Nuclear entry mechanism of rat PER2 (rPER2): Role of rPER2 in nuclear localization of CRY protein

Mammalian PERIOD2 protein (PER2) is the product of a clock gene that contro ls circadian rhythms, because PER2-deficient mice have an arrhythmic phenot ype. The nuclear entry regulation of clock gene products is a key step in p roper circadian rhythm formation in both Drosophila and mammals, because th e periodic transcription of clock genes is controlled by an intracellular, oscillating, negative feedback loop. The present study used deletion mutant s of rat PER2 (rPER2) to identify the functional nuclear localization signa l (NLS) in rPER2. The elimination of putative NLS (residues 778 to 794) fro m the rPER2 fragment resulted in the loss of nuclear entry activity. Adding the NLS to the cytosolic protein (bacterial alkaline phosphatase) transloc ates the fusion protein to the nuclei. The data indicate the presence of a functional NLS in rPER2. Furthermore, intact rPER2 was preferentially trans located from the cytoplasm to the nucleus when coexpressed with human CRY1 (hCRY1). However, rPER2 mutants lacking a carboxyl-terminal domain could no t enter the nucleus even in the presence of hCRY1. In addition, coexpressio n of the nuclear localization domain (residues 512 to 794) lacking rPER2 an d CRY1 changed the subcellular localization of CRY1 from the nucleus to the cytoplasm. In vitro protein interaction studies demonstrated that the carb oxyl-terminal domain of rPER2 is essential for binding to CRY1. The data su ggested that both the rPER2 NLS and carboxyl-terminal CRY binding domain ar e essential for nuclear entry of the rPER2-CRY1 complex.