Association of FHIT (fragile histidine triad), a candidate tumour suppressor gene, with the ubiquitin-conjugating enzyme hUBC9

FHIT (fragile histidine triad), a candidate tumour suppressor gene, has rec ently been identified at chromosomal region 3p14.2, and deletions of the ge ne have been reported in many types of human cancer. However, the biologica l function of the Fhit protein has not been fully characterized yet. Using the yeast two-hybrid screen to search for proteins that interact with Fhit in vivo, we identified a protein that is specifically associated with Fhit. This association was confirmed in both immunoprecipitation and glutathione S-transferase pull-down assays. The sequence of the protein is identical w ith that of human ubiquitin-conjugating enzyme 9 (hUBC9). The last 21 amino acids at the C-terminus of hUBC9 appear to be unimportant for its biologic al activity, since an hUBC9 mutant harbouring a deletion of these amino aci ds could still restore normal growth of yeast containing a temperature-sens itive mutation in the homologue UBC9 gene. Mutational analysis indicated th at hUBC9 was associated with the C-terminal portion of Fhit. Neither a sing le amino acid substitution at codon 96 (His --> Asn) nor triple amino acid substitutions (His --> Asn) at a histidine triad (codons 94, 96 and 98) aff ected the association, whereas Fhit triphosphate (diadenosine 5',5'''-P-1,P -3-triphosphate) hydrolase activity has been reported to be eliminated by e ither type of mutation, suggesting that the interaction between Fhit and hU BC9 is independent of Fhit enzymic activity. Given that yeast UBC9 is invol ved in the degradation of S- and M-phase cyclins, Fhit may be involved in c ell cycle control through its interaction with hUBC9.