Dimerization and nuclear entry of mPER proteins in mammalian cells

Nuclear entry of circadian oscillatory gene products is a key step for the generation of a 24-hr cycle of the biological clock. We have examined nucle ar import of clock proteins of the mammalian period gene family and the eff ect of serum shock, which induces a synchronous clock in cultured cells. Pr eviously, mCRY1 and mCRY2 have been found to complex with PER proteins lead ing to nuclear import. Here we report that nuclear translocation of mPER1 a nd mPER2 (1) involves physical interactions with mPER3, (2) is accelerated by serum treatment, and (3) still occurs in mCry1/mCry2 double-deficient ce lls lacking a functional biological clock. Moreover, nuclear localization o f endogenous mPER1 was observed in cultured mCry1/mCry2 double-deficient ce lls as well as in the liver and the suprachiasmatic nuclei (SCN) of mCry1/m Cry2 double-mutant mice. This indicates that nuclear translocation of at le ast mPER1 also can occur under physiological conditions (i.e., in the intac t mouse) in the absence of any CRY protein. The mPER3 amino acid sequence p redicts the presence of a cytoplasmic localization domain (CLD) and a nucle ar localization signal (NLS). Deletion analysis suggests that the interplay of the CLD and NLS proposed to regulate nuclear entry of PER in Drosophila is conserved in mammals, but with the novel twist that mPER3 can act as th e dimerizing partner.