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

In this study, the small heat shock protein (sHSP) chaperones, alpha B-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 alpha B-crystallin that could involve transcriptional splicing effects. Both alpha B-crystallin and HSP27 were immunolocalised to nuclear speckles (interchromatin granule clusters). While alpha B-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 alpha B-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 alpha B-crystallin as well as some cells with ectopic alpha B-crystallin expression. All consistently showed alpha B-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 alpha B-crystallin isoform. This suggested that phosphorylation was not required for nuclear localisation of alpha B-crystallin. This was confirmed by the transient transfection of HeLa cells with a phosphorylation-defective alpha B-crystallin. In contrast, the transfection of R120G alpha B-crystallin, the mutation that causes cardiomyopathy, inhibited the nuclear speckle localisation of alpha B-crystallin. These data suggest that the cardiomyopathy-causing mutation for alpha B-crystallin has nuclear as well as cytoplasmic consequences, suggesting an explanation for the difference in severity of the desmin and alpha B-crystallin transgenic models of their respective cardiomyopathies.

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