Dissecting the complex regulation of Mad4 in glioblastoma multiforme cells

Abstract

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-IAP 1 E3 ligase activity for degradation, the E3 ligase activity of c-IAP 1 was not required for downregulation of Mad4 expression. The association of c-IAP 1 with Sin3B or Mad4 suggested that Sin3B might interfere with the binding of c-IAP 1 to Mad4; however, overexpression of Sin3B did not affect the interaction between Mad4 and c-IAP 1. Instead, direct binding of Sin3B to c-IAP 1 may protect Mad4 from degradation by c-IAP 1, leading to enhanced stability of Mad4. Exogenous expression of Sin3B also inhibited c-IAP 1-mediated degradation of Mad1, TRAF2, c-IAP 2 and AS K1, known targets of c-IAP 1 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-IAP 1-mediated degradation of Mad4. © 2012 Landes Bioscience.