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

PLoS One. 2013 Sep 4;8(9):e73489. doi: 10.1371/journal.pone.0073489. eCollection 2013.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Active Transport, Cell Nucleus
  • Cell Nucleus / metabolism*
  • DEAD Box Protein 20 / metabolism
  • HeLa Cells
  • Humans
  • Phosphorylation
  • SMN Complex Proteins / metabolism*
  • alpha-Crystallin B Chain / metabolism*

Substances

  • SMN Complex Proteins
  • alpha-Crystallin B Chain
  • DEAD Box Protein 20

Grants and funding

CvdS was funded by KWF (Dutch Cancer Society, KUN 2007-3864, http://www.kwfkankerbestrijding.nl/) and JdE by Netherlands Organization for Scientific Research (NWO-MW 902-27-227, http://www.nwo.nl/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.