SAG/ROC-SCF beta-TrCP E3 ubiquitin ligase promotes pro-caspase-3 degradation as a mechanism of apoptosis protection.
Skp1-cullin-F-box protein (SCF) is a multicomponent E3 ubiquitin (Ub) ligase that ubiquitinates a number of important biologic molecules such as p27, beta-catenin, and IkappaB for proteasomal degradation, thus regulating cell proliferation and survival. One SCF component, SAG/ROC2/Rbx2/Hrt2, a RING finger protein, was first identified as a redox-inducible protein, which, when overexpressed, inhibited apoptosis both in vitro and in vivo. We report here that sensitive to apoptosis gene (SAG), as well as its family member ROC1/Rbx1, bound to the proinactive form of caspase-3 (pro-caspase-3). Binding was likely mediated through F-box protein, beta-transducin repeat-containing protein (beta-TrCP), which binds to the first 38 amino acids of pro-caspase-3. Importantly, beta-TrCP1 expression significantly shortened the protein half-life of pro-caspase-3, whereas expression of a dominant-negative beta-TrCP1 mutant with the F-box domain deleted extended it. An in vitro ubiquitination assay showed that SAG/ROC-SCF(beta-TrCP) promoted ubiquitination of pro-caspase-3. Furthermore, endogenous levels of pro-caspase-3 were decreased by overexpression of SAG/ROC-SCF(beta-TrCP) E3 Ub ligases, but increased on siRNA silencing of SAG, regulator of cullin-1 (ROC1), or beta-TrCPs, leading to increased apoptosis by etoposide and TNF-related apoptosis-inducing ligand through increased activation of caspase-3. Thus, pro-caspase-3 appears to be a substrate of SAG/ROC-SCF(beta-TrCP) E3 Ub ligase, which protects cells from apoptosis through increased apoptosis threshold by reducing the basal level of pro-caspase-3.
Tan
Mingjia
M
Department of Radiation Oncology, University of Michigan Comprehensive Cancer Center, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0936, USA.
Gallegos
Jayme R
JR
Gu
Qingyang
Q
Huang
Yuanhui
Y
Li
Jun
J
Jin
Yetao
Y
Lu
Hua
H
Sun
Yi
Y
D017209:Apoptosis / Q000502:physiology*; D002352:Carrier Proteins / Q000502:physiology*; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism*; D061945:Caspase Inhibitors; D002460:Cell Line; D045744:Cell Line, Tumor; D019610:Cytoprotection / Q000502:physiology; D006207:Half-Life; D006367:HeLa Cells; D006801:Humans; D016601:RNA-Binding Proteins / Q000502:physiology*; D044843:SKP Cullin F-Box Protein Ligases / Q000502:physiology*; D013379:Substrate Specificity / Q000502:physiology; D044767:Ubiquitin-Protein Ligases / Q000502:physiology*
D002352:Carrier Proteins; D061945:Caspase Inhibitors; C479529:RBX1 protein, human; D016601:RNA-Binding Proteins; C118307:RNF7 protein, human; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases; D053148:Caspase 3