PERIOD (PER) proteins are central components within the mammalian circadian oscillator, and are believed to form a negative feedback complex that inhibits their own transcription at a particular circadian phase. Phosphorylation of PER proteins regulates their stability as well as their subcellular localization. In a systematic screen, we have identified 21 phosphorylated residues of mPER2 including Ser 659, which is mutated in patients suffering from familial advanced sleep phase syndrome (FASPS). When expressing FASPS-mutated mPER2 in oscillating fibroblasts, we can phenocopy the short period and advanced phase of FASPS patients' behavior. We show that phosphorylation at Ser 659 results in nuclear retention and stabilization of mPER2, whereas phosphorylation at other sites leads to mPER2 degradation. To conceptualize our findings, we use mathematical modeling and predict that differential PER phosphorylation events can result in opposite period phenotypes. Indeed, interference with specific aspects of mPER2 phosphorylation leads to either short or long periods in oscillating fibroblasts. This concept explains not only the FASPS phenotype, but also the effect of the tau mutation in hamster as well as the doubletime mutants (dbt S and dbt L ) in Drosophila.[ These cell-autonomous oscillations are thought to be established by feedback loops involving transcription of clock genes and their subsequent autoregulatory transcriptional repression. In mammals, the transcription factor heterodimer CLOCK-BMAL1 activates the expression of Period (Per1, Per2, and Per3) and Cryptochrome (Cry1 and Cry2) genes via E-box enhancer elements in their promoters. PER and CRY proteins are believed to form complexes that translocate in the nucleus to inhibit their own transcription by directly interacting with the CLOCK-BMAL1 complex.Critical to the properties of this oscillator is the delay between the production of PER and CRY proteins and their autorepression. Post-translational events such as complex formation among clock proteins, nuclear import and export, regulated degradation, modulation of transcriptional activity, and chromatin modification have all been implicated in the generation of this delay (for a review, see Harms et al. 2004). In many cases, phosphorylation of clock proteins is the key step that both initiates these events and regulates their correct timing. In cyanobacteria, even the core of the circadian oscillator seems to be based on rhythmic phosphorylation and dephosphorylation of clock proteins rather than on a transcriptional-translational feedback loop (Nakajima et al.

Ubiquitination is a post-translational modification (PTM) that is essential for balancing numerous physiological processes. To enable delineation of protein ubiquitination at a site-specific level, we generated an antibody, denoted UbiSite, recognizing the C-terminal 13 amino acids of ubiquitin, which remain attached to modified peptides after proteolytic digestion with the endoproteinase LysC. Notably, UbiSite is specific to ubiquitin. Furthermore, besides ubiquitination on lysine residues, protein N-terminal ubiquitination is readily detected as well. By combining UbiSite enrichment with sequential LysC and trypsin digestion and high-accuracy MS, we identified over 63,000 unique ubiquitination sites on 9,200 proteins in two human cell lines. In addition to uncovering widespread involvement of this PTM in all cellular aspects, the analyses reveal an inverse association between protein N-terminal ubiquitination and acetylation, as well as a complete lack of correlation between changes in protein abundance and alterations in ubiquitination sites upon proteasome inhibition.

A common feature of animal circadian clocks is the progressive phosphorylation of PERIOD (PER) proteins, which is highly dependent on casein kinase I␦/ (CKI␦/; termed DOUBLETIME [DBT] in Drosophila) and ultimately leads to the rapid degradation of hyperphosphorylated isoforms via a mechanism involving the F-box protein, ␤-TrCP (SLIMB in Drosophila). Here we use the Drosophila melanogaster model system, and show that a key step in controlling the speed of the clock is phosphorylation of an N-terminal Ser (S47) by DBT, which collaborates with other nearby phosphorylated residues to generate a high-affinity atypical SLIMB-binding site on PER. DBT-dependent increases in the phospho-occupancy of S47 are temporally gated, dependent on the centrally located DBT docking site on PER and partially counterbalanced by protein phosphatase activity. We propose that the gradual DBT-mediated phosphorylation of a nonconsensus SLIMB-binding site establishes a temporal threshold for when in a daily cycle the majority of PER proteins are tagged for rapid degradation. Surprisingly, most of the hyperphosphorylation is unrelated to direct effects on PER stability. We also use mass spectrometry to map phosphorylation sites on PER, leading to the identification of a number of "phospho-clusters" that explain several of the classic per mutants.[Keywords: Circadian rhythms; Drosophila; PER; ␤-TrCP/SLIMB; CK1/DBT; F-box protein; phosphorylation] Supplemental material is available at http://www.genesdev.org.

Post-translational processes are essential for the generation and dynamics of mammalian circadian rhythms. In particular, phosphorylation of the key circadian protein PER2 precisely controls the period and phase of circadian oscillations. However, the mechanisms underlying that control are poorly understood. Here, we identified in a high-throughput RNAi-based genetic screen casein kinase 2 (CK2) as a PER2-phosphorylating kinase and novel component of the mammalian circadian clock. When CK2 subunits are silenced by RNAi or when CK2 activity is inhibited pharmacologically, circadian rhythms are disrupted. CK2 binds to PER2 in vivo, phosphorylates PER2 specifically at N-terminal residues in vitro, and supports normal nuclear PER2 accumulation. Mutation of CK2 phosphorylation sites decreases PER2 stability and copies CK2 inhibition regarding oscillation dynamics. We propose a new concept of how PER2 phosphorylation and stabilization can set the clock speed in opposite directions, dependent on the phase of action.[Keywords: Circadian clock; casein kinase 2; period 2; RNAi screen; phosphorylation] Supplemental material is available at http://www.genesdev.org.

While gene expression is a fundamental and tightly controlled cellular process that is regulated at multiple steps, the exact contribution of each step remains unknown in any organism. The absence of transcription initiation regulation for RNA polymerase II in the protozoan parasite Trypanosoma brucei greatly simplifies the task of elucidating the contribution of translation to global gene expression. Therefore, we have sequenced ribosome-protected mRNA fragments in T. brucei, permitting the genome-wide analysis of RNA translation and translational efficiency. We find that the latter varies greatly between life cycle stages of the parasite and ∼100-fold between genes, thus contributing to gene expression to a similar extent as RNA stability. The ability to map ribosome positions at sub-codon resolution revealed extensive translation from upstream open reading frames located within 5′ UTRs and enabled the identification of hundreds of previously un-annotated putative coding sequences (CDSs). Evaluation of existing proteomics and genome-wide RNAi data confirmed the translation of previously un-annotated CDSs and suggested an important role for >200 of those CDSs in parasite survival, especially in the form that is infective to mammals. Overall our data show that translational control plays a prevalent and important role in different parasite life cycle stages of T. brucei.