Abstract 1335: Lack of casein kinase 1 delta induces DNA damage, inhibition of mTORC1 signaling and nucleophagy

Abstract

Abstract Casein kinase 1 delta (CK1δ) is a conserved serine/threonine protein kinase that regulates diverse cellular processes including vesicle trafficking, cell cycle progression, chromosomal segregation, DNA repair, circadian rhythm, neurite outgrowth and ciliogenesis. CK1δ has been reported to phosphorylate Mdm2, the E3 ubiquitin ligase for p53, resulting in Mdm2 ubiquitination/degradation and concomitant accumulation of p53, the latter functioning as a mediator of cell cycle checkpoints and DNA repair. Mice that lack Csnk1d exhibit a perinatal lethal phenotype and typically weigh 30-50% less than their wild type littermates. The exact causes of death and small size are unknown. We hypothesized that the absence of CK1δ initiates cellular stresses that adversely affect cell survival. To examine this idea, we collected mouse embryonic fibroblasts (MEFs) from mice homozygous for a Csnk1d floxed allele, and generated MEF Csnk1d null cells by infection with adenovirus expressing Cre. Endogenous p53 protein level was significantly lower in MEF Csnk1d null cells relative to MEF Ctl cells infected with adenovirus-GFP. Substantial DNA damage also was detected in MEF Csnk1d null cells, as measured by γ-H2AX staining. Flow cytometric analysis revealed that early passage (P3) MEF Csnk1d null cells exhibited a larger sub-G0 fraction (64%) than MEF Ctl cells (34%). The sub-G0 fraction decreased sharply with increasing passage number, becoming negligible by P10. In addition to the decline in sub-G0 fraction, we observed a transient population of MEF Csnk1d null cells with multiple DAPI-stained micronuclei containing both γ-H2AX and membranes positive for the autophagosomal marker LC3 and lysosomal marker LAMP1. These features were preceded by and coincided with mTORC1 pathway inactivation, as indicated by low concentrations of phospho-Akt, phospho-mTOR and phospho-S6K, consistent with the view that mTORC1 inhibition facilitates the induction of autophagy. Subsequently, these differences between MEF Csnk1d null and MEF Ctl cells subsided. Our findings demonstrate that loss of CK1δ expression in proliferating MEF cells results in DNA damage and initially a large sub-G0 peak, followed by a wave of nucleophagy and ultimately cell survival in culture. Ongoing investigation will explore whether similar processes occur in embryonic tissues from Csnk1d null mice and contribute to their pathologic phenotype. Citation Format: Yoshimi Endo Greer, Bo Gao, Yingzi Yang, Jeffrey S. Rubin. Lack of casein kinase 1 delta induces DNA damage, inhibition of mTORC1 signaling and nucleophagy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1335. doi:10.1158/1538-7445.AM2014-1335