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.

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.

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 CLOCK⅐CYCLE (CLK⅐CYC) complex. Induction of CLK⅐CYC-dependent transcripts upon induction of nej expression from a heat-shock promoter showed that NEJ is limiting. Reduced CLK⅐CYC-mediated transcription in a nej hypomorphic mutant indicates an essential function of NEJ/CBP for CLK⅐CYC 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.The circadian clock controls genome wide transcription of many key regulatory components in a diverse selection of vital pathways (1-3) that ultimately allow a coordination of physiological and behavioral activities and their synchronization with the environmental cycles of day and night. The analogous and homologous clock mechanisms in Drosophila and mammals are based on two interconnected feedback loops (4, 5). In Drosophila, the heterodimeric complex of transcription factors CLOCK (CLK) 3 and CYCLE (CYC) (BMAL1 in mammals) activates expression of its own inhibitors PERIOD (PER) and TIME-LESS (TIM) forming the first feedback loop. This loop is interconnected with CLK⅐CYC-mediated expression of the transcription repressor vrille (vri) and the activator par-domain protein 1 (pdp1). VRI and PDP1 control the rhythmic transcription of Clk and contribute to the robustness of molecular oscillations (6, 7). Oscillations in cyclic nucleotide, calcium, and MAPK signaling (8 -10) likely contribute to a circadian control of physiological processes such as cell proliferation (11) and the sleep/wake cycle, which is important for memory formation (12). However, these pathways also feedback on the molecular oscillator at least in part through control of CLK⅐CYC activity (13). Cross-talk between circadian and cell signaling may increase the robustness of circadian oscillations and allow a coordination of circadian transcription with physiological requirements. Previous studies showed that recruitment of the CREB-binding protein (CBP) from a limiting cellular pool mediates cross-talk between the transcription factors E2F, JAK/STAT, AP1, and nuclear hormone receptors (14 -16) that control e.g. entry into the cell cycle and the immune response. Here we show that CLK⅐CYC-...

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.

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). Posttranslational 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 transactivation 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.The circadian clock provides a molecular mechanism that orchestrates behavior and physiology in a temporal fashion and synchronizes homeostatic functions with the environmental cycles of day and night (1-3). The central oscillating activity of the Drosophila and mammalian circadian clock is formed by the heterodimeric complex of the transcription factors CLOCK (CLK) 2 and CYCLE (CYC) that ultimately controls genome-wide transcription and activity states of key regulatory components (4 -7). Importantly, the circadian oscillator is synchronized by environmental cycles, primarily light/dark and temperature cycles (8, 9), but keeps time on its own in the absence of environmental cues, therefore representing a true molecular clock.Despite the crucial role of CLK/CYC for the circadian orchestration of physiology in Drosophila and mammals, little is known about their post-translational regulation (2, 10, 11). Transcript levels of Clk reveal robust circadian oscillations in Drosophila, due to rhythmic binding of the activator PAR-DOMAIN PROTEIN 1 and the repressor VRILLE to V/P-elements in the Clk-promoter (12-14). Rhythmic Clk transcription appears however not essential for self-sustained molecular oscillations, because expression of CLK from a perpromoter in anti-phase to its endogenous rhythm was found to support normal clock function (15). The post-translational regulation of CLK/CYC is however crucial for constituti...

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.

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.

Edited by Martha Merrow and Michael BrunnerKeywords: Drosophila Circadian clock Post-translational regulation Phosphorylation SUMO Nucleocytoplasmic transport a b s t r a c t 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.