Critical role of lysine 123 in the ubiquitin-mediated degradation of MDA-7/IL-24.
Melanoma differentiation-associated gene-7/interleukin-24 (MDA-7/IL-24) uniquely takes on multiple anticancer functions, such as direct tumor cell cytotoxicity, immune stimulation, and antiangiogenic activities. MDA-7/IL-24 protein levels depend on proteasome degradation. Western blotting and coimmunoprecipitation analyses verified that the MDA-7/IL-24 protein was ubiquitinated and degraded by the 26S proteasome in Hela cells, which was confirmed by protein accumulation treated with proteasome inhibitor MG132. MDA-7/IL-24 contains 10 lysine sites: K63, K69, K78, K119, K123, K136, K179, K189, K203, and K206. Site-directed mutagenesis in these sites reveals that lysine 123 is the major internal lysine involvement of MDA-7/IL-24 ubiquitination. Our results further demonstrated that the mutation of lysine 123 to arginine led to the inhibition of ubiquitin-mediated degradation and the recovery of MDA-7/IL-24 protein level. At the same time, the apoptosis-inducing activity of K123R mutant was obviously stronger than wild-type MDA-7/IL-24 detected by 4'-6-diamidino-2-phenylindole (DAPI) assay and annexin V analysis. In addition, K123R mutant tilted the balance between antiapoptotic protein Bcl-2 and proapoptotic protein Bax, which subsequently induced the cleavage of caspase signaling cascade and initiated cell apoptosis. Together, lysine 123 is mainly implicated in the ubiquitination and degradation of MDA-7/IL-24. Inhibition of degradation and ubiquitination of MDA-7/IL-24 through mutation of lysine 123 result in enhanced stability of MDA-7/IL-24 and exhibits persistent tumor suppression activity compared with the wild type.
Laboratory of Biological Cancer Therapy, Xuzhou Medical College, Xuzhou, Jiangsu 221002, P.R. China.
Hui
H
Tian
Liantao
L
Li
Baofu
B
Zhang
Jiehui
J
Di
Feifei
F
Chen
Huizhong
H
Li
Junjie
J
Liu
Dongsheng
D
Pei
Junnian
J
Zheng
D017209:Apoptosis; D001120:Arginine / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D005260:Female; D006367:HeLa Cells; D006801:Humans; D007378:Interleukins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects / Q000378:metabolism*; D055550:Protein Stability / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D054875:Ubiquitination / Q000187:drug effects; D002583:Uterine Cervical Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D000099180:Interleukin-24
D001120:Arginine; D053148:Caspase 3; D015853:Cysteine Proteinase Inhibitors; D007378:Interleukins; D008239:Lysine; D050505:Mutant Proteins; D009363:Neoplasm Proteins; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D046988:Proteasome Endopeptidase Complex; D011994:Recombinant Proteins; D000099180:Interleukin-24; C499320:PARP1 protein, human; C505407:CASP3 protein, human