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.
van den IJssel
Paul
P
School of Life Sciences, Medical Science Institute, The University, Dundee DD1 5EH, UK.
Wheelock
Robert
R
Prescott
Alan
A
Russell
Paul
P
Quinlan
Roy A
RA
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D009202:Cardiomyopathies / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002466:Cell Nucleolus / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D055551:HSP27 Heat-Shock Proteins; D006367:HeLa Cells; D006360:Heat-Shock Proteins; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism* / Q000473:pathology; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009363:Neoplasm Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D038203:alpha-Crystallin B Chain / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*
D000903:Antibiotics, Antineoplastic; D014408:Biomarkers, Tumor; D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D020033:Protein Isoforms; D038203:alpha-Crystallin B Chain; D003609:Dactinomycin