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Structural basis of protein phosphatase 2A stable latency.

Jiang L, Stanevich V, Satyshur KA, Kong M, Watkins GR, Wadzinski BE, Sengupta R, Xing Y - Nat Commun (2013)

Bottom Line: This structure suggests that α4 binding to the full-length PP2Ac requires local unfolding near the active site, which perturbs the scaffold subunit binding site at the opposite surface via allosteric relay.These changes stabilize an inactive conformation of PP2Ac and convert oligomeric PP2A complexes to the α4 complex upon perturbation of the active site.Our results show that α4 is a scavenger chaperone that binds to and stabilizes partially folded PP2Ac for stable latency, and reveal a mechanism by which α4 regulates cell survival, and biogenesis and surveillance of PP2A holoenzymes.

View Article: PubMed Central - PubMed

Affiliation: McArdle Laboratory, Department of Oncology, University of Wisconsin-Madison, School of Medicine and Public Health, Madison, Wisconsin 53706, USA.

ABSTRACT
The catalytic subunit of protein phosphatase 2A (PP2Ac) is stabilized in a latent form by α4, a regulatory protein essential for cell survival and biogenesis of all PP2A complexes. Here we report the structure of α4 bound to the N-terminal fragment of PP2Ac. This structure suggests that α4 binding to the full-length PP2Ac requires local unfolding near the active site, which perturbs the scaffold subunit binding site at the opposite surface via allosteric relay. These changes stabilize an inactive conformation of PP2Ac and convert oligomeric PP2A complexes to the α4 complex upon perturbation of the active site. The PP2Ac-α4 interface is essential for cell survival and sterically hinders a PP2A ubiquitination site, important for the stability of cellular PP2Ac. Our results show that α4 is a scavenger chaperone that binds to and stabilizes partially folded PP2Ac for stable latency, and reveal a mechanism by which α4 regulates cell survival, and biogenesis and surveillance of PP2A holoenzymes.

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The α4 interfaces with the phosphatase are essential for the role of α4 in cell survival.(a) Mutational analysis of α4 residues at the interface with both PP2A internal packing and surface residues. Pull-down assay determined the effect of α4 mutations on interaction between α4 and GST-nPP2Ac. Proteins bound to GS4B resin were visualized on SDS–PAGE by coomassie blue staining. (b) Co-IP showed defects of recombinant Flag-α4 mutants in binding to cellular PP2Ac in C6 glioma cells. (c) Western blot showing expression of wild-type (WT) and mutant Flag-α4 at endogenous levels in α4fl MEFs (left). Viable cell growth curves after induction of α4 knockout determined the ability of Flag-α4 mutants to reverse the cell death phenotype caused by α4 knockout (right). For all panels, experiments were repeated three times; representative results are shown. For panel c (right), experiments were performed in triplicate and expressed as mean±s.e.m.
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f6: The α4 interfaces with the phosphatase are essential for the role of α4 in cell survival.(a) Mutational analysis of α4 residues at the interface with both PP2A internal packing and surface residues. Pull-down assay determined the effect of α4 mutations on interaction between α4 and GST-nPP2Ac. Proteins bound to GS4B resin were visualized on SDS–PAGE by coomassie blue staining. (b) Co-IP showed defects of recombinant Flag-α4 mutants in binding to cellular PP2Ac in C6 glioma cells. (c) Western blot showing expression of wild-type (WT) and mutant Flag-α4 at endogenous levels in α4fl MEFs (left). Viable cell growth curves after induction of α4 knockout determined the ability of Flag-α4 mutants to reverse the cell death phenotype caused by α4 knockout (right). For all panels, experiments were repeated three times; representative results are shown. For panel c (right), experiments were performed in triplicate and expressed as mean±s.e.m.

Mentions: To determine if the PP2Ac–α4 interface predicted from the structure of nPP2Ac–α4 complex is essential for α4 function, we identified α4 mutations that disrupted PP2Ac interaction. As shown by pull-down assay (Fig. 6a), α4 mutations, R155E and K158D at the interface with PP2Ac internal packing and Y162D and E214R at the interface with PP2Ac surface residues, completely abolished the interaction between α4 and the GST-tagged nPP2Ac. In contrast, a mutation located outside the nPP2Ac–α4 interface, E206R, barely affected the interaction. Consistent with the in vitro binding results, recombinant Flag-tagged α4 harbouring mutations at either interface, R155E and E214R, had severely reduced interaction with the cellular full-length PP2Ac (Fig. 6b).


Structural basis of protein phosphatase 2A stable latency.

Jiang L, Stanevich V, Satyshur KA, Kong M, Watkins GR, Wadzinski BE, Sengupta R, Xing Y - Nat Commun (2013)

The α4 interfaces with the phosphatase are essential for the role of α4 in cell survival.(a) Mutational analysis of α4 residues at the interface with both PP2A internal packing and surface residues. Pull-down assay determined the effect of α4 mutations on interaction between α4 and GST-nPP2Ac. Proteins bound to GS4B resin were visualized on SDS–PAGE by coomassie blue staining. (b) Co-IP showed defects of recombinant Flag-α4 mutants in binding to cellular PP2Ac in C6 glioma cells. (c) Western blot showing expression of wild-type (WT) and mutant Flag-α4 at endogenous levels in α4fl MEFs (left). Viable cell growth curves after induction of α4 knockout determined the ability of Flag-α4 mutants to reverse the cell death phenotype caused by α4 knockout (right). For all panels, experiments were repeated three times; representative results are shown. For panel c (right), experiments were performed in triplicate and expressed as mean±s.e.m.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3644067&req=5

f6: The α4 interfaces with the phosphatase are essential for the role of α4 in cell survival.(a) Mutational analysis of α4 residues at the interface with both PP2A internal packing and surface residues. Pull-down assay determined the effect of α4 mutations on interaction between α4 and GST-nPP2Ac. Proteins bound to GS4B resin were visualized on SDS–PAGE by coomassie blue staining. (b) Co-IP showed defects of recombinant Flag-α4 mutants in binding to cellular PP2Ac in C6 glioma cells. (c) Western blot showing expression of wild-type (WT) and mutant Flag-α4 at endogenous levels in α4fl MEFs (left). Viable cell growth curves after induction of α4 knockout determined the ability of Flag-α4 mutants to reverse the cell death phenotype caused by α4 knockout (right). For all panels, experiments were repeated three times; representative results are shown. For panel c (right), experiments were performed in triplicate and expressed as mean±s.e.m.
Mentions: To determine if the PP2Ac–α4 interface predicted from the structure of nPP2Ac–α4 complex is essential for α4 function, we identified α4 mutations that disrupted PP2Ac interaction. As shown by pull-down assay (Fig. 6a), α4 mutations, R155E and K158D at the interface with PP2Ac internal packing and Y162D and E214R at the interface with PP2Ac surface residues, completely abolished the interaction between α4 and the GST-tagged nPP2Ac. In contrast, a mutation located outside the nPP2Ac–α4 interface, E206R, barely affected the interaction. Consistent with the in vitro binding results, recombinant Flag-tagged α4 harbouring mutations at either interface, R155E and E214R, had severely reduced interaction with the cellular full-length PP2Ac (Fig. 6b).

Bottom Line: This structure suggests that α4 binding to the full-length PP2Ac requires local unfolding near the active site, which perturbs the scaffold subunit binding site at the opposite surface via allosteric relay.These changes stabilize an inactive conformation of PP2Ac and convert oligomeric PP2A complexes to the α4 complex upon perturbation of the active site.Our results show that α4 is a scavenger chaperone that binds to and stabilizes partially folded PP2Ac for stable latency, and reveal a mechanism by which α4 regulates cell survival, and biogenesis and surveillance of PP2A holoenzymes.

View Article: PubMed Central - PubMed

Affiliation: McArdle Laboratory, Department of Oncology, University of Wisconsin-Madison, School of Medicine and Public Health, Madison, Wisconsin 53706, USA.

ABSTRACT
The catalytic subunit of protein phosphatase 2A (PP2Ac) is stabilized in a latent form by α4, a regulatory protein essential for cell survival and biogenesis of all PP2A complexes. Here we report the structure of α4 bound to the N-terminal fragment of PP2Ac. This structure suggests that α4 binding to the full-length PP2Ac requires local unfolding near the active site, which perturbs the scaffold subunit binding site at the opposite surface via allosteric relay. These changes stabilize an inactive conformation of PP2Ac and convert oligomeric PP2A complexes to the α4 complex upon perturbation of the active site. The PP2Ac-α4 interface is essential for cell survival and sterically hinders a PP2A ubiquitination site, important for the stability of cellular PP2Ac. Our results show that α4 is a scavenger chaperone that binds to and stabilizes partially folded PP2Ac for stable latency, and reveal a mechanism by which α4 regulates cell survival, and biogenesis and surveillance of PP2A holoenzymes.

Show MeSH
Related in: MedlinePlus