Expression of SUMO-2/3 induced senescence through p53- and pRB-mediated pathways.
Three highly homologous small ubiquitin-related modifier (SUMO) proteins have been identified in mammals. Modifications of proteins by SUMO-1 have been shown to regulate transcription, nucleocytoplasmic transport, protein stability, and protein-protein interactions. Relative to SUMO-1, little is known about the functions of SUMO-2 or SUMO-3 (referred to as SUMO-2/3). Here, stable cell lines overexpressing processed forms of SUMO-2/3 (SUMO-2/3GG) as well as their non-conjugatable derivatives, SUMO-2/3DeltaGG, were established. Cells overexpressing SUMO-2/3GG showed a premature senescence phenotype as revealed by cellular morphology and senescence-associated galactosidase activity. The senescence pathway protein p21 was up-regulated in cells overexpressing SUMO-2/3GG. In contrast, cells overexpressing non-conjugatable forms of SUMO-2/3DeltaGG showed neither an apparent senescent phenotype nor elevated p21. Both p53 and pRB were found to be modified by SUMO-2/3. Site-directed mutagenesis studies showed that Lys-386 of p53, the SUMO-1 modification site, is also the modification site for SUMO-2/3. In addition, H2O2 treatment of untransfected cells caused an increase in p53 sumoylation by SUMO-2/3, whereas that by SUMO-1 remained unchanged. Moreover, knocking down tumor suppressor proteins p53 or pRB using small interfering RNA significantly alleviated the premature senescence phenotypes in SUMO-2/3GG overexpressing cells. Together, our results reveal that p53 and pRB can be sumoylated by SUMO-2/3 in vivo, and such modification of p53 and pRB may play roles in premature senescence and stress response.
Li
Tianwei
T
Laboratory of Biochemistry and Proteomics Core Facility, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Santockyte
Rasa
R
Shen
Rong-Fong
RF
Tekle
Ephrem
E
Wang
Guanghui
G
Yang
David C H
DC
Chock
P Boon
PB
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D016922:Cellular Senescence; D003593:Cytoplasm / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D034741:RNA, Small Interfering / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000502:physiology*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D014452:Ubiquitins / Q000502:physiology*
D034741:RNA, Small Interfering; D016160:Retinoblastoma Protein; C117909:SUMO2 protein, human; C515972:SUMO2 protein, mouse; C492189:SUMO3 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; C117908:Sumo3 protein, mouse; D016159:Tumor Suppressor Protein p53; D014452:Ubiquitins