Cell Cycle-Regulated Inactivation of Endothelial NO Synthase through NOSIP-Dependent Targeting to the Cytoskeleton

Abstract

Nitric oxide (NO) plays a key role in vascular function, cell proliferation, and apoptosis. Proper subcellular localization of endothelial NO synthase (eNOS) is crucial for its activity; however, the role of eNOS trafficking for NO biosynthesis remains to be defined. Overexpression of NOS-interacting protein (NOSIP) induces translocation of eNOS from the plasma membrane to intracellular compartments, thereby impairing NO production. Here we report that endogenous NOSIP reduces the enzymatic capacity of eNOS, specifically in the G₂ phase of the cell cycle by targeting eNOS to the actin cytoskeleton. This regulation is critically dependent on the nucleocytoplasmic shuttling of NOSIP and its cytoplasmic accumulation in the G₂ phase. The predominant nuclear localization of NOSIP depends on a bipartite nuclear localization sequence (NLS) mediating interaction with importin α. Mutational destruction of the NLS abolishes nuclear import and interaction with importin α. Nuclear export is insensitive to leptomycin B and hence different from the CRM1-dependent default mechanism. Inhibition of NOSIP expression by RNA interference completely abolishes G₂-specific cytoskeletal association and inhibition of eNOS. These findings describe a novel cell cycle-dependent modulation of endogenous NO levels that are critical to the cell cycle-related actions of NO such as apoptosis or cell proliferation.

ACKNOWLEDGMENTS

We thank Günter Blobel and Tarik Soliman (Rockefeller University, New York, NY) for the generous gift of karyopherin cDNAs, Hans Clevers and Marc van de Wetering (Hubrecht Laboratory, Utrecht, The Netherlands) for the pTER⁺ vector, Ludger Hengst (Max Planck Institute, Munich, Germany) for the heterokaryon protocol, Sabine Graf (University of Frankfurt) for excellent technical assistance, and Daniela Höller and the Institute for Biochemistry II members for constructive criticism and helpful discussions.

This work was supported by a grant of the Deutsche Forschungsgemeinschaft through the Sonderforschungsbereich 553 (B3).