
<ArticleTitle>Nuclear entry mechanism of rat <prot>PER2</prot> (<prot>rPER2</prot>): role of <prot>rPER2</prot> in nuclear localization of <prot>CRY protein</prot>.</ArticleTitle>
<AbstractText>Mammalian <prot>PERIOD2 protein</prot> (<prot>PER2</prot>) is the product of a clock gene that controls circadian rhythms, because <prot>PER2</prot>-deficient mice have an arrhythmic phenotype. The nuclear entry regulation of clock gene products is a key step in proper circadian rhythm formation in both Drosophila and mammals, because the periodic transcription of clock genes is controlled by an intracellular, oscillating, negative feedback loop. The present study used deletion mutants of rat <prot>PER2</prot> (<prot>rPER2</prot>) to identify the functional nuclear localization signal (NLS) in <prot>rPER2</prot>. The elimination of putative NLS (residues 778 to 794) from the <prot>rPER2</prot> fragment resulted in the loss of nuclear entry activity. Adding the NLS to the cytosolic protein (<prot>bacterial alkaline phosphatase</prot>) translocates the fusion protein to the nuclei. The data indicate the presence of a functional NLS in <prot>rPER2</prot>. Furthermore, intact <prot>rPER2</prot> was preferentially translocated from the cytoplasm to the nucleus when coexpressed with human <prot>CRY1</prot> (<prot>hCRY1</prot>). However, <prot>rPER2</prot> mutants lacking a carboxyl-terminal domain could not enter the nucleus even in the presence of <prot>hCRY1</prot>. In addition, coexpression of the nuclear localization domain (residues 512 to 794) lacking <prot>rPER2</prot> and <prot>CRY1</prot> changed the subcellular localization of <prot>CRY1</prot> from the nucleus to the cytoplasm. In vitro protein interaction studies demonstrated that the carboxyl-terminal domain of <prot>rPER2</prot> is essential for binding to <prot>CRY1</prot>. The data suggested that both the <prot>rPER2</prot> NLS and carboxyl-terminal <prot>CRY</prot> binding domain are essential for nuclear entry of the <prot>rPER2</prot>-<prot>CRY1</prot> complex.</AbstractText>
