Cloning and characterization of mouse UBPy, a deubiquitinating enzyme thatinteracts with the Ras guanine nucleotide exchange factor CDC25(Mm)/Ras-GRF1

We used yeast "two-hybrid" screening to isolate cDNA-encoding proteins inte racting with the N-terminal domain of the Ras nucleotide exchange factor CD C25(Mm). Three independent overlapping clones were isolated from a mouse em bryo cDNA library. The full-length cDNA was cloned by RACE-polymerase chain reaction. It encodes a large protein (1080 amino acids) highly homologous to the human deubiquitinating enzyme hUBPy and contains a well conserved do main typical of ubiquitin isopeptidases. Therefore we called this new prote in mouse UBPy (mUBPy). Northern blot analysis revealed a 4-kilobase mRNA pr esent in several mouse tissues and highly expressed in testis; a good level of expression was also found in brain, where CDC25(Mm) is exclusively expr essed. Using a glutathione S-transferase fusion protein, we demonstrated an "in vitro" interaction between mUBPy and the N-terminal half (amino acids 1-625) of CDC25(Mm). In addition "in vivo" interaction was demonstrated aft er cotransfection in mammalian cells. We also showed that CDC25Mm, expresse d in HEK293 cells, is ubiquitinated and that the coexpression of mUBPy decr eases its ubiquitination. In addition the half-life of CDC25Mm protein was considerably increased in the presence of mUBPy. The specific function of t he human homolog hUBPy is not defined, although its expression was correlat ed with cell proliferation. Our results suggest that mUBPy may play a role in controlling degradation of CDC25(Mm), thus regulating the level of this Ras-guanine nucleotide exchange factor.