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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 PP2A–α4 interface is essential for the stability of total cellular PP2A.(a) Co-IP determined the interaction between wild-type and mutant V5-C and α4 in C6 glioma cells. (b) Cycloheximide (CHX) chasing and western blot using an antibody that specifically detects V5-tag showed that the turnover rate of V5-C is accelerated by mutations (Y130D/D131R) that disrupt α4 interaction. K29E, a mutation located outside the interface, exhibited no effect. For panels a and b, experiments were repeated three times; representative results are shown. (c) Exponential decay simulation of the results of CHX chasing described in b determined the half-life of wild-type and mutant V5-C. The results shown are the average of three experiments and expressed as mean±s.e.m. (d) Structure of the nPP2Ac–α4 complex highlights PP2A lysine residues (grey) at or near the interface to α4. The loop switch in active PP2Ac is shown (yellow). α4 and nPP2Ac are shown in ribbon and coloured magenta and blue, respectively. (e) CHX chasing showed that the turnover rate of V5-C is reduced by mutation K41R near the interface to α4, but not by K34R. (f) K41R mutation attenuated the loss of V5-C upon α4 knockout.
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f7: The PP2A–α4 interface is essential for the stability of total cellular PP2A.(a) Co-IP determined the interaction between wild-type and mutant V5-C and α4 in C6 glioma cells. (b) Cycloheximide (CHX) chasing and western blot using an antibody that specifically detects V5-tag showed that the turnover rate of V5-C is accelerated by mutations (Y130D/D131R) that disrupt α4 interaction. K29E, a mutation located outside the interface, exhibited no effect. For panels a and b, experiments were repeated three times; representative results are shown. (c) Exponential decay simulation of the results of CHX chasing described in b determined the half-life of wild-type and mutant V5-C. The results shown are the average of three experiments and expressed as mean±s.e.m. (d) Structure of the nPP2Ac–α4 complex highlights PP2A lysine residues (grey) at or near the interface to α4. The loop switch in active PP2Ac is shown (yellow). α4 and nPP2Ac are shown in ribbon and coloured magenta and blue, respectively. (e) CHX chasing showed that the turnover rate of V5-C is reduced by mutation K41R near the interface to α4, but not by K34R. (f) K41R mutation attenuated the loss of V5-C upon α4 knockout.

Mentions: The previous study showed that knockout of α4 led to a complete loss of total cellular PP2A and PP2A-like phosphatases23. To test whether the stability of cellular PP2Ac relies on its interaction with α4, we examined the turnover rate of V5-tagged PP2Ac (V5-C)-bearing mutations that disrupt interaction with α4. The level of V5-C was monitored at different times after addition of cycloheximide to inhibit de novo protein synthesis. Because a large area of the nPP2Ac–α4 interface involves PP2Ac internal packing (Fig. 4b), PP2Ac mutations were carefully chosen not to perturb internal packing and were limited to surface residues at the interface with α4. Recombinant wild-type V5-C interacted readily with α4 and exhibited a half-life of about 13 h (Fig. 7a–c). V5-C-bearing mutation of PP2Ac surface residues at the interface with α4, Y130D/D131R, failed to interact with α4 (Fig. 7a) and exhibited a higher turnover rate (Fig. 7b) and a minimum of sixfold reduction in the half-life (Fig. 7c). V5-C-bearing mutations outside the interface with α4, K29E, barely affected α4 binding or the cellular stability of PP2Ac (Fig. 7a–c). These data indicate that α4 interaction is required for the stability of cellular PP2Ac.


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 PP2A–α4 interface is essential for the stability of total cellular PP2A.(a) Co-IP determined the interaction between wild-type and mutant V5-C and α4 in C6 glioma cells. (b) Cycloheximide (CHX) chasing and western blot using an antibody that specifically detects V5-tag showed that the turnover rate of V5-C is accelerated by mutations (Y130D/D131R) that disrupt α4 interaction. K29E, a mutation located outside the interface, exhibited no effect. For panels a and b, experiments were repeated three times; representative results are shown. (c) Exponential decay simulation of the results of CHX chasing described in b determined the half-life of wild-type and mutant V5-C. The results shown are the average of three experiments and expressed as mean±s.e.m. (d) Structure of the nPP2Ac–α4 complex highlights PP2A lysine residues (grey) at or near the interface to α4. The loop switch in active PP2Ac is shown (yellow). α4 and nPP2Ac are shown in ribbon and coloured magenta and blue, respectively. (e) CHX chasing showed that the turnover rate of V5-C is reduced by mutation K41R near the interface to α4, but not by K34R. (f) K41R mutation attenuated the loss of V5-C upon α4 knockout.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: The PP2A–α4 interface is essential for the stability of total cellular PP2A.(a) Co-IP determined the interaction between wild-type and mutant V5-C and α4 in C6 glioma cells. (b) Cycloheximide (CHX) chasing and western blot using an antibody that specifically detects V5-tag showed that the turnover rate of V5-C is accelerated by mutations (Y130D/D131R) that disrupt α4 interaction. K29E, a mutation located outside the interface, exhibited no effect. For panels a and b, experiments were repeated three times; representative results are shown. (c) Exponential decay simulation of the results of CHX chasing described in b determined the half-life of wild-type and mutant V5-C. The results shown are the average of three experiments and expressed as mean±s.e.m. (d) Structure of the nPP2Ac–α4 complex highlights PP2A lysine residues (grey) at or near the interface to α4. The loop switch in active PP2Ac is shown (yellow). α4 and nPP2Ac are shown in ribbon and coloured magenta and blue, respectively. (e) CHX chasing showed that the turnover rate of V5-C is reduced by mutation K41R near the interface to α4, but not by K34R. (f) K41R mutation attenuated the loss of V5-C upon α4 knockout.
Mentions: The previous study showed that knockout of α4 led to a complete loss of total cellular PP2A and PP2A-like phosphatases23. To test whether the stability of cellular PP2Ac relies on its interaction with α4, we examined the turnover rate of V5-tagged PP2Ac (V5-C)-bearing mutations that disrupt interaction with α4. The level of V5-C was monitored at different times after addition of cycloheximide to inhibit de novo protein synthesis. Because a large area of the nPP2Ac–α4 interface involves PP2Ac internal packing (Fig. 4b), PP2Ac mutations were carefully chosen not to perturb internal packing and were limited to surface residues at the interface with α4. Recombinant wild-type V5-C interacted readily with α4 and exhibited a half-life of about 13 h (Fig. 7a–c). V5-C-bearing mutation of PP2Ac surface residues at the interface with α4, Y130D/D131R, failed to interact with α4 (Fig. 7a) and exhibited a higher turnover rate (Fig. 7b) and a minimum of sixfold reduction in the half-life (Fig. 7c). V5-C-bearing mutations outside the interface with α4, K29E, barely affected α4 binding or the cellular stability of PP2Ac (Fig. 7a–c). These data indicate that α4 interaction is required for the stability of cellular PP2Ac.

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