Abstract 562: The Hippo pathway component LATS1 phosphorylates MYPT1 to counteract PLK1 and regulate G2 DNA damage checkpoint.

Abstract

Abstract Background: LATS1 participates in the Hippo pathway, which is best characterized in Drosophila melanogaster. Hippo pathway regulates cell proliferation and cell death to ensure appropriate organ size is maintained. LATS1 knockout mice develop ovarian tumor and soft tissue sarcoma, indicating that LATS1 functions as a tumor suppressor. Indeed, LATS1 expression is reduced in breast cancers due to hypermethylation of its promoter region, which is well correlated with poor prognosis of the patients. However in budding yeast, Dbf2 kinase which is the yeast counterpart of LATS1 phosphorylates and activates Cdc14 phosphatase to faithfully execute mitotic exit. This signaling pathway is known as mitotic exit network (MEN). Since there has been reported no phosphatase substrate of LATS1, we pursued the novel target of LATS1 to elucidate the significance of MEN in mammals and clarified a novel role of LATS1. Methods: We screened LATS1 substrates using phosphopeptide enrichment techniques coupled with high-accuracy mass spectrometry. We verified that the identified peptides are substrates of LATS1 both in vitro and in vivo. After determining the phosphorylation sites of the novel substrate, we analyzed functional significance of this phosphorylation-mediated signaling pathway. Results: Phosphoproteomic screening identified myosin phosphatase-targeting subunit1 (MYPT1) as a new substrate for LATS1. LATS1 directly and preferentially phosphorylated serine 445 (S445) of MYPT1. An MYPT1 mutant (S445A) failed to dephosphorylate threonine 210 of polo-like kinase1 (PLK1), thereby activating PLK1. This suggests that LATS1 promotes MYPT1 to antagonize PLK1 activity. Consistent with this, LATS1-depleted HeLa cells or fibroblasts from LATS1 knock-out mice showed increased PLK1 activity. We also found that DNA damage-induced LATS1 activation caused PLK1 suppression via the phosphorylation of MYPT1 S445. Furthermore, LATS1 knock-down caused impairment of G2 checkpoint arrest after DNA damage. Conclusions: These results indicate that LATS1 phosphorylates a phosphatase as does the yeast Dbf2 and demonstrate a novel link between two mitotic kinases, LATS1 and PLK1. PLK1 is overexpressed in many cancers and its expression levels often correlate with poor prognosis of the patients. The oncogenic properties of PLK1, such as inducing genomic instability by incomplete G2 checkpoint, would be enhanced under the condition of reduced LATS1 expression. Thus, our data raise the intriguing possibility that cancer patients with reduced LATS1 expression in their tumors are potential candidates for treatment with PLK inhibitors. Our data also suggests that mammalian MEN signaling pathway plays an important role for the G2 DNA damage checkpoint response other than mitotic exit. Citation Format: Tatsuyuki Chiyoda, Shinji Kuninaka, Kenta Masuda, Takatsune Shimizu, Yoshimi Arima, Daisuke Aoki, Hideyuki Saya. The Hippo pathway component LATS1 phosphorylates MYPT1 to counteract PLK1 and regulate G2 DNA damage checkpoint. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 562. doi:10.1158/1538-7445.AM2013-562