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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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Selective binding of α4 to the partially folded PP2Ac.(a) Pull-down of PP2Ac by GST-α4 after co-incubation at 37 °C in the presence of 50 μM Mn++ or 2 mM PPi (left panel), the level of the phosphatase activity measured at the end of incubation (middle panel) and changes in the level of Mn++ ions stably bound to PP2Ac before and after PPi treatment (right panel). Experiments were repeated three times; representative results are shown. Phosphatase assay was performed in triplicates and mean±s.e.m. were calculated and shown. (b) Light scattering detected aggregation of PP2Ac upon PPi treatment (PP2Ac+PPi), which is suppressed by the presence of α4 (PP2Ac+PPi+α4). (c) Illustration of nPP2Ac using the structure of active PP2Ac. (d) Determination of the binding affinity between nPP2Ac and α4 using ITC.
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f1: Selective binding of α4 to the partially folded PP2Ac.(a) Pull-down of PP2Ac by GST-α4 after co-incubation at 37 °C in the presence of 50 μM Mn++ or 2 mM PPi (left panel), the level of the phosphatase activity measured at the end of incubation (middle panel) and changes in the level of Mn++ ions stably bound to PP2Ac before and after PPi treatment (right panel). Experiments were repeated three times; representative results are shown. Phosphatase assay was performed in triplicates and mean±s.e.m. were calculated and shown. (b) Light scattering detected aggregation of PP2Ac upon PPi treatment (PP2Ac+PPi), which is suppressed by the presence of α4 (PP2Ac+PPi+α4). (c) Illustration of nPP2Ac using the structure of active PP2Ac. (d) Determination of the binding affinity between nPP2Ac and α4 using ITC.

Mentions: Initial efforts to determine the crystal structure of the PP2Ac–α4 complex were hampered by the lack of interaction between α4 and the purified recombinant active PP2Ac in vitro (Supplementary Fig. S2a,b). Based on reports that the interaction between PP2Ac and α4 was increased by heat shock513 and our recent data that suggested a malleable nature of PP2Ac following eviction of catalytic metal ions19, we tested the possibility that α4 binds selectively to the partially folded PP2Ac. Co-incubation with pyrophosphate (PPi) at 37 °C rapidly dislodges PP2A catalytic metal ions as indicated by ICP-MS (inductively coupled plasma mass spectrometry), resulting in rapid formation of a stoichiometric complex between PP2Ac and α4 accompanied by a nearly complete loss of phosphatase activity (Fig. 1a). α4 binding in turn prevented rapid aggregation of PP2Ac upon dislodging of catalytic metal ions (Fig. 1b). These results are consistent with our notion that PP2Ac is partially folded in the absence of catalytic metal ions, which allows α4 binding that in turn prevents aggregation, presumably by shielding exposed PP2Ac internal packing. The resulting PP2Ac–α4 complex, however, eluded crystallization despite considerable effort.


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)

Selective binding of α4 to the partially folded PP2Ac.(a) Pull-down of PP2Ac by GST-α4 after co-incubation at 37 °C in the presence of 50 μM Mn++ or 2 mM PPi (left panel), the level of the phosphatase activity measured at the end of incubation (middle panel) and changes in the level of Mn++ ions stably bound to PP2Ac before and after PPi treatment (right panel). Experiments were repeated three times; representative results are shown. Phosphatase assay was performed in triplicates and mean±s.e.m. were calculated and shown. (b) Light scattering detected aggregation of PP2Ac upon PPi treatment (PP2Ac+PPi), which is suppressed by the presence of α4 (PP2Ac+PPi+α4). (c) Illustration of nPP2Ac using the structure of active PP2Ac. (d) Determination of the binding affinity between nPP2Ac and α4 using ITC.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Selective binding of α4 to the partially folded PP2Ac.(a) Pull-down of PP2Ac by GST-α4 after co-incubation at 37 °C in the presence of 50 μM Mn++ or 2 mM PPi (left panel), the level of the phosphatase activity measured at the end of incubation (middle panel) and changes in the level of Mn++ ions stably bound to PP2Ac before and after PPi treatment (right panel). Experiments were repeated three times; representative results are shown. Phosphatase assay was performed in triplicates and mean±s.e.m. were calculated and shown. (b) Light scattering detected aggregation of PP2Ac upon PPi treatment (PP2Ac+PPi), which is suppressed by the presence of α4 (PP2Ac+PPi+α4). (c) Illustration of nPP2Ac using the structure of active PP2Ac. (d) Determination of the binding affinity between nPP2Ac and α4 using ITC.
Mentions: Initial efforts to determine the crystal structure of the PP2Ac–α4 complex were hampered by the lack of interaction between α4 and the purified recombinant active PP2Ac in vitro (Supplementary Fig. S2a,b). Based on reports that the interaction between PP2Ac and α4 was increased by heat shock513 and our recent data that suggested a malleable nature of PP2Ac following eviction of catalytic metal ions19, we tested the possibility that α4 binds selectively to the partially folded PP2Ac. Co-incubation with pyrophosphate (PPi) at 37 °C rapidly dislodges PP2A catalytic metal ions as indicated by ICP-MS (inductively coupled plasma mass spectrometry), resulting in rapid formation of a stoichiometric complex between PP2Ac and α4 accompanied by a nearly complete loss of phosphatase activity (Fig. 1a). α4 binding in turn prevented rapid aggregation of PP2Ac upon dislodging of catalytic metal ions (Fig. 1b). These results are consistent with our notion that PP2Ac is partially folded in the absence of catalytic metal ions, which allows α4 binding that in turn prevents aggregation, presumably by shielding exposed PP2Ac internal packing. The resulting PP2Ac–α4 complex, however, eluded crystallization despite considerable effort.

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