A Kruppel zinc finger of ZNF 146 interacts with the SUMO-1 conjugating enzyme UBC9 and is sumoylated in vivo.
The OZF (ZNF146) protein is a 33 kDa Kruppel protein, composed solely of 10 zinc finger motifs. It is overexpressed in the majority of pancreatic cancers and in more than 80% of colorectal cancers. We have identified OZF interacting factors with a yeast two-hybrid screen. Half of the positive clones characterized encoded UBC9, the E2 enzyme involved in the covalent conjugation of the small ubiquitin-like modifier 1 (SUMO-1). SUMO-1 is a 17 kDa migrating protein that is conjugated to several proteins and has been reported to exhibit multiple effects, including modulation of protein stability, subcellular localization, and gene expression. In HeLa cells transfected with OZF and SUMO-1 expression vectors, immunoblot revealed a major band migrating at 50 kDa and a minor band at 67 kDa, corresponding to the attachment to OZF of one and two SUMO-1 proteins, respectively. The relative amount of the sumoylated proteins increased following transfection with a UBC9 expression vector. The presence of the sumoylated form in HeLa cells solely transfected by OZF indicates the physiological activity of the endogenous SUMO-1 conjugation pathway. Using deletion mutants, we showed that two SUMO-1 modification sites are located on the sixth zinc finger. Mutation of two lysine residues greatly reduced the amount of the sumoylated form of OZF though their surrounding sequences differ from the consensus sequence reported for most proteins modified by SUMO-1 conjugation. Despite the presence of the sixth zinc finger, an OZF mutant containing zinc fingers 1-6 was not modified by SUMO-1 and failed to interact with UBC9. Addition of zinc finger 7 restored SUMO-1 modification and UBC9 interaction and provides evidence that a region downstream of the target lysines is required for interaction with UBC9, in order to achieve SUMO-1 modification. This is the first report of in vivo conjugation of a SUMO-1 protein to a Kruppel zinc finger motif.
Antoine
Karène
K
Laboratoire d'Oncogenèse, FRE 2584 CNRS, Institut Curie, 26 rue d'Ulm, 75248 Paris 05, France.
Prosperi
Marie-Thérèse
MT
Ferbus
Didier
D
Boule
Catherine
C
Goubin
Gérard
G
D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051741:Kruppel-Like Transcription Factors; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry; D025842:SUMO-1 Protein / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D004268:DNA-Binding Proteins; D051741:Kruppel-Like Transcription Factors; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D025842:SUMO-1 Protein; D014157:Transcription Factors; C085944:ZNF146 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9; D008239:Lysine