Nuclear Export of Mammalian PERIOD Proteins

Abstract

The timing of mammalian circadian rhythm is determined by interlocking negative and positive transcriptional feedback loops that govern the cyclic expression of both clock regulators and output genes. In mammals, nuclear localization of the circadian regulators PER1-3 is controlled by multiple mechanisms, including multimerization with PER and CRY proteins. In addition, nuclear entry of mammalian PER1 (mPER1) can be regulated by a phosphorylation-dependent masking of its nuclear localization signal. Here we present evidence suggesting that nuclear localization of PER proteins is a dynamic process determined by both nuclear import and previously unrecognized nuclear export pathways. Examination of the subcellular localization of a series of truncated mPER1 proteins demonstrated that cytoplasmic localization is mediated by an 11-amino acid region with homology to leucine-rich nuclear export signals (NESs). Similar sequences were identified in mPER2 and mPER3 as well as in several insect PER proteins. The putative NESs from mPER1 and mPER2 were able to direct cytoplasmic accumulation when fused to a heterologous protein. Mutations in conserved NES residues and the nuclear export inhibitor leptomycin B each blocked the function of the NES. Full-length mPER1 was also exported from microinjected Xenopus laevis oocyte nuclei in an NES-dependent manner. The presence of a functional NES in mPER1 and mPER2 as well as related sequences in a variety of other PER proteins suggests that nuclear export may be a conserved and important feature of circadian regulators.Circadian rhythm is a ubiquitous process that orders physiologic events in a 24-h day/night cycle (recently reviewed in Refs. 1-3). The timing of circadian clocks is established in a cell autonomous manner by interacting negative and positive transcription/translation-based negative feedback loops. The negative feedback loop begins by activating transcription of clock genes such as Period (Per) and Cryptochrome (Cry) family members. The products of these clock genes then negatively regulate their own expression, thus setting up the rhythmic oscillations of gene expression that drive the circadian clock. To produce stable oscillations of gene expression with a period of 24 h, there must also be a delay between the production and the action of the inhibitory clock gene products. Several mechanisms may contribute to delayed repression of the circadian promoter, including proteolysis of the repressors, alterations in repressor activity by post-translational modification (e.g. phosphorylation), and regulation of nuclear accumulation by alterations in rates of nuclear entry and/or nuclear egress.

Unwinding the differences of the mammalian PERIOD clock proteins from crystal structure to cellular function

“…S1), which corresponds well to the consensus sequence L- (28). Whereas Leu489 mPER1 ∕Leu460 mPER2 ∕Leu399 mPER3 , Ile493 mPER1 ∕Ile464 mPER2 ∕Ile403 mPER3 , and Leu496 mPER1 ∕ Leu467 mPER2 ∕Leu406 mPER3 pack against the αC' helix of PAS-B, the C-terminal residues Leu498 mPER1 ∕Met469 mPER2 ∕ Leu408 mPER3 point to the molecule surface (Fig.…”

The intricate dance of post-translational modifications in the rhythm of life

“…A mechanism for PER1 nuclear export has been proposed, in which the mouse PER1 nuclear localization signal (NLS, amino acids 824-851) is masked by CKIε-mediated phosphorylation of the serine cluster from amino acids 902 to 916. In addition, PER1-3 proteins contain nuclear export signals (NESs, amino acids 484-495 for mPER1) 42 . However, other studies have reported that expression of CKIε or CKIδ promotes nuclear accumulation of mouse PER1 and PER3 in COS-7 cells 16,43 .…”

Casein Kinase I in the Mammalian Circadian Clock

“…Examination of protein localization and nuclear export is described elsewhere (Vielhaber et al 2000;Vielhaber et al 2001). Several approaches to examining the effect of phosphorylation on protein stability are available.…”