title: Pseudophosphorylated αB-crystallin is a nuclear chaperone imported into the nucleus with help of the SMN complex.
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.
authors.affiliation: Department of Biomolecular Chemistry, Institute for Molecules and Materials and Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen, The Netherlands.
authors.forename: John
authors.initials: J
authors.lastname: den Engelsman
authors.forename: Chantal
authors.initials: C
authors.lastname: van de Schootbrugge
authors.forename: Jeongsik
authors.initials: J
authors.lastname: Yong
authors.forename: Ger J M
authors.initials: GJ
authors.lastname: Pruijn
authors.forename: Wilbert C
authors.initials: WC
authors.lastname: Boelens
mesh_terms: D021581:Active Transport, Cell Nucleus; D002467:Cell Nucleus / Q000378:metabolism*; D055541:DEAD Box Protein 20 / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D055532:SMN Complex Proteins / Q000378:metabolism*; D038203:alpha-Crystallin B Chain / Q000378:metabolism*
chemical_list: D055532:SMN Complex Proteins; D038203:alpha-Crystallin B Chain; D055541:DEAD Box Protein 20
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