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

John Den Engelsman, Erik J. Bennink, Linda Doerwald, Carla Onnekink, Lisa Wunderink, Usha P. Andley, Kanefusa Kato, Wilfried W. De Jong, Wilbert C. Boelens

Research output: Contribution to journalArticlepeer-review

65 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)4195-4203
Number of pages9
JournalEuropean Journal of Biochemistry
Volume271
Issue number21
DOIs
StatePublished - Nov 1 2004

Keywords

  • Desmin-related myopathy
  • Phosphorylation
  • SC35
  • Small heat-shock protein
  • Ubiquitin isopeptide ligase

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