The tumour suppressor PTEN mediates a negative regulation of the E3 ubiquitin-protein ligase Nedd4.
The tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10; a phosphatidylinositol 3-phosphatase) is a multifunctional protein deregulated in many types of cancer. It is suggested that a number of proteins that relate with PTEN functionally or physically have not yet been found. In order to search for PTEN-interacting proteins that might be crucial in the regulation of PTEN, we exploited a proteomics-based approach. PTEN-expressing NIH 3T3 cell lysates were used in affinity chromatography and then analysed by LC-ESI-MS/MS (liquid chromatography-electrospray ionization-tandem MS). A total of 93 proteins were identified. Among the proteins identified, we concentrated on the E3 ubiquitin-protein ligase Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated gene 4), and performed subsequent validation experiments using HeLa cells. Nedd4 inhibited PTEN-induced apoptotic cell death and, conversely, the Nedd4 level was down-regulated by PTEN. The down-regulation effect was diminished by a mutation (C124S) in the catalytic site of PTEN. Nedd4 expression was also decreased by a PI3K (phosphoinositide 3-kinase) inhibitor, LY294002, suggesting that the regulation is dependent on the phosphatase-kinase activity of the PTEN-PI3K/Akt pathway. Semi-quantitative real-time PCR analysis revealed that Nedd4 was transcriptionally regulated by PTEN. Thus our results have important implications regarding the roles of PTEN upon the E3 ubquitin ligase Nedd4 as a negative feedback regulator as well as a substrate.
Ahn
Younghee
Y
Life Sciences Division, Korea Institute of Science and Technology, Seoul 136-791, Korea.
Hwang
Chae Young
CY
Lee
Seung-Rock
SR
Kwon
Ki-Sun
KS
Lee
Cheolju
C
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D020169:Caspases / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D000075702:Nedd4 Ubiquitin Protein Ligases; D051059:PTEN Phosphohydrolase / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D020543:Proteome / Q000032:analysis; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014158:Transcription, Genetic; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000235:genetics / Q000378:metabolism
D056827:Endosomal Sorting Complexes Required for Transport; D020543:Proteome; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618461:Nedd4l protein, mouse; D044767:Ubiquitin-Protein Ligases; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human; D020169:Caspases