Mimicking phosphorylation of the small heat-shock protein alphaB-crystallin recruits the F-box protein FBX4 to nuclear SC35 speckles.
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 and all possible pseudophosphorylation mutants of alphaB-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 alphaB-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 alphaB-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 alphaB-crystallin, was also recruited to SC35 speckles when cotransfected with the pseudophosphorylation mutants. Because SC35 speckles also react with an antibody against alphaB-crystallin endogenously phosphorylated at Ser45, our findings suggest that alphaB-crystallin has a phosphorylation-dependent role in the ubiquitination of a component of SC35 speckles.
den Engelsman
John
J
Department of Biochemistry 161, Nijmegen Center for Molecular Life Sciences, University of Nijmegen, the Netherlands.
Bennink
Erik J
EJ
Doerwald
Linda
L
Onnekink
Carla
C
Wunderink
Lisa
L
Andley
Usha P
UP
Kato
Kanefusa
K
de Jong
Wilfried W
WW
Boelens
Wilbert C
WC
D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism; D044783:F-Box Proteins / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D000068103:Serine-Arginine Splicing Factors; D013347:Subcellular Fractions / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D038203:alpha-Crystallin B Chain / Q000737:chemistry* / Q000378:metabolism
D003902:Detergents; D044783:F-Box Proteins; D009687:Nuclear Proteins; D012261:Ribonucleoproteins; D025801:Ubiquitin; D038203:alpha-Crystallin B Chain; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors; D012694:Serine; D003850:Deoxyribonuclease I; D012259:Ribonuclease, Pancreatic; D010450:Endopeptidases; C091939:ubiquitin isopeptidase