TRIP12 functions as an E3 ubiquitin ligase of APP-BP1.
The NEDD8 pathway plays an essential role in various physiological processes, such as cell cycle progression and signal transduction. The conjugation of NEDD8 to target proteins is initiated by the NEDD8-activating enzyme composed of APP-BP1 and Uba3. In the present study, we show that APP-BP1 is degraded by ubiquitin-dependent proteolysis. To study biological functions of TRIP12, a HECT domain-containing E3 ubiquitin ligase, we used the yeast two-hybrid system and identified APP-BP1 as its binding partner. Immunoprecipitation analysis showed that TRIP12 specifically interacts with the APP-BP1 monomer but not with the APP-BP1/Uba3 heterodimer. Overexpression of TRIP12 enhanced the degradation of APP-BP1, whereas knockdown of TRIP12 stabilized it. In vitro ubiquitination assays revealed that TRIP12 functions as an E3 enzyme of APP-BP1 and additionally requires an E4 activity for polyubiquitination of APP-BP1. Moreover, neddylation of endogenous CUL1 was increased in TRIP12 knockdown cells, while complementation of the knockdown cells with TRIP12 lowered neddylated CUL1. Our data suggest that that TRIP12 promotes degradation of APP-BP1 by catalyzing its ubiquitination, which in turn modulates the neddylation pathway.
Park
Yoon
Y
Department of Biochemistry and Protein Network Research Center, Yonsei University, 134 Shinchon-Dong, Seodaemoon-Gu, Seoul 120-749, Republic of Korea.
Yoon
Sungjoo Kim
SK
Yoon
Jong-Bok
JB
D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D044842:Cullin Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D006801:Humans; D047468:Immunoprecipitation; D000075687:NEDD8 Protein; D017434:Protein Structure, Tertiary; D020798:Two-Hybrid System Techniques; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D014452:Ubiquitins / Q000378:metabolism
D002352:Carrier Proteins; C100318:Cullin 1; D044842:Cullin Proteins; D004268:DNA-Binding Proteins; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D014452:Ubiquitins; C530813:TRIP12 protein, human; D044767:Ubiquitin-Protein Ligases; D044764:Ubiquitin-Activating Enzymes; C491802:NAE protein, human