Identification of Mechanism That Couples Multisite Phosphorylation of Yes-associated Protein (YAP) with Transcriptional Coactivation and Regulation of Apoptosis

Abstract

Background:The transcriptional coactivator YAP has a dual role, stimulating cell proliferation and promoting apoptosis. Results: YAP is phosphorylated and activated in response to genotoxic stress. Conclusion: The phosphorylation of YAP at multiple sites activates transcription and protects against apoptosis. Significance: Hyperphosphorylation provides a mechanism by which YAP regulates transcription and apoptosis.The transcriptional coactivator Yes-associated protein (YAP) has been implicated in tumorigenesis by regulating cell proliferation and apoptosis. YAP interacts with the transcription factor TEAD and is essential in mediating TEAD-dependent gene expression. Here we show that YAP is hyperphosphorylated and activated in response to genotoxic stress such as UV irradiation and cisplatin treatment. Using high resolution mobility shift assay for phosphorylated proteins, we identified multiple sites of phosphorylation induced by UV irradiation. Pretreatment with p38 and JNK inhibitors completely suppressed the mobility retardation of phosphorylated YAP in UV-irradiated cells. Coimmunoprecipitation experiments showed that the physical interaction of YAP with TEAD was markedly enhanced by UV irradiation or CDDP treatment but suppressed by pretreatment with p38 and JNK inhibitors. Similarly, pretreatment with p38 and JNK inhibitors suppressed the expression of YAP/TEAD target genes, which were elevated on exposure to genotoxic stress. Using phosphomimetic and phosphorylation-deficient YAP mutants, we showed that the coactivator activity of YAP correlated with its state of phosphorylation and sensitivity to cisplatin-induced apoptosis. Our results demonstrate that multisite phosphorylation of YAP induces YAP/TEAD-dependent gene expression and provides a mechanism by which YAP regulates apoptosis differently depending on cellular context.The opposing actions of proliferation and apoptosis control cell numbers in particular tissues and organs and the coordination of cell fate is fundamental to animal development (1). The Hippo signaling pathway plays a key role in controlling both cell proliferation and apoptosis (2). Components of the Hippo pathway such as Merlin, Lats, and MST, are known to contribute to tumorigenesis (3-5). The MST-SAV complex phosphorylates and activates Lats, an NDR family kinase. Lats inhibits YAP, 2 a transcriptional coactivator, via phosphorylation of HXRXX(S/T) motif. This mechanism of regulation is involved in cell contact inhibition and tissue growth control and epithelial-mesenchymal transition. YAP binds to and activates TEAD, a transcription factor essential to the biological functions of . YAP is the candidate oncogene in the human chromosome 11q22 amplicon that is evident in several cancers (9, 10). Elevated YAP levels and increased nuclear localization have been reported in multiple cancerous tissues (11). Moreover, YAP overexpression in MCF10A induces epithelial-mesenchymal transition, a hallmark of tumorigenic transformation (10). YAP overexpression stimulates proliferation and in...

Osmotic stress‐induced phosphorylation by NLK at Ser128 activates YAP

“…It is possible that YAP Ser128 phosphorylation may regulate YAP localization under such conditions. It is worth noting that YAP Ser128 phosphorylation has been observed in many phosphoproteomic studies [25,37,38]. We speculate that YAP Ser128 may be a common regulatory phosphorylation site for proline-directed kinases, such as CDK family and MAP kinase family.…”

“…Notably, the NLK consensus site Ser128 in YAP is adjacent to the Ser127 site and is within the 14-3-3 binding pocket. Previous phosphoproteomic studies also showed that YAP Ser128 is a phosphorylation site [25,37,38]. Thus, we hypothesized that NLK phosphorylates YAP Ser128 to disrupt YAP-14-3-3 binding, leading to the uncoupling event between S127 phosphorylation and cytoplasmic localization.…”

JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships

“…Specifically, the coactivator protein YAP1 is also subject to multisite phosphorylation that can be considered a complex phosphoswitch. At least four sites (of which two are highly conserved) were identified as JNK targets in response to genotoxic stress (351), and all play a role in transactivation by YAP1, albeit by different mechanisms. Ser128 (underlined in the following sequence; it is also conserved in TAZ) lies near the TEAD-binding region of YAP1, directly inside one of the two LATS-phosphorylated 14-3-3 binding sites (HVRAHSSP).…”

Yap1 is dispensable for self‐renewal but required for proper differentiation of mouse embryonic stem ( ES ) cells

“…The Hippo signaling pathway, modulated by cell density and cellcell contact, is implicated in diverse cellular processes including cell proliferation [1][2][3][4][5], apoptosis [5,6], and organ size control [7,8]. Yap1 is a transcriptional co-activator of the Hippo pathway and is known to play a crucial role in the segregation of inner cell mass (ICM) and trophectoderm (TE) during early embryogenesis [9][10][11][12][13].…”