The CREB-binding protein affects the circadian regulation of behaviour.

Rhythmic changes in light and temperature conditions form the primary environmental cues that synchronize the molecular circadian clock of most species with the external cycles of day and night. Previous studies established a role for the CREB-binding protein (CBP) in molecular clock function by coactivation of circadian transcription. Here, we report that moderately increased levels of CBP strongly dampen circadian behavioural rhythms without affecting molecular oscillations of circadian transcription. Interestingly, light-dark cycles as well as high temperature facilitated a circadian control of behavioural activity. Based on these observations we propose that in addition to its coactivator function for circadian transcription, CBP is involved in the regulation of circadian behaviour down-stream of the circadian clock.

The E3 ubiquitin ligase CTRIP controls CLOCK levels and PERIOD oscillations in Drosophila.

In the Drosophila circadian clock, the CLOCK/CYCLE complex activates the period and timeless genes that negatively feedback on CLOCK/CYCLE activity. The 24-h pace of this cycle depends on the stability of the clock proteins. RING-domain E3 ubiquitin ligases have been shown to destabilize PERIOD or TIMELESS. Here we identify a clock function for the circadian trip (ctrip) gene, which encodes a HECT-domain E3 ubiquitin ligase. ctrip expression in the brain is mostly restricted to clock neurons and its downregulation leads to long-period activity rhythms in constant darkness. This altered behaviour is associated with high CLOCK levels and persistence of phosphorylated PERIOD during the subjective day. The control of CLOCK protein levels does not require PERIOD. Thus, CTRIP seems to regulate the pace of the oscillator by controlling the stability of both the activator and the repressor of the feedback loop.

Post-translational timing mechanisms of the Drosophila circadian clock.

Circadian clocks allow a temporal coordination and segregation of physiological, metabolic, and behavioural processes as well as their synchronization with the environmental cycles of day and night. Circadian regulation thereby provides a vital advantage, improving an organisms' adaptation to its environment. The molecular clock can be synchronized with environmental cycles of day and night, but is able to maintain a self-sustained molecular oscillation also in the absence of environmental stimuli. Interlocked transcriptional-translational feedback loops were shown to form the basis of circadian clock function in all phyla from bacteria, fungi, plants, insects to humans. More recently post-translational regulation was identified to be equally important, if not sufficient for molecular clock function and accurate timing of circadian transcription. Here we review recent insights into post-translational timing mechanisms that control the circadian clock, with a particular focus on Drosophila. Analogous to transcriptional feedback regulation, circadian clock function in Drosophila appears to rely on inter-connected post-translational timers. Post-translational regulation of clock proteins illustrates mechanisms that allow a precise temporal control of transcription factors in general and of circadian transcription in particular.

Sequential and compartment-specific phosphorylation controls the life cycle of the circadian CLOCK protein.

The circadian clock facilitates a temporal coordination of most homeostatic activities and their synchronization with the environmental cycles of day and night. The core oscillating activity of the circadian clock is formed by a heterodimer of the transcription factors CLOCK (CLK) and CYCLE (CYC). Post-translational regulation of CLK/CYC has previously been shown to be crucial for clock function and accurate timing of circadian transcription. Here we report that a sequential and compartment-specific phosphorylation of the Drosophila CLK protein assigns specific localization and activity patterns. Total and nuclear amounts of CLK protein were found to oscillate over the course of a day in circadian neurons. Detailed analysis of the cellular distribution and phosphorylation of CLK revealed that newly synthesized CLK is hypophosphorylated in the cytoplasm prior to nuclear import. In the nucleus, CLK is converted into an intermediate phosphorylation state that correlates with trans-activation of circadian transcription. Hyperphosphorylation and degradation are promoted by nuclear export of the CLK protein. Surprisingly, CLK localized to discrete nuclear foci in cell culture as well as in circadian neurons of the larval brain. These subnuclear sites likely contain a storage form of the transcription factor, while homogeneously distributed nuclear CLK appears to be the transcriptionally active form. These results show that sequential post-translational modifications and subcellular distribution regulate the activity of the CLK protein, indicating a core post-translational timing mechanism of the circadian clock.

HSP90, a capacitor of behavioral variation.

Many aspects of behavior such as aggression, courtship, sexual orientation, and the sleep-wake cycle are determined by specific genes. Although point mutations in these genes predictably change characteristics of behavior, substantial variation can be observed among a population as well as during the lifetime of individuals. The origin of variation in behavior, however, is largely unknown. Here the authors investigated the role of HSP90 for the circadian control of behavior in Drosophila. They found that a partial loss of HSP90 function, either by mutagenesis or by pharmacological inhibition, did not affect the circadian clock itself, but the translation of molecular oscillations into behavioral rhythms. In HSP90-deficient flies behavioral activity was no longer stringently coupled to molecular oscillations giving rise to a large variation in individual behavioral activity patterns. The results show that HSP90 is a potent capacitor of behavioral variation, analogous to its role in morphology. Decreased HSP90 activity not only increases behavioral variability among a population, but interestingly also during the lifetime of individuals.

Cytoplasmic interaction with CYCLE promotes the post-translational processing of the circadian CLOCK protein.

Post-translational regulation of the transcription factor CLOCK (CLK) is crucial for circadian clock function. The contribution of the hetero-dimerization partner CYCLE (CYC) to the post-translational regulation of CLK is largely unknown. Here we report that Drosophila CLK and CYC proteins not only interact in the nucleus, where they activate circadian transcription, but also in the cytoplasm of Drosophila S2R+ cells. Cytoplasmic CLK accumulates in a hypo-phosphorylated state. Impairment of CYC-binding caused a further reduction in CLK phosphorylation, while over-expression of CYC enhanced the phosphorylation of cytoplasmic CLK towards a hypo-phosphorylated state. CYC also promotes nuclear import of CLK, which is required for hyper-phosphorylation of the CLK protein. Our results indicate a role of CYC in the post-translational regulation of the CLK protein.

Circadian transcription depends on limiting amounts of the transcription co-activator nejire/CBP.

The circadian clock orchestrates physiological and behavioral activities, including metabolism, neuronal activity, and cell proliferation in synchrony with the environmental cycle of day and night. Here we show that the Drosophila ortholog of the CBP/p300 family of transcription co-activators, nejire (nej), is an intrinsic component of the circadian clock that performs regulatory functions for circadian controlled transcription. Screening of overexpression mutants revealed that gain of nej function was associated with a loss of behavioral and molecular rhythms. Overexpression of NEJ suppresses the long period phenotype of a mutation in the clock gene period (per). NEJ physically interacts through two binding sites with CLOCK and the CLOCK. CYCLE (CLK.CYC) complex. Induction of CLK.CYC-dependent transcripts upon induction of nej expression from a heat-shock promoter showed that NEJ is limiting. Reduced CLK.CYC-mediated transcription in a nej hypomorphic mutant indicates an essential function of NEJ/CBP for CLK.CYC activity and a regulation of circadian transcription by availability of the co-activator. Competition for recruitment of NEJ/CBP provides a potential mechanism for cross-talk between circadian transcription and other CBP-dependent physiological processes.

Second messenger and Ras/MAPK signalling pathways regulate CLOCK/CYCLE-dependent transcription.

The heterodimeric complex of the transcription factors CLOCK (CLK) and CYCLE (CYC) constitutes the positive element of the circadian clock in Drosophila and mammals. Phosphorylation of clock proteins represents an essential mechanism for promotion and control of the molecular oscillator. However, the kinases and signalling pathways that regulate CLK/CYC function remain largely elusive. In the present study we performed a chemical screen of kinase inhibitors in a cell culture reporter assay to identify functional regulators of CLK/CYC-dependent gene expression. These studies and analysis of constitutively active forms of kinases revealed that cyclic nucleotide/protein kinase A (PKA), calcium/calmodulin-dependent kinase (CaMK) II and Ras/mitogen-activated protein kinase (MAPK) regulate CLK/CYC activity. In vitro phosphorylation analysis showed a direct phosphorylation of CLK by CaMK II and p42 MAPK [extracellular signal-regulated kinase (ERK) 2], suggesting that these kinases regulate CLK/CYC-dependent transcription by direct phosphorylation of CLK.

Role of the T cell receptor alpha chain in the development and phenotype of naturally arising CD4+CD25+ T cells.

The T cell receptor alpha chain repertoire and the possible influence of the alpha chain on the development and phenotype of naturally arising mouse CD4+CD25+ T cells have not been extensively analysed. We used all available Valpha-specific monoclonal antibodies and a sensitive multiplex genomic DNA PCR assay to study the Valpha repertoire of CD4+CD25+ T cells in normal mice. To address whether CD4+CD25+ T cells express two TCR alpha chains, we have carried out four-colour flow cytometry using combinations of the available anti-Valpha reagents in mice where one allele of the TCRA locus had been inactivated. Results indicate that the Valpha repertoire of CD4+CD25+ T cells is as diverse as their CD25- partners. In addition, CD4+CD25+ T cells develop normally in Tcralpha+/- mice and we show for the first time that despite expressing only one TCRalpha chain, they retain their characteristic CD4(low), CD3(low), TCRbeta(low), CD5(high), CD45RB(low) and cytoplasmic CD152(high) phenotype.

Syndecan captures, protects, and transmits HIV to T lymphocytes.

This study demonstrates that syndecan functions as an in trans HIV receptor. We show that syndecan, when expressed in nonpermissive cells, becomes the major mediator for HIV adsorption. This adsorption is mediated by the binding of gp120 to the heparan sulfate chains of syndecan. Although syndecan does not substitute for HIV entry receptors, it enhances the in trans infectivity of a broad range of primate lentiviruses including primary viruses produced from PBMCs. Furthermore, syndecan preserves virus infectivity for a week, whereas unbound virus loses its infectivity in less than a day. Moreover, we obtain evidence suggesting that the vast syndecan-rich endothelial lining of the vasculature can provide a microenvironment which boosts HIV replication in T cells.

Active site residues of cyclophilin A are crucial for its signaling activity via CD147.

Cyclophilin A (CyPA), a ubiquitously distributed intracellular protein, is a peptidylprolyl cis-trans-isomerase and the major target of the potent immunosuppressive drug cyclosporin A. Although expressed predominantly as an intracellular molecule, CyPA is secreted by cells in response to inflammatory stimuli and is a potent neutrophil and eosinophil chemoattractant in vitro and in vivo. The mechanisms underlying CyPA-mediated signaling and chemotaxis are unknown. Here, we identified CD147 as a cell surface receptor for CyPA and demonstrated that CD147 is an essential component in the CyPA-initiated signaling cascade that culminates in ERK activation. Both signaling and chemotactic activities of CyPA depended also on the presence of heparans, which served as primary binding sites for CyPA on target cells. The proline 180 and glycine 181 residues in the extracellular domain of CD147 were critical for signaling and chemotactic activities mediated by CD147. Also crucial were active site residues of CyPA, because rotamase-defective CyPA mutants failed to initiate signaling events. These results establish cyclophilins as natural ligands for CD147 and suggest an unusual, rotamase-dependent mechanism of signaling.

Functional equivalence of late gene promoters in bean golden mosaic virus with those in tomato golden mosaic virus.

In the bipartite geminivirus tomato golden mosaic virus (TGMV), the activity of late gene promoters is up-regulated by the multifunctional viral protein AL2. Cis-acting sequences required for AL2-mediated promoter responses have not been well characterized. However, nucleotide sequence analysis has implicated a motif termed the conserved late element (CLE). The CLE is present in TGMV and many other begomoviruses, although it is not ubiquitous. Here we analysed the regulation of late gene expression in bean golden mosaic virus (BGMV), one of the begomoviruses which lacks the CLE. Transient reporter gene assays showed that BGMV late gene promoters were trans-activated in Nicotiana benthamiana protoplasts, both by the homologous BGMV AL2 protein and by the heterologous TGMV AL2 protein. The BGMV AL2 protein also trans-activated TGMV late gene promoters. Consistent with these results, we found that hybrid viruses with the late gene promoters exchanged between BGMV and TGMV were viable in planta.