Pseudophosphorylated αB-crystallin is a nuclear chaperone imported into the nucleus with help of the SMN complex.

John Den Engelsman,Jeongsik Yong,Wilbert C Boelens,Ger J M Pruijn,Chantal Van De Schootbrugge

doi:10.1371/journal.pone.0073489

Abstract

The human small heat shock protein αB-crystallin (HspB5) is a molecular chaperone which is mainly localized in the cytoplasm. A small fraction can also be found in nuclear speckles, of which the localization is mediated by successional phosphorylation at Ser-59 and Ser-45. αB-crystallin does not contain a canonical nuclear localization signal sequence and the mechanism by which αB-crystallin is imported into the nucleus is not known. Here we show that after heat shock pseudophosphorylated αB-crystallin mutant αB-STD, in which all three phosphorylatable serine residues (Ser-19, Ser-45 and Ser-59) were replaced by negatively charged aspartate residues, is released from the nuclear speckles. This allows αB-crystallin to chaperone proteins in the nucleoplasm, as shown by the ability of αB-STD to restore nuclear firefly luciferase activity after a heat shock. With the help of a yeast two-hybrid screen we found that αB-crystallin can interact with the C-terminal part of Gemin3 and confirmed this interaction by co-immunoprecipitation. Gemin3 is a component of the SMN complex, which is involved in the assembly and nuclear import of U-snRNPs. Knockdown of Gemin3 in an in situ nuclear import assay strongly reduced the accumulation of αB-STD in nuclear speckles. Furthermore, depletion of SMN inhibited nuclear import of fluorescently labeled recombinant αB-STD in an in vitro nuclear import assay, which could be restored by the addition of purified SMN complex. These results show that the SMN-complex facilitates the accumulation of hyperphosphorylated αB-crystallin in nuclear speckles, thereby creating a chaperone depot enabling a rapid chaperone function in the nucleus in response to stress.

Highlights

ΑB-Crystallin is a member of the small heat shock protein family and is regarded as a molecular chaperone with an important role in cellular protection against diverse stress stimuli [1,2,3,4,5]
To investigate whether αB-crystallin remains in the nuclear speckles upon heat shock, the pseudophosphorylated αBcrystallin mutant αB-STD was used
Speckle localization was restored 24 hours after heat shock. These results suggest that during heat stress nuclear αB-crystallin exerts its chaperone function in the nucleoplasm

Summary

Introduction

ΑB-Crystallin is a member of the small heat shock protein (sHSP) family and is regarded as a molecular chaperone with an important role in cellular protection against diverse stress stimuli [1,2,3,4,5]. This is consistent with its high expression in stress-sensitive tissues, like eye lens and muscle [6]. The changes in αB-crystallin increase the chaperone activity by preventing heat shock- and reduction-induced aggregation of target proteins, for some substrates the chaperone activity might be decreased depending on the nature and concentration of the substrates [12,13,14,15]

Methods

Results

Conclusion