CHARACTERIZATION OF NEDD8, A DEVELOPMENTALLY DOWN-REGULATED UBIQUITIN-LIKE PROTEIN

NEDD8 is a novel 81 amino acid polypeptide which is 60% identical and 80% homologous to ubiquitin, Northern blot analysis showed that the NE DD8 message was developmentally down-regulated, In adult tissues, NEDD 8 expression was mostly restricted to the heart and skeletal muscle. A ntiserum specific for NEDD8 detected a 6-kDa monomer in SK-N-SH, BJAB, and HL60 cell lysates, A 14-kDa band was also detected in BJAB, HL60, and SK-MEL28 but not in SK-N-SH and K562 cell lysates, An approximate ly 90-kDa band was detected in all cell lines tested, Thus, NEDD8 is l ikely to be conjugated to other proteins in a manner analogous to ubiq uitination. However, the conjugation pattern of NEDD8 is entirely diff erent from that of ubiquitin in all cell lines tested, To study NEDD8 conjugation in more detail, hemagglutinin-epitope-tagged NEDD8 was exp ressed in COS cells. Western blot analysis revealed an NEDD8 monomer a nd a series of higher molecular weight NEDD8-conjugated proteins or NE DD8 multimers, Immunocytochemical analysis showed that NEDD8 expressio n was highly enriched in the nucleus and was much weaker in the cytoso l, In contrast, ubiquitin expression was detectable equally well in th e nucleus and cytosol, Mutational analysis showed that the C terminus of NEDD8 was efficiently cleaved and that Gly-76 was required for conj ugation of NEDD8 to other proteins, Taken together, NEDD8 provides ano ther substrate for covalent protein modification and may play a unique role during development.