SIM-dependent enhancement of substrate-specific SUMOylation by a ubiquitin ligase in vitro.
SIMs (SUMO-interaction motifs), which mediate the non-covalent binding of SUMO (small ubiquitin-related modifier) to other proteins, are usually involved in the recognition of SUMOylated substrates by downstream effectors that transmit the biological signal of the modification. In ubiquitin ligase Rad18 (radiation-sensitive 18) from Saccharomyces cerevisiae, a SIM, contributes to the recognition of SUMOylated PCNA (proliferating-cell nuclear antigen) as its physiological ubiquitylation target. In the present study we show that Rad18 is also capable of enhancing PCNA SUMOylation in a SIM-dependent manner in vitro, most probably by means of directing SUMO-loaded Ubc9 (ubiquitin-conjugating enzyme 9) towards the substrate. The process shares important features with Rad18-dependent ubiquitylation, such as an exquisite specificity for the modification site on PCNA and the requirement of DNA, and the reaction proceeds under conditions that are widely used in other in vitro assays for SUMO ligase activity. However, there is no evidence that Rad18 contributes to PCNA SUMOylation in vivo. The findings of the present study therefore illustrate the problematic nature of in vitro SUMOylation assays and highlight the danger of extrapolating from this type of experiment to the biological function of a SUMO-interacting protein.
*Cancer Research UK London Research Institute, Clare Hall Laboratories, Blanche Lane, South Mimms EN6 3LD, U.K.
Joanne L
JL
Parker
Helle D
HD
Ulrich
D019943:Amino Acid Substitution; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry; D008954:Models, Biological; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D025842:SUMO-1 Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013379:Substrate Specificity; D058207:Sumoylation; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D004268:DNA-Binding Proteins; D050505:Mutant Proteins; C546594:POL30 protein, S cerevisiae; D018809:Proliferating Cell Nuclear Antigen; C063607:RAD18 protein, S cerevisiae; D011994:Recombinant Proteins; D025842:SUMO-1 Protein; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D000098843:Ubiquitin-Conjugating Enzyme UBC9; D008239:Lysine