Cloning and characterization of mouse UBPy, a deubiquitinating enzyme that interacts with the ras guanine nucleotide exchange factor CDC25(Mm)/Ras-GRF1.
We used yeast "two-hybrid" screening to isolate cDNA-encoding proteins interacting with the N-terminal domain of the Ras nucleotide exchange factor CDC25(Mm). Three independent overlapping clones were isolated from a mouse embryo 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 domain typical of ubiquitin isopeptidases. Therefore we called this new protein mouse UBPy (mUBPy). Northern blot analysis revealed a 4-kilobase mRNA present in several mouse tissues and highly expressed in testis; a good level of expression was also found in brain, where CDC25(Mm) is exclusively expressed. 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 after cotransfection in mammalian cells. We also showed that CDC25(Mm), expressed in HEK293 cells, is ubiquitinated and that the coexpression of mUBPy decreases its ubiquitination. In addition the half-life of CDC25Mm protein was considerably increased in the presence of mUBPy. The specific function of the human homolog hUBPy is not defined, although its expression was correlated 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.
Gnesutta
N
N
Dipartimento di Fisiologia e Biochimica Generali, Università di Milano, Via Celoria 26, Milano, Italy.
Ceriani
M
M
Innocenti
M
M
Mauri
I
I
Zippel
R
R
Sturani
E
E
Borgonovo
B
B
Berruti
G
G
Martegani
E
E
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics*; D056827:Endosomal Sorting Complexes Required for Transport; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013737:Testis / Q000378:metabolism; D013997:Time Factors; D014018:Tissue Distribution; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism; D043222:Ubiquitin Thiolesterase; D020666:ras-GRF1 / Q000737:chemistry* / Q000378:metabolism*
D018076:DNA, Complementary; D056827:Endosomal Sorting Complexes Required for Transport; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D025801:Ubiquitin; D020666:ras-GRF1; D005982:Glutathione Transferase; D010450:Endopeptidases; C541534:USP8 protein, human; D043222:Ubiquitin Thiolesterase; C541536:Usp8 protein, mouse; C091939:ubiquitin isopeptidase