Covalent modification of p73alpha by SUMO-1. Two-hybrid screening with p73 identifies novel SUMO-1-interacting proteins and a SUMO-1 interaction motif.

Two-hybrid screening in yeast with p73alpha isolated SUMO-1 (small ubiquitin-like modifier 1), the enzyme responsible for its conjugation, Ubc-9, and a number of novel SUMO-1-interacting proteins, including thymine DNA glycosylase, PM-Scl75, PIASx, PKY, and CHD3/ZFH. A subset of these proteins contain a common motif, hhXSXS/Taaa, where h is a hydrophobic amino acid and a is an acidic amino acid, that is shown to interact with SUMO-1 in the two-hybrid system. We show here that p73alpha, but not p73beta, can be covalently modified by SUMO-1. The major SUMO-1-modified residue in p73alpha is the C-terminal lysine (Lys(627)). The sequence surrounding this lysine conforms to a consensus SUMO-1 modification site b(X)XXhKXE, where b is a basic amino acid. SUMO-1-modified p73 is more rapidly degraded by the proteasome than unmodified p73, although SUMO-1 modification is not required for p73 degradation. SUMO-1 modification does not affect the transcriptional activity of p73alpha on an RGC-luciferase reporter gene in SK-N-AS cells. Instead, SUMO-1 modification may alter the subcellular localization of p73, because SUMO-1-modified p73 is preferentially found in detergent-insoluble fractions. Alternatively, it may modulate the interaction of p73 with other proteins that are substrates for SUMO-1 modification or which interact with SUMO-1, such as those identified here.