Dissecting the complex regulation of Mad4 in glioblastoma multiforme cells.
Among proteins in the c-Myc/Max/Mad/Sin3 regulatory complex, Mad4 and Sin3B are routinely detected in human glioblastoma multiforme (GBM) cell lines. In response to gamma radiation, the expression of Sin3B and Mad4 in GBM cells was upregulated in parallel over time, suggesting that Sin3B may play a role in the regulation of Mad4 stability. In agreement with this hypothesis, exogenously expressed Sin3B significantly stabilized co-transfected Mad4 and, to a lesser extent, endogenous Mad4. In addition, siRNA silencing of Sin3B induced an increase in the expression of c-Myc and Sin3A, which contributed to increased expression of Mad4. Simultaneous silencing of Sin3B, Sin3A and c-Myc decreased Mad4 stability to a greater extent than silencing of Sin3B alone. Although Mad1 was reported to be a target of c-IAP1 E3 ligase activity for degradation, the E3 ligase activity of c-IAP1 was not required for downregulation of Mad4 expression. The association of c-IAP1 with Sin3B or Mad4 suggested that Sin3B might interfere with the binding of c-IAP1 to Mad4; however, overexpression of Sin3B did not affect the interaction between Mad4 and c-IAP1. Instead, direct binding of Sin3B to c-IAP1 may protect Mad4 from degradation by c-IAP1, leading to enhanced stability of Mad4. Exogenous expression of Sin3B also inhibited c-IAP1-mediated degradation of Mad1, TRAF2, c-IAP2 and ASK1, known targets of c-IAP1 E3 ligase activity. These results indicate that Sin3B, together with other c-Myc regulatory members, maintain the steady-state level of Mad4, in part through inhibition of c-IAP1-mediated degradation of Mad4.
Department of Radiation Oncology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. wensheny@mail.med.upenn.edu
Wensheng
W
Yang
Xiaolu
X
Yang
Gregory
G
David
Jay F
JF
Dorsey
D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics* / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D005909:Glioblastoma / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism*; D056264:Sin3 Histone Deacetylase and Corepressor Complex; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism
D051792:Basic Helix-Loop-Helix Proteins; D018797:Cell Cycle Proteins; D051034:Inhibitor of Apoptosis Proteins; D009687:Nuclear Proteins; D016271:Proto-Oncogene Proteins c-myc; D012097:Repressor Proteins; D056264:Sin3 Histone Deacetylase and Corepressor Complex; D044767:Ubiquitin-Protein Ligases; C501384:MAD1L1 protein, human; C484347:MXD4 protein, human; C099683:SIN3A transcription factor; C492042:SIN3B protein, human