Abstract
Overexpression of Neu (ErbB-2/HER2) is found in approximately 20% of breast tumors. Activation of Neu by a point mutation (NeuT) causes constitutive tyrosine kinase activity of this transmembrane receptor and transforming activity in fibroblasts. To identify downstream targets of Neu, we have analyzed the ability of Neu to activate gene expression. Expression of NeuT, but not normal Neu, caused transcriptional activation of Ets, AP-1, or NF-kappaB-dependent reporter genes. Dominant inhibitory Ras or Raf mutants blocked the Neu-mediated transcriptional activation, confirming that Ras signaling pathways were required for this activation. Analysis with Ets2 mutants indicated that activation of Ets2 transcriptional activity mediated by NeuT or oncogenic Ras required phosphorylation of the same Ets2 residue, threonine 72. Cotransfection of dominant inhibitory Ets2 mutants specifically blocked NeuT-mediated activation of Ets-dependent reporter genes. Furthermore, in focus formation assays using NIH 3T3 cells, the transforming activity of NeuT was inhibited 5-fold when NeuT was cotransfected with a dominant negative Ets2 mutant. However, parallel colony formation assays showed that the Ets2 dominant negative mutant did not inhibit the growth of normal cells. Together, these data show that NeuT activates a variety of transcription factor families via the Ras signaling pathway and that Ets activation is required for NeuT-mediated cellular transformation. Thus, downstream targets of Neu, including Ets transcription factors, may be useful points for therapeutic intervention in Neu/ErbB-2-associated cancers.
Highlights
The c-neu oncogene product is a 185-kDa transmembrane receptor tyrosine kinase that belongs to the epidermal growth factor family [1,2,3]
Transforming Neu Activates Transcription of Ets, AP-1, and NF-B-dependent Reporter Genes—To test the effects of normal and activated Neu/ErbB-2 on transcription factor activation, transient cotransfection experiments were performed in NIH
The Neu by a point mutation (NeuT)-mediated superactivation of Ets2 activity resulted from altered transactivation activity and not increases in Ets2 protein levels. Because this pattern of NeuT-mediated transcriptional activation in combination with Ets2 mutants was the same as that we previously found mediated by oncogenic Ras [40], these results strongly suggest that phosphorylation of Ets2 Thr72 is a common molecular target of both NeuT and oncogenic Ras signaling
Summary
The c-neu oncogene product ( called ErbB-2 or HER2) is a 185-kDa transmembrane receptor tyrosine kinase that belongs to the epidermal growth factor family [1,2,3]. Similar to the results we previously found for oncogenic Ras [29], reporter genes containing the minimal fos promoter with only a single added binding site for Ets, AP-1, or NF-B family members were not significantly transactivated by expression of NeuT (data not shown), indicating that the presence of a single binding site for any of these transcription factors was not sufficient to create an Neu responsive promoter element.
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