Covalent modification of p73α 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 p73 alpha isolated SUMO-1 (small ubiquitin-like modifier (1) over bar), the enzyme responsible for its conjugation, Ubc-9, and a number of novel SUMO-l-interacting proteins, including thymine DNA glycosylase, PM-Sc175, PIASx, PKY, and CHD3/ZFH, A subset: of these proteins contain a common motif, hhX-SXS/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 p73 alpha, but not p73 beta, can be covalently modified by SUMO-I, The major SUMO-l-modified residue in p73 alpha 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-l-modified p73 is more rapidly degraded by the proteasme than unmodified p73, although SUMO-1 modification:is not required for p73 degradation. SUMO-I modification does not affect the transcriptional activity of p73 alpha 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.