Transforming growth factor beta facilitates beta-TrCP-mediated degradation of Cdc25A in a Smad3-dependent manner.
Ubiquitin-dependent degradation of Cdc25A is a major mechanism for damage-induced S-phase checkpoint. Two ubiquitin ligases, the Skp1-cullin-beta-TrCP (SCFbeta-TrCP) complex and the anaphase-promoting complex (APCCdh1), are involved in Cdc25A degradation. Here we demonstrate that the transforming growth factor beta (TGF-beta)-Smad3 pathway promotes SCF(beta-TrCP)-mediated Cdc25A ubiquitination. Cells treated with TGF-beta, as well as cells transfected with Smad3 or a constitutively active type I TGF-beta receptor, exhibit increased ubiquitination and markedly shortened half-lives of Cdc25A. Furthermore, Cdc25A is stabilized in cells transfected with Smad3 small interfering RNA (siRNA) and cells from Smad3-null mice. TGF-beta-induced ubiquitination is associated with Cdc25A phosphorylation at the beta-TrCP docking site (DS82G motif) and physical association of Cdc25A with Smad3 and beta-TrCP. Cdc25A mutant proteins deficient in DS82G phosphorylation are resistant to TGF-beta-Smad3-induced degradation, whereas a Cdc25A mutant protein defective in APCCdh1 recognition undergoes efficient degradation. Smad3 siRNA inhibits beta-TrCP-Cdc25A interaction and Cdc25A degradation in response to TGF-beta. beta-TrCP2 siRNA also inhibits Smad3-induced Cdc25A degradation. In contrast, Cdh1 siRNA had no effect on Cdc25A down-regulation by Smad3. These data suggest that Smad3 plays a key role in the regulation of Cdc25A ubiquitination by SCFbeta-TrCP and that Cdc25A stabilization observed in various cancers could be associated with defects in the TGF-beta-Smad3 pathway.
Ray
Dipankar
D
Department of Biochemistry and Molecular Genetics, University of Illinois College of Medicine, Chicago, IL 60607, USA.
Terao
Yasuhisa
Y
Nimbalkar
Dipali
D
Chu
Li-Hao
LH
Donzelli
Maddalena
M
Tsutsui
Tateki
T
Zou
Xianghong
X
Ghosh
Asish K
AK
Varga
John
J
Draetta
Giulio F
GF
Kiyokawa
Hiroaki
H
D000818:Animals; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006207:Half-Life; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D009154:Mutation / Q000235:genetics; D009369:Neoplasms / Q000378:metabolism*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism; D051900:Smad3 Protein; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology / Q000502:physiology*; D025801:Ubiquitin / Q000378:metabolism; D044844:beta-Transducin Repeat-Containing Proteins / Q000378:metabolism / Q000502:physiology*; D020687:cdc25 Phosphatases / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; D018125:Receptors, Transforming Growth Factor beta; C497790:SMAD3 protein, human; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D025801:Ubiquitin; D044844:beta-Transducin Repeat-Containing Proteins; C517004:CDC25A protein, human; C517005:Cdc25a protein, mouse; D020687:cdc25 Phosphatases; D046988:Proteasome Endopeptidase Complex