ZNF198, a zinc finger protein rearranged in myeloproliferative disease, localizes to the PML nuclear bodies and interacts with SUMO-1 and PML.
The ZNF198/FGFR1 fusion gene in atypical myeloproliferative disease produces a constitutively active cytoplasmic tyrosine kinase, unlike ZNF198 which is normally a nuclear protein. We have now shown that the ZNF198/FGFR1 fusion kinase interacts with the endogenous ZNF198 protein suggesting that the function of ZNF198 may be compromised in cells expressing it. Little is currently known about the endogenous function of ZNF198 and to investigate this further we performed a yeast two-hybrid analysis and identified SUMO-1 as a binding partner of ZNF198. These observations were confirmed using co-immunoprecipitation which demonstrated that ZNF198 is covalently modified by SUMO-1. Since many of the SUMO-1-modified proteins are targeted to the PML nuclear bodies we used confocal microscopy to show that SUMO-1, PML and ZNF198 colocalize to punctate structures, shown by immunocytochemistry to be PML bodies. Using co-immunoprecipitation we now show that PML and sumoylated ZNF198 can be found in a protein complex in the cell. Mutation of the SUMO-1 binding site in wild-type ZNF198 resulted in loss of distinct PML bodies, reduced PML levels and a more dispersed nuclear localization of the PML protein. In cells expressing ZNF198/FGFR1, which also lack the SUMO-1 binding site, SUMO-1 is preferentially localized in the cytoplasm, which is associated with loss of distinct PML bodies. Recently, arsenic trioxide (ATO) was proposed as an alternative therapy for APL that was resistant to traditional therapy. Treatment of cells expressing ZNF198/FGFR1 with ATO demonstrated reduced autophosphorylation of the ZNF198/FGFR1 protein and induced apoptosis, which is not seen in cells expressing wild-type ZNF198. Overall our results suggest that the sumoylation of ZNF198 is important for PML body formation and that the abrogation of sumoylation of ZNF198 in ZNF198/FGFR1 expressing cells may be an important mechanism in cellular transformation.
Kunapuli
Padmaja
P
Department of Cancer Genetics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA.
Kasyapa
Chitta S
CS
Chin
Suet-Feung
SF
Caldas
Carlos
C
Cowell
John K
JK
D019943:Amino Acid Substitution; D017209:Apoptosis / Q000187:drug effects; D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D022003:Cell Nucleus Structures / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019281:Dimerization; D050939:Gene Fusion; D015321:Gene Rearrangement; D006801:Humans; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D050506:Mutant Chimeric Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009196:Myeloproliferative Disorders / Q000235:genetics* / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D010087:Oxides / Q000494:pharmacology; D017396:Plasminogen Activator Inhibitor 2 / Q000235:genetics; D000071156:Promyelocytic Leukemia Protein; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025842:SUMO-1 Protein / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D016335:Zinc Fingers / Q000235:genetics
D001152:Arsenicals; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D050506:Mutant Chimeric Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D010087:Oxides; D017396:Plasminogen Activator Inhibitor 2; D000071156:Promyelocytic Leukemia Protein; D025842:SUMO-1 Protein; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C111777:ZMYM2 protein, human; C070576:PML protein, human; C496348:FGFR1 protein, human; D051496:Receptor, Fibroblast Growth Factor, Type 1; D000077237:Arsenic Trioxide