Pseudophosphorylated αB-Crystallin Is a Nuclear Chaperone Imported into the Nucleus with Help of the SMN Complex

<div><p>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 <i>in situ</i> 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 <i>in vitro</i> 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. </p> </div

Co-immunoprecipitation of Gemin3 with αB-crystallin.

<p>Extracts of HeLa cells, transfected with pIRES as a control or pIRES constructs coding for wild-type (WT), non-phosphorylatable αB-crystallin (αB-STA) or pseudophosphorylated αB-crystallin (αB-STD) were subjected to immunoprecipitation with a monoclonal antibody against αB-crystallin. The immunoprecipitates were analyzed by immunoblotting using a mouse monoclonal antibody against Gemin3 and rabbit polyclonal antibodies to αB-crystallin. Note that Gemin3 shows two bands, of which the lower band is likely a degradation product.</p

<i>In vitro</i> nuclear import of pseudophosphorylated αB-crystallin is dependent on the SMN complex.

<p><i>In vitro</i> nuclear import assays were conducted by incubating digitonin-permeabilized HeLa cells for 1 hour at room temperature with reticulocyte lysates containing ATP and GTP. (A) Import reactions were performed with FITC-labeled recombinant wild-type αB-crystallin (WT), non-phosphorylatable αB-crystallin (αB-STA) and pseudophosphorylated αB-crystallin (αB-STD). As a negative control the import assay with αB-STD was conducted at 4°C (STD 4°C). (B) Import reactions of αB-STD with rabbit reticulocyte lysates immunodepleted with anti-Gemin3 antibodies (ΔGemin3), anti-SMN antibodies (ΔSMN) and control-depleted (Cntr). The impaired import of αB-STD with SMN-depleted reticulocyte lysate could be rescued by addition of purified SMN complex (ΔSMN+ SMN complex). (C) The immunodepleted reticulocyte lysates were analyzed by western blot to assess depletion. Skp1 was used as a loading control. Dotted circles indicate the position of the nuclei.</p

Pseudophosphorylated αB-crystallin enhances refolding of heat-inactivated nuclear luciferase.

<p>Transiently transfected HeLa cells expressing nuclear luciferase were heated (45 min at 45^oC) and subsequently lysed directly or 6 hours thereafter. Cycloheximide was added just prior to heating to prevent de novo luciferase synthesis. Recovery of luciferase activity is indicated for cells cotransfected with empty pIRES vector (pIRES), or constructs encoding wild-type (WT), non-phosphorylatable αB-crystallin (αB-STA) or pseudophosphorylated αB-crystallin (αB-STD). The luciferase activity was determined and compared with the initial luciferase activity before heat shock. All transfections were corrected for the transfection efficiency as measured by the β-galactosidase activity resulting from a cotransfected β-galactosidase expression vector. The results represent the mean values of 6 independent experiments; error bars indicate the standard deviation. A western blot illustrating the expression of αB-crystallin is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073489#pone.0073489.s001" target="_blank">Figure S1</a>.</p

alphaB-crystallin stimulates VEGF secretion and tumor cell migration and correlates with enhanced distant metastasis in head and neck squamous cell carcinoma

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