Nedd4 regulates ubiquitination and stability of the guanine-nucleotide exchange factor CNrasGEF.
Cyclic nucleotide ras GEF (CNrasGEF) is a guanine-nucleotide exchange factor previously isolated in a screen for Nedd4-WW domain interacting proteins (Pham, N., Cheglakov, I., Koch, C. A., de Hoog, C. L., Moran, M. F., and Rotin, D. (2000) Curr. Biol. 10, 555-558). It activates Ras in a cAMP-dependent manner and Rap-1 independent of cAMP. Here we show that CNrasGEF is a likely substrate of the ubiquitin protein ligase Nedd4. CNrasGEF possesses two PY motifs at its C terminus that are responsible for binding to Nedd4 in vitro. Moreover, Nedd4 and CNrasGEF co-immunoprecipitate from 293T cells expressing ectopic CNrasGEF and endogenous Nedd4, and this co-immunoprecipitation is abrogated in PY motif-mutated CNrasGEF (CNrasGEFDelta2PY). CNrasGEF is ubiquitinated in cells, and this ubiquitination is augmented upon overexpression of wt-Nedd4 but is inhibited in cells overexpressing a catalytically inactive Nedd4 (Nedd4(CS)) or in cells expressing CNrasGEFDelta2PY, which cannot bind Nedd4. Moreover, pulse-chase experiments have demonstrated that the half-life of CNrasGEF is reduced 5-fold (from approximately 10 to approximately 2 h) in cells co-expressing Nedd4 with CNrasGEF but not with CNrasGEFDelta2PY (t(0.5) approximately 14 h). CNrasGEF is also stabilized in cells co-expressing Nedd4(CS) or following treatment with lactacystin, indicating proteasomal degradation of this protein. Deletion/mutation of the CDC25 domain to abrogate Ras (or Rap-1) binding leads to impaired ubiquitination of CNrasGEF, suggesting that such binding is critical for ubiquitination. Treatment of cells with the cAMP analogue 8-bromo-cAMP does not affect the ability of CNrasGEF to bind Nedd4 nor its level of ubiquitination, suggesting that Ras binding per se and not its activation is the critical step in triggering ubiquitination of CNrasGEF. These results suggest that CNrasGEF is a substrate for Nedd4, which regulates its ubiquitination and stability in cells.
Pham
N
N
Program in Cell Biology, Hospital for Sick Children, University of Toronto, 555 University Ave., Toronto, Ontario M5G 1X8, Canada.
Rotin
D
D
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000378:metabolism; D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000378:metabolism* / Q000502:physiology*; D002384:Catalysis; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport; D004789:Enzyme Activation; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008025:Ligases / Q000378:metabolism* / Q000502:physiology*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D000075702:Nedd4 Ubiquitin Protein Ligases; D009419:Nerve Tissue Proteins; D009712:Nucleotides, Cyclic / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D020663:ras Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism
D002135:Calcium-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D020662:Guanine Nucleotide Exchange Factors; D009097:Multienzyme Complexes; D009419:Nerve Tissue Proteins; D009712:Nucleotides, Cyclic; C403525:RAPGEF2 protein, human; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D020663:ras Guanine Nucleotide Exchange Factors; C067713:lactacystin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000242:Cyclic AMP; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D018631:ras Proteins; D008025:Ligases; D000111:Acetylcysteine