HER4 Cyt1 and Cyt2 Isoforms Regulate Transcription through Differential Interaction with a Transcriptional Regulator, Yap

Our laboratory has previously shown that two isoforms of Epidermal Growth Factor Receptor family member, HER4 Cyt1 and Cyt2 exhibit opposing effects on mammary epithelial cells in vitro and in vivo. In our hands, Cyt1 attenuated growth and promoted differentiation, while Cyt2 promoted cell proliferation of mammary epithelial cells. The two isoforms differ by presence of additional 16 amino acids in Cyt1, which introduces a phosphoinositide- 3 kinase- and third WW- domain binding motives however, do not explain the different biological effects of the isoforms. We have focused our studies on a transcriptional regulator and an oncogene, Yap and have shown that Cyt1 preferentially binds Yap as compared to Cyt2. We confirmed these results in COS7 and 293T cells and using mutational in vitro studies and mass spectrometry identified tyrosines 341 and 394 of Yap as phosphorylation targets by HER4. However, we were unable to replicate these results in mammary epithelial cells, HC11 and MCF7. We also found that HER4 cytoplasmic domain s80 does not modulate localization of Yap in COS7 cells, although Yap promotes nuclear localization of s80 and these effects are not dependent on isoform nor kinase activity. HER4 expression also did not affect Yap cellular localization in vivo in a transgenic mouse model and human breast carcinoma. Additionally, we evaluated whether HER4 interacts with TEAD, transcriptional factor regulated by Yap, and have found that HER4 forms complex with TEAD however, this interaction is likely mediated by Yap and we could not find any evidence for HER4- mediated phosphorylation of TEAD. We were also unable to find any transcriptional consequences of HER4 interaction with or tyrosine phosphorylation of Yap. Taken together, our data suggest that HER4Yap interaction observed in COS7 and 293T cells is nonspecific and due to overexpression of the proteins and either does not occur in mammary epithelial cells or is highly regulated.