Role for Slimb in the degradation of Drosophila Period protein phosphorylated by Doubletime

Protein phosphorylation has a key role in modulating the stabilities of circadian clock proteins in a manner specific to the time of day. A conserved feature of animal clocks is that Period (Per) proteins undergo daily rhythms in phosphorylation and levels, events that are crucial for normal clock progression. Casein kinase Iepsilon (CKIepsilon) has a prominent role in regulating the phosphorylation and abundance of Per proteins in animals. This was first shown in Drosophila with the characterization of Doubletime (Dbt), a homologue of vertebrate casein kinase Iepsilon. However, it is not clear how Dbt regulates the levels of Per. Here we show, using a cell culture system, that Dbt promotes the progressive phosphorylation of Per, leading to the rapid degradation of hyperphosphorylated isoforms by the ubiquitin-proteasome pathway. Slimb, an F-box/WD40-repeat protein functioning in the ubiquitin-proteasome pathway interacts preferentially with phosphorylated Per and stimulates its degradation. Overexpression of slimb or expression in clock cells of a dominant-negative version of slimb disrupts normal rhythmic activity in flies. Our findings suggest that hyperphosphorylated Per is targeted to the proteasome by interactions with Slimb.     

Shaggy/GSK3 antagonizes Hedgehog signalling by regulating Cubitus interruptus

The Drosophila protein Shaggy (Sgg, also known as Zeste-white3, Zw3) and its vertebrate orthologue glycogen synthase kinase 3 (GSK3) are inhibitory components of the Wingless (Wg) and Wnt pathways. Here we show that Sgg is also a negative regulator in the Hedgehog (Hh) pathway. In Drosophila, Hh acts both by blocking the proteolytic processing of full-length Cubitus interruptus, Ci (Ci155), to generate a truncated repressor form (Ci75), and by stimulating the activity of accumulated Ci155 (refs 2-6). Loss of sgg gene function results in a cell-autonomous accumulation of high levels of Ci155 and the ectopic expression of Hh-responsive genes including decapentaplegic (dpp) and wg. Simultaneous removal of sgg and Suppressor of fused, Su(fu), results in wing duplications similar to those caused by ectopic Hh signalling. Ci is phosphorylated by GSK3 after a primed phosphorylation by protein kinase A (PKA), and mutating GSK3-phosphorylation sites in Ci blocks its processing and prevents the production of the repressor form. We propose that Sgg/GSK3 acts in conjunction with PKA to cause hyperphosphorylation of Ci, which targets it for proteolytic processing, and that Hh opposes Ci proteolysis by promoting its dephosphorylation.     

The mPer2 gene encodes a functional component of the mammalian circadian clock.

Circadian rhythms are driven by endogenous biological clocks that regulate many biochemical, physiological and behavioural processes in a wide range of life forms. In mammals, there is a master circadian clock in the suprachiasmatic nucleus of the anterior hypothalamus. Three putative mammalian homologues (mPer1, mPer2 and mPer3) of the Drosophila circadian clock gene period (per) have been identified. The mPer genes share a conserved PAS domain (a dimerization domain found in Per, Arnt and Sim) and show a circadian expression pattern in the suprachiasmatic nucleus. To assess the in vivo function of mPer2, we generated and characterized a deletion mutation in the PAS domain of the mouse mPer2 gene. Here we show that mice homozygous for this mutation display a shorter circadian period followed by a loss of circadian rhythmicity in constant darkness. The mutation also diminishes the oscillating expression of both mPer1 and mPer2 in the suprachiasmatic nucleus, indicating that mPer2 may regulate mPer1 in vivo. These data provide evidence that an mPer gene functions in the circadian clock, and define mPer2 as a component of the mammalian circadian oscillator.     

An inducible promoter fused to the period gene in Drosophila conditionally rescues adult per-mutant arrhythmicity

The period (per) gene of Drosophila melanogaster is involved in the expression of circadian rhythms of locomotor activity in adult flies. Molecular studies of per (reviewed in ref. 2) have shown that the transcribed and translated products of this gene are present primarily at the embryonic, pupal and adult stages. Here we describe experiments with arrhythmic per mutants bearing an inducible form of this gene which indicate that strongly rhythmic adult behaviour can be obtained only if per expression is induced in the adult, independent of its history of expression earlier in development. Thus per-mutant locomotor-activity phenotypes seem not to result from abnormalities in the development of neural structures or in physiological processes that may be required at pre-adult stages for the expression of this circadian rhythm. Moreover, the action of per after light:dark cycle entrainment seems to be sufficient for activity rhythms to be exhibited in constant darkness; this suggests further that the per product is required only during the time that the rhythmic behaviour is being manifested. Our strategy used a heat-shock gene promotor fused to per coding sequences to obtain conditional gene expression. Heat-shock promoter-driven genes have previously been used to study the mode of action and tissue specificity of a variety of Drosophila genes; our experiments on circadian rhythms demonstrate the use of such gene constructions for the temporal manipulation of genes whose phenotypes, behavioural and otherwise, affect whole organisms.     

Stress response genes protect against lethal effects of sleep deprivation in Drosophila

Sleep is controlled by two processes: a homeostatic drive that increases during waking and dissipates during sleep, and a circadian pacemaker that controls its timing. Although these two systems can operate independently, recent studies indicate a more intimate relationship. To study the interaction between homeostatic and circadian processes in Drosophila, we examined homeostasis in the canonical loss-of-function clock mutants period (per(01)), timeless (tim(01)), clock (Clk(jrk)) and cycle (cyc(01)). cyc(01) mutants showed a disproportionately large sleep rebound and died after 10 hours of sleep deprivation, although they were more resistant than other clock mutants to various stressors. Unlike other clock mutants, cyc(01) flies showed a reduced expression of heat-shock genes after sleep loss. However, activating heat-shock genes before sleep deprivation rescued cyc(01) flies from its lethal effects. Consistent with the protective effect of heat-shock genes, was the observation that flies carrying a mutation for the heat-shock protein Hsp83 (Hsp83(08445)) showed exaggerated homeostatic response and died after sleep deprivation. These data represent the first step in identifying the molecular mechanisms that constitute the sleep homeostat.     

Circadian rhythms in olfactory responses of Drosophila melanogaster.

The core mechanism of circadian timekeeping in arthropods and vertebrates consists of feedback loops involving several clock genes, including period (per) and timeless (tim). In the fruitfly Drosophila, circadian oscillations in per expression occur in chemosensory cells of the antennae, even when the antennae are excised and maintained in isolated organ culture. Here we demonstrate a robust circadian rhythm in Drosophila in electrophysiological responses to two classes of olfactory stimuli. These rhythms are observed in wild-type flies during light-dark cycles and in constant darkness, but are abolished in per or tim null-mutant flies (per01 and tim01) which lack rhythms in adult emergence and locomotor behaviour. Olfactory rhythms are also abolished in the per 7.2:2 transgenic line in which per expression is restricted to the lateral neurons of the optic lobe. Because per 7.2:2 flies do not express per in peripheral oscillators, our results provide evidence that peripheral circadian oscillators are necessary for circadian rhythms in olfactory responses. As olfaction is essential for food acquisition, social interactions and predator avoidance in many animals, circadian regulation of olfactory systems could have profound effects on the behaviour of organisms that rely on this sensory modality.     

A role for Drosophila LKB1 in anterior-posterior axis formation and epithelial polarity

The PAR-4 and PAR-1 kinases are necessary for the formation of the anterior-posterior (A-P) axis in Caenorhabditis elegans. PAR-1 is also required for A-P axis determination in Drosophila. Here we show that the Drosophila par-4 homologue, lkb1, is required for the early A-P polarity of the oocyte, and for the repolarization of the oocyte cytoskeleton that defines the embryonic A-P axis. LKB1 is phosphorylated by PAR-1 in vitro, and overexpression of LKB1 partially rescues the par-1 phenotype. These two kinases therefore function in a conserved pathway for axis formation in flies and worms. lkb1 mutant clones also disrupt apical-basal epithelial polarity, suggesting a general role in cell polarization. The human homologue, LKB1, is mutated in Peutz-Jeghers syndrome and is regulated by prenylation and by phosphorylation by protein kinase A. We show that protein kinase A phosphorylates Drosophila LKB1 on a conserved site that is important for its activity. Thus, Drosophila and human LKB1 may be functional homologues, suggesting that loss of cell polarity may contribute to tumour formation in individuals with Peutz-Jeghers syndrome.     

Functional consequences of a CKIdelta mutation causing familial advanced sleep phase syndrome

Familial advanced sleep phase syndrome (FASPS) is a human behavioural phenotype characterized by early sleep times and early-morning awakening. It was the first human, mendelian circadian rhythm variant to be well-characterized, and was shown to result from a mutation in a phosphorylation site within the casein kinase I (CKI)-binding domain of the human PER2 gene. To gain a deeper understanding of the mechanisms of circadian rhythm regulation in humans, we set out to identify mutations in human subjects leading to FASPS. We report here the identification of a missense mutation (T44A) in the human CKIdelta gene, which results in FASPS. This mutant kinase has decreased enzymatic activity in vitro. Transgenic Drosophila carrying the human CKIdelta-T44A gene showed a phenotype with lengthened circadian period. In contrast, transgenic mice carrying the same mutation have a shorter circadian period, a phenotype mimicking human FASPS. These results show that CKIdelta is a central component in the mammalian clock, and suggest that mammalian and fly clocks might have different regulatory mechanisms despite the highly conserved nature of their individual components.     

Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction

Signalling by Wnt proteins (Wingless in Drosophila) has diverse roles during embryonic development and in adults, and is implicated in human diseases, including cancer. LDL-receptor-related proteins 5 and 6 (LRP5 and LRP6; Arrow in Drosophila) are key receptors required for transmission of Wnt/beta-catenin signalling in metazoa. Although the role of these receptors in Wnt signalling is well established, their coupling with the cytoplasmic signalling apparatus remains poorly defined. Using a protein modification screen for regulators of LRP6, we describe the identification of Xenopus Casein kinase 1 gamma (CK1gamma), a membrane-bound member of the CK1 family. Gain-of-function and loss-of-function experiments show that CK1gamma is both necessary and sufficient to transduce LRP6 signalling in vertebrates and Drosophila cells. In Xenopus embryos, CK1gamma is required during anterio-posterior patterning to promote posteriorizing Wnt/beta-catenin signalling. CK1gamma is associated with LRP6, which has multiple, modular CK1 phosphorylation sites. Wnt treatment induces the rapid CK1gamma-mediated phosphorylation of these sites within LRP6, which, in turn, promotes the recruitment of the scaffold protein Axin. Our results reveal an evolutionarily conserved mechanism that couples Wnt receptor activation to the cytoplasmic signal transduction apparatus.     

Unexpected features of Drosophila circadian behavioural rhythms under natural conditions

Circadian clocks have evolved to synchronize physiology, metabolism and behaviour to the 24-h geophysical cycles of the Earth. Drosophila melanogaster's rhythmic locomotor behaviour provides the main phenotype for the identification of higher eukaryotic clock genes. Under laboratory light-dark cycles, flies show enhanced activity before lights on and off signals, and these anticipatory responses have defined the neuronal sites of the corresponding morning (M) and evening (E) oscillators. However, the natural environment provides much richer cycling environmental stimuli than the laboratory, so we sought to examine fly locomotor rhythms in the wild. Here we show that several key laboratory-based assumptions about circadian behaviour are not supported by natural observations. These include the anticipation of light transitions, the midday 'siesta', the fly's crepuscular activity, its nocturnal behaviour under moonlight, and the dominance of light stimuli over temperature. We also observe a third major locomotor component in addition to M and E, which we term 'A' (afternoon). Furthermore, we show that these natural rhythm phenotypes can be observed in the laboratory by using realistic temperature and light cycle simulations. Our results suggest that a comprehensive re-examination of circadian behaviour and its molecular readouts under simulated natural conditions will provide a more authentic interpretation of the adaptive significance of this important rhythmic phenotype. Such studies should also help to clarify the underlying molecular and neuroanatomical substrates of the clock under natural protocols.     

A new role for cryptochrome in a Drosophila circadian oscillator

Cryptochromes are flavin/pterin-containing proteins that are involved in circadian clock function in Drosophila and mice. In mice, the cryptochromes Cry1 and Cry2 are integral components of the circadian oscillator within the brain and contribute to circadian photoreception in the retina. In Drosophila, cryptochrome (CRY) acts as a photoreceptor that mediates light input to circadian oscillators in both brain and peripheral tissue. A Drosophila cry mutant, cryb, leaves circadian oscillator function intact in central circadian pacemaker neurons but renders peripheral circadian oscillators largely arrhythmic. Although this arrhythmicity could be caused by a loss of light entrainment, it is also consistent with a role for CRY in the oscillator. A peripheral oscillator drives circadian olfactory responses in Drosophila antennae. Here we show that CRY contributes to oscillator function and physiological output rhythms in the antenna during and after entrainment to light-dark cycles and after photic input is eliminated by entraining flies to temperature cycles. These results demonstrate a photoreceptor-independent role for CRY in the periphery and imply fundamental differences between central and peripheral oscillator mechanisms in Drosophila.     

A resetting signal between Drosophila pacemakers synchronizes morning and evening activity

The biochemical machinery that underlies circadian rhythms is conserved among animal species and drives self-sustained molecular oscillations and functions, even within individual asynchronous tissue-culture cells. Yet the rhythm-generating neural centres of higher eukaryotes are usually composed of interconnected cellular networks, which contribute to robustness and synchrony as well as other complex features of rhythmic behaviour. In mammals, little is known about how individual brain oscillators are organized to orchestrate a complex behavioural pattern. Drosophila is arguably more advanced from this point of view: we and others have recently shown that a group of adult brain clock neurons expresses the neuropeptide PDF and controls morning activity (small LN(v) cells; M-cells), whereas another group of clock neurons controls evening activity (CRY+, PDF- cells; E-cells). We have generated transgenic mosaic animals with different circadian periods in morning and evening cells. Here we show, by behavioural and molecular assays, that the six canonical groups of clock neurons are organized into two separate neuronal circuits. One has no apparent effect on locomotor rhythmicity in darkness, but within the second circuit the molecular and behavioural timing of the evening cells is determined by morning-cell properties. This is due to a daily resetting signal from the morning to the evening cells, which run at their genetically programmed pace between consecutive signals. This neural circuit and oscillator-coupling mechanism ensures a proper relationship between the timing of morning and evening locomotor activity.     

HP1-beta mobilization promotes chromatin changes that initiate the DNA damage response

Minutes after DNA damage, the variant histone H2AX is phosphorylated by protein kinases of the phosphoinositide kinase family, including ATM, ATR or DNA-PK. Phosphorylated (gamma)-H2AX-which recruits molecules that sense or signal the presence of DNA breaks, activating the response that leads to repair-is the earliest known marker of chromosomal DNA breakage. Here we identify a dynamic change in chromatin that promotes H2AX phosphorylation in mammalian cells. DNA breaks swiftly mobilize heterochromatin protein 1 (HP1)-beta (also called CBX1), a chromatin factor bound to histone H3 methylated on lysine 9 (H3K9me). Local changes in histone-tail modifications are not apparent. Instead, phosphorylation of HP1-beta on amino acid Thr 51 accompanies mobilization, releasing HP1-beta from chromatin by disrupting hydrogen bonds that fold its chromodomain around H3K9me. Inhibition of casein kinase 2 (CK2), an enzyme implicated in DNA damage sensing and repair, suppresses Thr 51 phosphorylation and HP1-beta mobilization in living cells. CK2 inhibition, or a constitutively chromatin-bound HP1-beta mutant, diminishes H2AX phosphorylation. Our findings reveal an unrecognized signalling cascade that helps to initiate the DNA damage response, altering chromatin by modifying a histone-code mediator protein, HP1, but not the code itself.     

Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane

The receptor for epidermal growth factor (EGF) is a 170,000-180,000 molecular weight single-chain glycoprotein of 1,186 amino acids. Its sequence suggests that it has an external EGF-binding domain, formed by the NH2-terminal 621 amino acids, linked to a cytoplasmic region by a single membrane-spanning segment. In the cytoplasmic portion, starting 50 residues from the membrane, there is a 250-residue stretch similar to the catalytic domain of the src gene family of retroviral tyrosine protein kinases, and, indeed, a tyrosine-specific protein kinase activity intrinsic to the receptor is stimulated when EGF is bound. Increased tyrosine phosphorylation of cellular proteins, detected in A431 cells following EGF binding, may be important in the mitogenic signal pathway. Tumour promoters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), counteract this increase, as well as causing loss of a high affinity class of EGF binding sites. The major receptor for TPA has been identified as the serine/threonine-specific Ca2+/phospholipid-dependent diacylglycerol-activated protein kinase, protein kinase C. By substituting for diacylglycerol, TPA stimulates protein kinase C. Protein kinase C phosphorylates purified EGF receptor at specific sites, and this reduces EGF-stimulated tyrosine protein kinase activity. TPA treatment of A431 cells increases serine and threonine phosphorylation of the EGF receptor at the same sites, which suggests that the reduction of EGF receptor kinase activity in TPA-treated cells is a consequence of the receptor's phosphorylation by the kinase. We have attempted to identify these phosphorylation sites and show here that protein kinase C phosphorylates threonine 654 in the human EGF receptor. This threonine is in a very basic sequence nine residues from the cytoplasmic face of the plasma membrane in the region before the protein kinase domain; it is thus in a position to modulate signalling between this internal domain and the external EGF-binding domain.