Nuclear speckle localisation of the small heat shock protein αB-crystallin and its inhibition by the R120G cardiomyopathy-linked mutation

In this study, the small heat shock protein (sHSP) chaperones, alphaB-crystallin and HSP27, are identified as nuclear speckle components in unstressed cells in tissue culture. This new finding suggests a constitutive function for these sHSP chaperones in the nucleus and suggests a new perspective on the cardiomyopathy-causing mutation for alphaB-crystallin that could involve transcriptional splicing effects. Both alphaB-crystallin and HSP27 were immunolocalised to nuclear speckles (interchromatin granule clusters). While alphaB-crystallin was preferentially localised to speckles as shown by colocalisation with non-snRNP, SC35, as well as the snRNP components Sm and U1A, HSP27 was also seen associated with the nucleolar compartment, indicating a subtle difference between these closely related sHSPs. Actinomycin D treatment caused the relocalisation of alphaB-crystallin along with Sm and SC35 to a smaller number of more distinct spots, suggesting a link between speckle localisation and the transcriptional status of the cells. We then examined several transformed, immortalised, and primary cells expressing endogenous alphaB-crystallin as well as some cells with ectopic alphaB-crystallin expression. All consistently showed alphaB-crystallin in nuclear speckles. The nuclear localisation of the sHSPs was also confirmed biochemically and 2D gel electrophoresis revealed that there was only one major nuclear alphaB-crystallin isoform. This suggested that phosphorylation was not required for nuclear localisation of alphaB-crystallin. This was confirmed by the transient transfection of HeLa cells with a phosphorylation-defective alphaB-crystallin. In contrast, the transfection of R120G alphaB-crystallin, the mutation that causes cardiomyopathy, inhibited the nuclear speckle localisation of alphaB-crystallin. These data suggest that the cardiomyopathy-causing mutation for alphaB-crystallin has nuclear as well as cytoplasmic consequences, suggesting an explanation for the difference in severity of the desmin and alphaB-crystallin transgenic models of their respective cardiomyopathies. (C) 2003 Elsevier Science (USA). All rights reserved.