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

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