Association of v-ErbA with Smad4 Disrupts TGF-β Signaling

Abstract

Disruption of the transforming growth factor-␤ (TGF-␤) pathway is observed in the majority of cancers. To further understand TGF-␤ pathway inactivation in cancer, we stably expressed the v-ErbA oncoprotein in TGF-␤ responsive cells. v-ErbA participates in erythroleukemic transformation of cells induced by the avian erythroblastosis virus (AEV).Here we demonstrate that expression of v-ErbA was sufficient to antagonize TGF-␤-induced cell growth inhibition and that dysregulation of TGF-␤ signaling required that v-ErbA associate with the Smad4 which sequesters Smad4 in the cytoplasm. We also show that AEV-transformed erythroleukemia cells were resistant to TGF-␤-induced growth inhibition and that TGF-␤ sensitivity could be recovered by reducing v-ErbA expression. Our results reveal a novel mechanism for oncogenic disruption of TGF-␤ signaling and provide a mechanistic explanation of v-ErbA activity in AEV-induced erythroleukemia. INTRODUCTIONThe transforming growth factor-␤ (TGF-␤) signaling pathway is a major signaling network that controls cell proliferation, differentiation, and tumor suppression (Massague et al., 2000;Attisano and Wrana, 2002;Wakefield and Roberts, 2002). The TGF-␤ cytokine signals through hetero-oligomeric complexes of type I and type II serine/threonine kinase receptors, TGF-␤ type I (T␤RI) and type II (T␤RII) receptors (Heldin et al., 1997;Massague et al., 2000). On ligand binding to the T␤RII receptor, T␤RII phosphorylates T␤RI and activates the Tß␤RI kinase (Wrana et al., 1992;Chen and Weinberg, 1995). The signal is further propagated by phosphorylation of the downstream signal transducers Smad2 and Smad3 by the type I receptor kinase (Heldin et al., 1997). Phosphorylated Smad2 and Smad3 form a complex with Smad4 and translocate into the nucleus (Massague et al., 2000). These Smad complexes then interact with other transcription factors and bind the promoter regions of TGF-␤-responsive genes to regulate gene expression (Massague et al., 2000).Insensitivity to antigrowth signals such as TGF-␤ is common in cancer cells (Hanahan and Weinberg, 2000;Massague et al., 2000). Multiple genetic mutations are observed in TGF-␤-insensitive tumor cells, and mechanisms have been identified that explain the disruption of TGF-␤'s cytostatic activity. These mechanisms include the down-regulation of TGF-␤ receptors, mutations in TGF-␤ signaling components such as Smad4, and mutations in other downstream targets (Hanahan and Weinberg, 2000;Massague et al., 2000). Overexpression of oncoproteins is also implicated in the disruption of TGF-␤ signaling. Tumor cells insensitive to TGF-␤ are known to overexpress diverse oncoproteins such as Cdk4, Ski, Evi-1, Ras, Mdm2, E6/E7, E1A, and PLZF-RAR␣ (Pietenpol et al., 1990;Ewen et al., 1993;Datto et al., 1997;Kurokawa et al., 1998;Sun et al., 1998;Hanahan and Weinberg, 2000;Massague et al., 2000;La et al., 2003;Luo, 2004). Overexpression of HPV-16 E7 and E1A, for example, is shown to cause TGF-␤ resistance in skin keratinocytes by preventing TGF-␤-induced down-regulatio...

Recruitment of the oncoprotein v-ErbA to aggresomes

“…These results defined a new mode of action of v-ErbA, and illustrated the importance of cellular compartmentalization in transcriptional regulation (Bonamy and Allison, 2006). Our findings were closely followed by a report defining a cytoplasmic function for v-ErbA, whereby the oncoprotein sequesters Smad4 into the cytoplasm and disrupts the transforming growth factor-β (TGF-β) pathway (Erickson and Liu, 2009). To further explore the cytoplasmic activities of v-ErbA, we sought to ascertain the nature of the cytoplasmic foci formed by a subpopulation of v-ErbA.…”

“…Since v-ErbA was originally isolated from chicken erythroblast cells, we used HD3 cells to investigate the distribution of native v-ErbA. HD3 cells are chicken erythroblasts transformed by AEV that express both native v-ErbA and v-ErbB (Erickson and Liu, 2009). After immunostaining with anti-v-ErbA/c-ErbA antibodies, HD3 cells were visualized by fluorescence microscopy.…”

“…As noted earlier, v-ErbA is expressed by AEV as a Gag-v-ErbA fusion protein (Sap et al, 1986). The Gag sequence is required for the biological activity of the oncoprotein (Erickson and Liu, 2009), and it contains a strong nuclear export sequence (DeLong et al, 2004). In a virus, Gag is involved in forming the viral capsid.…”

“…v-ErbA is a highly mobile protein that trafficks between multiple cellular compartments and carries out distinct activities within each compartment. The first two compartments are well-documented in the literature: a diffuse cytosolic location where v-ErbA is thought to dysregulate the mTOR (mammalian target of rapamycin) and TGF-β pathways (Erickson and Liu, 2009; Gonin-Giraud et al, 2008) and a nuclear location where it interacts with target genes (Bresson et al, 2007; Ventura-Holman et al, 2008). The third compartment, described here, is a perinuclear inclusion characterized as an aggresome.…”

“…In addition to rapid nucleocytoplasmic shuttling, the oncoprotein is trafficked along the aggresomal pathway, forming aggresomes that sequester and mislocalize nuclear receptors. In addition to mislocalizing nuclear receptors (Bonamy et al, 2005), v-ErbA also forms complexes with Smad4 and disrupts Smad4 nuclear localization, which is necessary for a functional TGF-β pathway (Erickson and Liu, 2009). Whether Smad4 is sequestered in aggresomes will be of interest to investigate.…”