probed with anti-ZAP, p99 or NIPP1 antibodies [31]. To test for the salt dependence of peptide displacement, columns were eluted with peptide plus or minus NaCl as indicated and samples blotted for NIPP1 (d). To determine optimal peptide concentration for displacement from the column, the protein loaded beads were divided into 3 equal parts and eluted with 0.1, 0.5 and 2 mM peptide (e). To test for the specificity of the peptide displacement, the beads were divided into 3 equal parts and eluted with either GKKRVRWADLE, the GKKRVRWADLE peptide with the key interacting residues changed to A (GKKRARAADLE), or a scrambled version of the GKKRVRWADLE peptide (KLRGEVAKDWR) and blotted for ZAP, p99 and NIPP1. Glycogen particles were isolated from rabbit skeletal muscle and PP1GM bound to the microcystin matrix followed by peptide elution first with the GKKRARAADLE peptide, then GKKRVRWADLE, followed by 3 M NaSCN.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 1: A peptide based on the PP1 interaction motif RVXF/W displaces PP1 regulatory subunits from the affinity matrix microcystin-Sepharose. Proteins extracted from isolated rat liver nuclei were incubated with the affinity (MC) or control (Con) matrix (Tris coupled). Proteins were displaced with the RPKRKRKNSRVTFSEDDEII peptide (a), while in (b), elution was performed with either RPKRKRKNSRVTFSEDDEII or GKKRVRWADLE prior to the 3 M NaSCN elution. The control matrix is only used in the panel (a) experiment while microcystin-Sepharose is used in all others. After concentration, samples were run on 10% SDS-PAGE, blotted to nitrocellulose and membranes were probed with anti-PR65 and PP1 antibodies. In panel (c) the membrane was probed with anti-ZAP, p99 or NIPP1 antibodies [31]. To test for the salt dependence of peptide displacement, columns were eluted with peptide plus or minus NaCl as indicated and samples blotted for NIPP1 (d). To determine optimal peptide concentration for displacement from the column, the protein loaded beads were divided into 3 equal parts and eluted with 0.1, 0.5 and 2 mM peptide (e). To test for the specificity of the peptide displacement, the beads were divided into 3 equal parts and eluted with either GKKRVRWADLE, the GKKRVRWADLE peptide with the key interacting residues changed to A (GKKRARAADLE), or a scrambled version of the GKKRVRWADLE peptide (KLRGEVAKDWR) and blotted for ZAP, p99 and NIPP1. Glycogen particles were isolated from rabbit skeletal muscle and PP1GM bound to the microcystin matrix followed by peptide elution first with the GKKRARAADLE peptide, then GKKRVRWADLE, followed by 3 M NaSCN.
Mentions: We initiated our PP1 peptide displacement study selecting RVXF/W containing peptides from the PP1 targeting subunits NIPP1 [25,30] and ZAP (ZAP3) [31,32]. They were synthesized and tested for their ability to displace PP1 binding proteins from complexes retained on the microcystin matrix. In brief, we isolated rat liver nuclei, extracted proteins and incubated extracts with Microcystin-Sepharose to bind the microcystin-sensitive protein phosphatases [31]. After extensive column washing, we incubated the matrix with the NIPP1 or ZAP peptides (RPKRKRKNSRVTFSEDDEII and GKKRVRWADLE, respectively) to selectively displace proteins retained on the matrix through PP1. This implies that PP1 itself and other microcystin-sensitive phosphatase complexes should be retained on the matrix and can subsequently be eluted with the chaotrophic agent sodium thiocyanate (NaSCN), which will also displace other bound proteins [9] including some that may be retained by binding non-specifically to the Sepharose bead. Blotting for PP1 and the PP2A regulatory subunit PR65 show this to be true (Figure 1a and 1b) with no retention of these phosphatase subunits on a control matrix coupled with Tris alone. The NIPP1 peptide (RPKRKRKNSRVTFSEDDEII) readily displaced PP1 regulatory subunits (data not shown), yet we chose to continue working with the ZAP peptide (GKKRVRWADLE, later also referred to as RVRW peptide) because it is small, readily soluble and an excellent match to the optimal PP1 binding peptide discovered through panning a random peptide library [24]. Indeed, the ZAP peptide RVRW motif and the additional C-terminal amino acids ADL and N-terminal basic amino acids (KK) were most frequently obtained in the random peptide library screen (Figure 2 in [24]). We thus predict that the ZAP sequence GKKRVRWADLE is the most suitable, high affinity peptide that will compete and displace most other proteins docked to PP1 through variations of the RVXF/W-motif. We acknowledge that even with an 'optimal binding peptide' there are certainly other PP1 interacting proteins on the matrix that may not be displaced because of completely novel PP1 interaction sites and/or additional interaction sites outside of the RVXF/W-motif [20] that could maintain the interaction with PP1 even if the RVXF/W site is displaced. Thus, the PP1 binding partners uncovered here still most likely only represent a sub-population of the total nuclear PP1 binding partners.

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, University of Calgary, 2500 University Dr, NW Calgary, AB T2N 1N4, Canada. moorhead@ucalgary.ca

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 IIalpha, 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.

Show MeSH
Related in: MedlinePlus