Mechanistic Analysis of a DNA Damage-Induced, PTEN-Dependent Size Checkpoint in Human Cells
AbstractMechanistic Analysis of a DNA Damage-Induced, PTEN-Dependent Size Checkpoint in Human Cells ▿ Jung-Sik Kim 1 , Xuehua Xu 3 , Huifang Li 1 , David Solomon 1 , 2 , William S. Lane 4 , Tian Jin 3 and Todd Waldman 1 , * 1 Department of Oncology 2 Tumor Biology Training Program, Lombardi Comprehensive Cancer Center, Georgetown University School of Medicine, Washington, DC 20057 3 Chemotaxis Signal Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland 20852 4 Mass Spectrometry and Proteomics Resource Laboratory, FAS Center for Systems Biology, Harvard University, Cambridge, Massachusetts 02138 ABSTRACT Following DNA damage, human cells undergo arrests in the G 1 and G 2 phases of the cell cycle and a simultaneous arrest in cell size. We previously demonstrated that the cell size arrest can be uncoupled from the cell cycle arrest by mutational inactivation of the PTEN tumor suppressor gene. Here we show that the cell size checkpoint is inducible by DNA-damaging chemotherapeutic agents as well as by ionizing radiation and is effectively regulated by PTEN but not by its oncogenic counterpart, PIK3CA. Mutational analysis of PTEN and pharmacological inhibition of Akt revealed that modulation of Akt phosphorylation is unnecessary for cell size checkpoint control. To discover putative PTEN regulators and/or effectors involved in size checkpoint control, we employed a novel endogenous epitope tagging (EET) approach, which revealed that endogenous PTEN interacts at the membrane with an actin-remodeling complex that includes actin, gelsolin, and EPLIN. Pharmacological inhibition of actin remodeling in PTEN +/+ cells recapitulated the lack of size checkpoint control seen in PTEN −/− cells. Taken together, these results provide further support for the existence of a DNA damage-inducible size checkpoint that is regulated by a major tumor suppressor, and they provide a novel Akt-independent mechanism by which PTEN controls cell size.