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The mammalian small heat shock proteinaB-crystallin can be phosphorylated at three different sites, Ser19, Ser45 and Ser59. We compared the intracellular distribution of wild-type, nonphosphorylatable and all possible pseudophos-phorylation mutants ofaB-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 translocationofaB-crystallin from the detergent-soluble to the detergent-insoluble frac-tion | Eur. J. Biochem. 271 4195-4203 2004 FEBS 2004 doi 10.1111 j.1432-1033.2004.04359.x Mimicking phosphorylation of the small heat-shock protein aB-crystallin recruits the F-box protein FBX4 to nuclear SC35 speckles John den Engelsman1 Erik J. Bennink1 Linda Doerwald1 Carla Onnekink1 Lisa Wunderink1 Usha P. Andley2 Kanefusa Kato3 Wilfried W. de Jong1 and Wilbert C. Boelens1 1 Department of Biochemistry 161 Nijmegen Center for Molecular Life Sciences University of Nijmegen the Netherlands department of Ophthalmology and Visual Sciences Washington University School of Medicine St. Louis MO USA 3Institute for Developmental Research Aichi Human Service Center Kasugai Aichi Japan The mammalian small heat shock protein aB-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 aB-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 aB-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 aB-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-pro-tein isopeptide ligase SKP1 CUL1 F-box known to interact with pseudophosphorylated aB-crystallin was also recruited to SC35 speckles when cotransfected with the pseudophosphorylation mutants. Because SC35 speckles also react with an antibody against aB-crystallin endogenously phosphorylated at Ser45 our .
