Abstract B77: Chk1-Lats2-14-3-3 is a novel signaling pathway that regulates the P-body formation in response to DNA damage

Abstract

Abstract Purpose: Lats2 (Large tumor suppressor 2), a member of the conserved Lats kinase family, is a human tumor suppressor gene. We previously reported that Lats2 knockout in mice displays embryonic lethality and the MEFs from Lats2−/− mice causes mitotic defects including centrosome fragmentation and cytokinesis defects associated with nuclear enlargement and multinucleation. DNA damage response (DDR) plays an essential role in maintaining the integrity of the genome and is crucial for cancer avoidance. The purpose of this study is to investigate the role of Lats2 in DDR, which prevents chromosome instability and malignant progression of human cancer by keeping the chromosome stability. MicroRNAs (miRNAs), endogenous RNAs of approximately 22 nucleotides, play an important role in the regulation of gene expression: miRNAs are incorporated into miRNA-induced silencing complexes (miRISCs), which leads to either cleavage or translational repression of targeted mRNAs. mRNA processing bodies (P-bodies) are sites where untranslated mRNAs accumulate, and mRNA degradation, translational repression, and mRNA surveillance take place. Methods: We raised two phospho-specific antibodies against the phosphorylation sites of Lats2 by Chk1 and 14-3-3 by Lats2 to analyze the phosphorylation status and localization by Western blot and immunocytochemical analyses. Moreover, we established myc-tagged U2OS/siLats2 strains that stably expressed Lats2 mutated the phosphorylation site to alanine or aspartic acid, and examined the effects of those on DNA damage signaling pathway. Results: We found that Lats2 is phophorylated by Chk1 and/or Chk2 in response to DNA damage, which induced the phosphorylation of 14-3-3 by Lats2. Immunocytochemical analysis using a phospho-specific antibody against 14-3-3 revealed that the phosphorylated 14-3-3 localized to the P-body, and these signals are enhanced in response to DNA damage. We propose here that 14-3-3 is a novel regulatory protein for P-body formation that promotes the miRNA-mediated gene silencing. Indeed, depletion of 14-3-3 or Lats2 reduced the localization to the P-body of both 14-3-3 and a putative P-body scaffold protein, GW182. Eventually, this pathway regulates the translesion synthesis (TLS), a process that synthesizes DNA across DNA lesion, through p21 translational repression by miRNA. Conclusion: We conclude that Chk1-Lats2-14-3-3 axis is a novel DNA damage signaling pathway that regulates the P-body assembly, thereby causing the translational repression of target genes via miRNA. These results provide the first evidence for a mechanism in which the DNA damage response pathway regulates miRNA-mediated translational repression through P-body assembly. Citation Information: Cancer Res 2009;69(23 Suppl):B77.