Mimicking phosphorylation of the small heat-shock protein αB-crystallin recruits the F-box protein FBX4 to nuclear SC35 speckles

The mammalian small heat shock protein αB-crystallin can be phosphorylated at three different sites, Ser19, Ser45 and Ser59. We compared the intracellular distribution of wildtype, nonphosphorylatable and all possible pseudophosphorylation mutants of αB-crystallin by immunoblot and immunocytochemical analyses of stable and transiently transfected cells. We observed that pseudophosphorylation at two (especially S19D/S45D) or all three (S19D/S45D/ S59D) sites induced the partial translocation of αB-crystallin from the detergent-soluble to the detergent-insoluble fraction. Double immunofluorescence studies showed that the pseudophosphorylation mutants localized in nuclear speckles containing the splicing factor SC35. The αB-crystallin mutants in these speckles were resistant to mild detergent treatment, and also to DNase I or RNase A digestion, indicating a stable interaction with one or more speckle proteins, not dependent on intact DNA or RNA. We further found that FBX4, an adaptor protein of the ubiquitin-protein isopeptide ligase SKP1/CUL1/F-box known to interact with pseudophosphorylated αB-crystallin, was also recruited to SC35 speckles when cotransfected with the pseudophosphorylation mutants. Because SC35 speckles also react with an antibody against αB-crystallin endogenously phosphorylated at Ser45, our findings suggest that αB-crystallin has a phosphorylation-dependent role in the ubiquitination of a component of SC35 speckles.