Abstract
Phosphorylation modulates the functioning of alphaB-crystallin as a molecular chaperone. We here explore the role of phosphorylation in the nuclear import and cellular localization of alphaB-crystallin in HeLa cells. Inhibition of nuclear export demonstrated that phosphorylation of alphaB-crystallin is required for import into the nucleus. As revealed by mutant analysis, phosphorylation at Ser-59 is crucial for nuclear import, and phosphorylation at Ser-45 is required for speckle localization. Co-immunoprecipitation experiments suggested that the import of alphaB-crystallin is possibly regulated by its phosphorylation-dependent interaction with the survival motor neuron (SMN) protein, an important factor in small nuclear ribonucleoprotein nuclear import and assembly. This interaction was supported by co-localization of endogenous phosphorylated alphaB-crystallin with SMN in nuclear structures. The cardiomyopathy-causing alphaB-crystallin mutant R120G was found to be excessively phosphorylated, which disturbed SMN interaction and nuclear import, and resulted in the formation of cytoplasmic inclusions. Like for other protein aggregation disorders, hyperphosphorylation appears as an important aspect of the pathogenicity of alphaB-crystallin R120G.
Highlights
␣B-crystallin, one of the most prominent human small heat-shock proteins [1,2,3], has its highest expression in eye lens and muscle cells [4]
Phosphorylation was associated with the localization of ␣B-crystallin during interphase in nuclear splicing speckles and survival motor neuron (SMN)-positive structures, and during cell division in mitotic interchromatin granule clusters (MIGs), which are the mitotic counterparts of nuclear speckles [25]. ␣B-Crystallin R120G was found to be hyperphosphorylated, which disturbed the normal interactions with binding partners such as SMN and splicing components, resulting in impaired nuclear import and accumulation in cytoplasmic inclusions
Nuclear Import of ␣B-crystallin Is Phosphorylation-dependent—We previously showed that transfected pseudophosphorylated ␣B-crystallin as well as endogenous ␣B-crystallin phosphorylated at Ser-45 are found in nuclear speckles in interphase cells [24]
Summary
␣B-crystallin, one of the most prominent human small heat-shock proteins [1,2,3], has its highest expression in eye lens and muscle cells [4]. The phosphorylation dependence of nuclear import was further supported by staining untransfected HeLa cells, with and without leptomycin B treatment, for endogenous ␣B-crystallin phosphorylated at Ser-45 and Ser-59 (Fig. 1b). Cells transfected with ␣B-crystallin S45A revealed a nuclear accumulation upon leptomycin B treatment, but no speckle localization (Fig. 1e, panels A and B).
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