ABSTRACT
All eukaryotic cells secrete a range of proteins in a constitutive or regulated manner through the conventional or canonical exocytic/secretory pathway characterized by vesicular traffic from the endoplasmic reticulum, through the Golgi apparatus, and towards the plasma membrane. However, a number of proteins are secreted in an unconventional manner, which are insensitive to inhibitors of conventional exocytosis and use a route that bypasses the Golgi apparatus. These include cytosolic proteins such as fibroblast growth factor 2 (FGF2) and interleukin-1ß (IL-1ß), and membrane proteins that are known to also traverse to the plasma membrane by a conventional process of exocytosis, such as α integrin and the cystic fibrosis transmembrane conductor (CFTR). Mechanisms underlying unconventional protein secretion (UPS) are actively being analyzed and deciphered, and these range from an unusual form of plasma membrane translocation to vesicular processes involving the generation of exosomes and other extracellular microvesicles. In this chapter, we provide an overview on what is currently known about UPS in animal cells.
Subject(s)
Proteins/metabolism , Secretory Pathway , Animals , Autophagy , Biomarkers , Carrier Proteins , Cell Membrane/metabolism , Endoplasmic Reticulum/metabolism , Exosomes/metabolism , Golgi Apparatus/metabolism , Protein Binding , Protein Transport , Transport Vesicles/metabolism , Yeasts/metabolismABSTRACT
Unlike founding members of the Ras superfamily of small GTPases that are prominently known for oncogenic signaling, members of the Rab subfamily are key regulators of cellular membrane traffic. However, a number of Rabs have in recent years also been strongly implicated as tumorigenic or metastatic biomarkers. Rab23 is an emerging example whose differential expression in tumor cells and functional association with proliferation and invasiveness is attracting attention as a useful cancer marker and a potential therapeutic target. Rab23 is ubiquitously expressed but appears to be particularly enriched in the adult brain. It has important developmental functions in vertebrates and has been shown to modulate Sonic hedgehog (Shh) and Nodal signaling. Although its exact cellular role in membrane traffic regulation remains elusive, its known role in Shh signaling, in conjunction with several recent findings, has clearly implicated a role for Rab23 in transport processes to the primary cilium. In this review, we summarize what is currently known about Rab23 as a cancer marker and discuss possible mechanism by which this Rab GTPase may act as an oncogenic or metastatic driver, while exhibiting tumor suppressive activity in some cases.
Subject(s)
Neoplasms/metabolism , Neoplasms/pathology , rab GTP-Binding Proteins/metabolism , Adult , HumansABSTRACT
Although, glial cells have well characterized functions in the developing and mature brain, it is only in the past decade that roles for these cells in behavior and plasticity have been delineated. Glial astrocytes and glia-neuron signaling, for example, are now known to have important modulatory functions in sleep, circadian behavior, memory and plasticity. To better understand mechanisms of glia-neuron signaling in the context of behavior, we have conducted cell-specific, genome-wide expression profiling of adult Drosophila astrocyte-like brain cells and performed RNA interference (RNAi)-based genetic screens to identify glial factors that regulate behavior. Importantly, our studies demonstrate that adult fly astrocyte-like cells and mouse astrocytes have similar molecular signatures; in contrast, fly astrocytes and surface glia-different classes of glial cells-have distinct expression profiles. Glial-specific expression of 653 RNAi constructs targeting 318 genes identified multiple factors associated with altered locomotor activity, circadian rhythmicity and/or responses to mechanical stress (bang sensitivity). Of interest, 1 of the relevant genes encodes a vesicle recycling factor, 4 encode secreted proteins and 3 encode membrane transporters. These results strongly support the idea that glia-neuron communication is vital for adult behavior.
ABSTRACT
We previously showed that endocytosis and/or vesicle recycling mechanisms are essential in adult Drosophila glial cells for the neuronal control of circadian locomotor activity. In this study, our goal was to identify specific glial vesicle trafficking, recycling, or release factors that are required for rhythmic behavior. From a glia-specific, RNAi-based genetic screen, we identified eight glial factors that are required for normally robust circadian rhythms in either a light-dark cycle or in constant dark conditions. In particular, we show that conditional knockdown of the ROP vesicle release factor in adult glial cells results in arrhythmic behavior. Immunostaining for ROP reveals reduced protein in glial cell processes and an accumulation of the Par Domain Protein 1ε (PDP1ε) clock output protein in the small lateral clock neurons. These results suggest that glia modulate rhythmic circadian behavior by secretion of factors that act on clock neurons to regulate a clock output factor.
ABSTRACT
The silent mating type information regulation 2 proteins (sirtuins) 1 of class III histone deacetylases (HDACs) have been associated with health span and longevity. SIRT1, the best studied member of the mammalian sirtuins, has a myriad of roles in multiple tissues and organs. However, a significant part of SIRT1's role that impinges on aging and lifespan may lie in its activities in the central nervous system (CNS) neurons. Systemically, SIRT1 influences energy metabolism and circadian rhythm through its activity in the hypothalamic nuclei. From a cell biological perspective, SIRT1 is a crucial component of multiple interconnected regulatory networks that modulate dendritic and axonal growth, as well as survival against stress. This neuronal cell autonomous activity of SIRT1 is also important for neuronal plasticity, cognitive functions, as well as protection against aging-associated neuronal degeneration and cognitive decline. We discuss recent findings that have shed light on the various activities of SIRT1 in the brain, which collectively impinge on aging-associated disorders and lifespan.
ABSTRACT
The analysis of adult astrocyte glial cells has revealed a remarkable heterogeneity with regard to morphology, molecular signature, and physiology. A key question in glial biology is how such heterogeneity arises during brain development. One approach to this question is to identify genes with differential astrocyte expression during development; certain genes expressed later in neural development may contribute to astrocyte differentiation. We have utilized the Drosophila model and Translating Ribosome Affinity Purification (TRAP)-RNA-seq methods to derive the genome-wide expression profile of Drosophila larval astrocyte-like cells (hereafter referred to as astrocytes) for the first time. These studies identified hundreds of larval astrocyte-enriched genes that encode proteins important for metabolism, energy production, and protein synthesis, consistent with the known role of astrocytes in the metabolic support of neurons. Comparison of the larval profile with that observed for adults has identified genes with astrocyte-enriched expression specific to adulthood. These include genes important for metabolism and energy production, translation, chromatin modification, protein glycosylation, neuropeptide signaling, immune responses, vesicle-mediated trafficking or secretion, and the regulation of behavior. Among these functional classes, the expression of genes important for chromatin modification and vesicle-mediated trafficking or secretion is overrepresented in adult astrocytes based on Gene Ontology analysis. Certain genes with selective adult enrichment may mediate functions specific to this stage or may be important for the differentiation or maintenance of adult astrocytes, with the latter perhaps contributing to population heterogeneity.
Subject(s)
Astrocytes/metabolism , Drosophila/genetics , Transcriptome , Animals , Drosophila/growth & development , Genes, Reporter , Genome , Immunohistochemistry , Larva/genetics , Life Cycle Stages/genetics , Microscopy, Fluorescence , Nervous System/metabolism , Nervous System/pathology , Sequence Analysis, RNAABSTRACT
Brain glial cells, in particular astrocytes and microglia, secrete signaling molecules that regulate glia-glia or glia-neuron communication and synaptic activity. While much is known about roles of glial cells in nervous system development, we are only beginning to understand the physiological functions of such cells in the adult brain. Studies in vertebrate and invertebrate models, in particular mice and Drosophila, have revealed roles of glia-neuron communication in the modulation of complex behavior. This chapter emphasizes recent evidence from studies of rodents and Drosophila that highlight the importance of glial cells and similarities or differences in the neural circuits regulating circadian rhythms and sleep in the two models. The chapter discusses cellular, molecular, and genetic approaches that have been useful in these models for understanding how glia-neuron communication contributes to the regulation of rhythmic behavior.
Subject(s)
Circadian Rhythm , Drosophila/physiology , Neuroglia/physiology , Animals , Neuroglia/metabolism , Neurons/metabolism , Signal Transduction , SleepABSTRACT
Studies on mitochondria protein import had revealed in detail molecular mechanisms of how peptides and proteins could be selectively targeted and translocated across membrane bound organelles. The opposite process of mitochondrial export, while known to occur in various aspects of cellular physiology and pathology, is less well understood. Two very recent reports have indicated that a large mitochondrial matrix protein complex, the pyruvate dehydrogenase complex (PDC) (or its component subunits), could be exported to the lysosomes and the nucleus, respectively. In the case of the latter, evidence was presented to suggest that the entire complex of 8-10 MDa could translocate in its entirety from the mitochondrial matrix to the nucleus upon mitogenic or stress stimuli. We discuss these findings in perspective to what is currently known about the processes of transport in and out of the mitochondrion.
Subject(s)
Eukaryotic Cells/metabolism , Mitochondria/metabolism , Pyruvate Dehydrogenase Complex/metabolism , Cell Nucleus/metabolism , Lysosomes/metabolism , Protein Transport , Stress, PhysiologicalABSTRACT
Transcriptional/translational feedback loops drive daily cycles of expression in clock genes and clock-controlled genes, which ultimately underlie many of the overt circadian rhythms manifested by organisms. Moreover, phosphorylation of clock proteins plays crucial roles in the temporal regulation of clock protein activity, stability and subcellular localization. dCLOCK (dCLK), the master transcription factor driving cyclical gene expression and the rate-limiting component in the Drosophila circadian clock, undergoes daily changes in phosphorylation. However, the physiological role of dCLK phosphorylation is not clear. Using a Drosophila tissue culture system, we identified multiple phosphorylation sites on dCLK. Expression of a mutated version of dCLK where all the mapped phospho-sites were switched to alanine (dCLK-15A) rescues the arrythmicity of Clk(out) flies, yet with an approximately 1.5 hr shorter period. The dCLK-15A protein attains substantially higher levels in flies compared to the control situation, and also appears to have enhanced transcriptional activity, consistent with the observed higher peak values and amplitudes in the mRNA rhythms of several core clock genes. Surprisingly, the clock-controlled daily activity rhythm in dCLK-15A expressing flies does not synchronize properly to daily temperature cycles, although there is no defect in aligning to light/dark cycles. Our findings suggest a novel role for clock protein phosphorylation in governing the relative strengths of entraining modalities by adjusting the dynamics of circadian gene expression.
Subject(s)
CLOCK Proteins/genetics , Circadian Rhythm/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Alanine/genetics , Animals , CLOCK Proteins/biosynthesis , Drosophila Proteins/biosynthesis , Drosophila melanogaster/growth & development , Gene Expression Regulation, Developmental , Mutation , Phosphorylation/genetics , RNA, Messenger/biosynthesisABSTRACT
Circadian (â 24 h) clocks control daily rhythms in metabolism, physiology, and behavior in animals, plants, and microbes. In Drosophila, these clocks keep circadian time via transcriptional feedback loops in which clock-cycle (CLK-CYC) initiates transcription of period (per) and timeless (tim), accumulating levels of PER and TIM proteins feed back to inhibit CLK-CYC, and degradation of PER and TIM allows CLK-CYC to initiate the next cycle of transcription. The timing of key events in this feedback loop are controlled by, or coincide with, rhythms in PER and CLK phosphorylation, where PER and CLK phosphorylation is high during transcriptional repression. PER phosphorylation at specific sites controls its subcellular localization, activity, and stability, but comparatively little is known about the identity and function of CLK phosphorylation sites. Here we identify eight CLK phosphorylation sites via mass spectrometry and determine how phosphorylation at these sites impacts behavioral and molecular rhythms by transgenic rescue of a new Clk null mutant. Eliminating phosphorylation at four of these sites accelerates the feedback loop to shorten the circadian period, whereas loss of CLK phosphorylation at serine 859 increases CLK activity, thereby increasing PER levels and accelerating transcriptional repression. These results demonstrate that CLK phosphorylation influences the circadian period by regulating CLK activity and progression through the feedback loop.
Subject(s)
CLOCK Proteins/metabolism , Circadian Rhythm/physiology , Drosophila Proteins/metabolism , ARNTL Transcription Factors/genetics , ARNTL Transcription Factors/metabolism , Animals , CLOCK Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster , Period Circadian Proteins/genetics , Period Circadian Proteins/metabolism , Phosphorylation/physiologyABSTRACT
Sirt1, the class III histone deacetylase, is generally associated with increased life span and with a pro-survival effect in neurons stressed by pathological factors. Recent work, however, suggests that Sirt1 silencing could also promote neuronal survival. A possible reason suggested is Sirt1 silencing enhanced expression of both IGF-1 and IGF-1 receptor, signaling from which promotes survival. This work adds to the small but steady stream of findings that are diametrically opposite to the overwhelmingly large amount of evidence supporting a beneficial effect of sustaining or enhancing Sirt1 activity in neuronal injuries and diseases. We attempt to reconcile this discrepancy below by noting evidence that elevated Sirt1 levels and/or activity may not help, and could even adversely exacerbates demise, during events of acute neuronal damage or death. However, sustained Sirt1 activation will be beneficial in situations of chronic and long-term sub-lethal stresses, and the status of IGF-1 signaling may influence Sirt1 action in a context dependent manner.
ABSTRACT
The sirtuin family of class III histone deacetylases has been extensively implicated in modulating a myriad of cellular processes, including energy metabolism, stress response, cell/tissue survival and malignancy. Recent studies have also identified multifaceted roles for Sirt1 and Sirt2 in the regulation of autophagy. Sirt1 could influence autophagy directly via its deacetylation of key components of the autophagy induction network, such as the products of autophagy genes (Atg) 5, 7, and 8. Nucleus-localized Sirt1 is also known to induce the expression of autophagy pathway components through the activation of FoxO transcription factor family members. The perception of a linear Sirt1-FoxO axis in autophagy induction is complicated by recent findings that acetylated FoxO1 could bind to Atg7 in the cytoplasm and affect autophagy directly. This occurs with prolonged stress signaling, with FoxO1's continuous dissociation from cytoplasmic Sirt2 and its consequential hyperacetylation. FoxO-mediated nuclear transcription may induce/enhance autophagy in ways that are different compared to cytoplasmic FoxO, thereby leading to contrasting (cell survival versus cell death) outcomes. FoxO and Sirt1 are both subjected to regulation by stress signaling (e.g., through the c-Jun N-terminal kinases (JNK)) in the context of autophagy induction, which are also critical in determining between cell survival and death in a context-dependent manner. We discussed here the emerging molecular intricacies of sirtuins' connections with autophagy. A good understanding of these connections would serve to consolidate a framework of mechanisms underlying Sirt1's protective effects in multiple physiological systems.
Subject(s)
Autophagy/physiology , Sirtuin 1/genetics , Sirtuin 1/metabolism , Animals , Autophagy/genetics , Humans , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
To investigate the regulation of Drosophila melanogaster behavior by biogenic amines, we have exploited the broad requirement of the vesicular monoamine transporter (VMAT) for the vesicular storage and exocytotic release of all monoamine neurotransmitters. We used the Drosophila VMAT (dVMAT) null mutant to globally ablate exocytotic amine release and then restored DVMAT activity in either individual or multiple aminergic systems, using transgenic rescue techniques. We find that larval survival, larval locomotion, and female fertility rely predominantly on octopaminergic circuits with little apparent input from the vesicular release of serotonin or dopamine. In contrast, male courtship and fertility can be rescued by expressing DVMAT in octopaminergic or dopaminergic neurons, suggesting potentially redundant circuits. Rescue of major aspects of adult locomotion and startle behavior required octopamine, but a complementary role was observed for serotonin. Interestingly, adult circadian behavior could not be rescued by expression of DVMAT in a single subtype of aminergic neurons, but required at least two systems, suggesting the possibility of unexpected cooperative interactions. Further experiments using this model will help determine how multiple aminergic systems may contribute to the regulation of other behaviors. Our data also highlight potential differences between behaviors regulated by standard exocytotic release and those regulated by other mechanisms.
Subject(s)
Dopamine/metabolism , Drosophila melanogaster/metabolism , Neurotransmitter Agents/metabolism , Octopamine/metabolism , Serotonin/metabolism , Animals , Animals, Genetically Modified , Circadian Rhythm/genetics , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Female , Larva/metabolism , Locomotion , Male , Mutation , Reflex, Startle/genetics , Reproduction , Sexual Behavior, AnimalABSTRACT
As frontline nurses, we know firsthand the many challenges of renal disease faced by our patients and the impact on their lives and their families. How can we help them cope with their illness? How can we improve their quality of life? How can we prevent the complications inherent to the disease? How do we know we are doing a good job? Where do we start? The purpose of this presentation is to showcase the global management of the hemodialysis (HD) patient. It provides a collaborative and systematic approach to assessing, implementing, evaluating and coordinating the physiologic and the psychosocial aspects of their care. It is a model of case management followed by the Southern Alberta Renal Program (SARP) in meeting the many and complex needs of our hemodialysis patients. The quality indicators, to name a few, that relate to the physiologic aspects of their care are dialysis adequacy and fluid removal, improved blood pressure (BP) control, maintenance and improved vascular access function, anemia, bone and mineral disease management, nutritional, and diabetes management. The psychosocial aspects of care encompass goals of care, residential support, transportation, and mobility programs in the community. There may be positive implications resulting from our practice that we believe would be invaluable in terms of improved patient care, increased adherence to therapeutic regimens, improved mortality and morbidity and overall enhanced quality of life. Moreover, better communication would possibly be fostered and wise and prompt use of resources may be a result. To date, we have not done studies to prove or disprove these outcomes.
Subject(s)
Case Management/organization & administration , Disease Management , Renal Dialysis/nursing , Renal Insufficiency/nursing , Renal Insufficiency/therapy , Advance Care Planning , Alberta , Humans , Patient Care Team/organization & administration , Quality Indicators, Health Care , Renal Dialysis/psychology , Renal Dialysis/standards , Renal Insufficiency/psychologyABSTRACT
RNA-binding proteins mediate posttranscriptional functions in the circadian systems of multiple species. A conserved RNA recognition motif (RRM) protein encoded by the lark gene is postulated to serve circadian output and molecular oscillator functions in Drosophila and mammals, respectively. In no species, however, has LARK been eliminated, in vivo, to determine the consequences for circadian timing. The present study utilized RNA interference (RNAi) techniques in Drosophila to decrease LARK levels in clock neurons and other cell types in order to evaluate the circadian functions of the protein. Knockdown of LARK in timeless (TIM)- or pigment dispersing factor (PDF)-containing clock cells caused a significant number of flies to exhibit arrhythmic locomotor activity, demonstrating a requirement for the protein in pacemaker cells. There was no obvious effect on PER protein cycling in lark interference (RNAi) flies, but a knockdown within the PDF neurons was associated with increased PDF immunoreactivity at the dorsal termini of the small ventral lateral neuronal (s-LNv) projections, suggesting an effect on neuropeptide release. The expression of lark RNAi in multiple neurosecretory cell populations demonstrated that LARK is required within pacemaker and nonpacemaker cells for the manifestation of normal locomotor activity rhythms. Interestingly, decreased LARK function in the prothoracic gland (PG), a peripheral organ containing a clock required for the circadian control of eclosion, was associated with weak population eclosion rhythms or arrhythmicity.
Subject(s)
Circadian Rhythm , Drosophila Proteins/metabolism , Drosophila Proteins/physiology , RNA-Binding Proteins/physiology , Animals , Animals, Genetically Modified , Female , Gene Expression Regulation , Locomotion , Male , Models, Biological , Neurons/metabolism , Phenotype , RNA Interference , RNA Processing, Post-Transcriptional , RNA-Binding Proteins/metabolismABSTRACT
Lysosomes serve key degradative functions for the turnover of membrane lipids and protein components. Its biogenesis is principally dependent on exocytic traffic from the late endosome via the trans-Golgi network, and it also receives cargo to be degraded from the endocytic pathway. Membrane trafficking to the late endosome-lysosome is tightly regulated to maintain the amplitude of signalling events and cellular homeostasis. Key coordinators of lysosomal traffic include members of the Rab small GTPase family. Amongst these, Rab7, Rab9 and the more recently studied Rab22B/31 have all been reported to regulate membrane trafficking processed at the late endosome-lysosome system. We discuss what is known about the roles of these Rab proteins and their interacting partners on the regulation of traffic of important receptor proteins such as the epidermal growth factor receptor (EGFR) and the mannose 6-phosphate receptor (M6PR), in association with the late endosome-lysosome system. Better knowledge of EGFR and M6PR traffic in this regard may aid in understanding the pathological processes, such as oncogenic transformations associated with these receptors.
Subject(s)
Endosomes/metabolism , ErbB Receptors/metabolism , Intracellular Membranes/metabolism , Lysosomes/metabolism , Receptor, IGF Type 2/metabolism , rab GTP-Binding Proteins/metabolism , Animals , Humans , Protein TransportABSTRACT
A detailed structure/function analysis of Drosophila p90 ribosomal S6 kinase (S6KII) or its mammalian homolog RSK has not been performed in the context of neuronal plasticity or behavior. We previously reported that S6KII is required for normal circadian periodicity. Here we report a site-directed mutagenesis of S6KII and analysis of mutants, in vivo, that identifies functional domains and phosphorylation sites critical for the regulation of circadian period. We demonstrate, for the first time, a role for the S6KII C-terminal kinase that is independent of its known role in activation of the N-terminal kinase. Both S6KII C-terminal kinase activity and its ERK-binding domain are required for wild-type circadian period and normal phosphorylation status of the protein. In contrast, the N-terminal kinase of S6KII is dispensable for modulation of circadian period and normal phosphorylation of the protein. We also show that particular sites of S6KII phosphorylation, Ser-515 and Thr-732, are essential for normal circadian behavior. Surprisingly, the phosphorylation of S6KII residues, in vivo, does not follow a strict sequential pattern, as implied by certain cell-based studies of mammalian RSK protein.
Subject(s)
Behavior, Animal/physiology , Circadian Clocks/physiology , Drosophila Proteins/metabolism , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Animals , Drosophila Proteins/genetics , Drosophila melanogaster , Mutagenesis, Site-Directed , Mutation , Phosphorylation/genetics , Protein Structure, Tertiary , Ribosomal Protein S6 Kinases, 90-kDa/geneticsABSTRACT
BACKGROUND: An important goal of contemporary neuroscience research is to define the neural circuits and synaptic interactions that mediate behavior. In both mammals and Drosophila, the neuronal circuitry controlling circadian behavior has been the subject of intensive investigation, but roles for glial cells in the networks controlling rhythmic behavior have only begun to be defined in recent studies. RESULTS: Here, we show that conditional, glial-specific genetic manipulations affecting membrane (vesicle) trafficking, the membrane ionic gradient, or calcium signaling lead to circadian arrhythmicity in adult behaving Drosophila. Correlated and reversible effects on a clock neuron peptide transmitter (PDF) and behavior demonstrate the capacity for glia-to-neuron signaling in the circadian circuitry. These studies also reveal the importance of a single type of glial cell-the astrocyte-and glial internal calcium stores in the regulation of circadian rhythms. CONCLUSIONS: This is the first demonstration in any system that adult glial cells can physiologically modulate circadian neuronal circuitry and behavior. A role for astrocytes and glial calcium signaling in the regulation of Drosophila circadian rhythms emphasizes the conservation of cellular and molecular mechanisms that regulate behavior in mammals and insects.
Subject(s)
Astrocytes/physiology , CLOCK Proteins/genetics , Circadian Rhythm , Drosophila/physiology , Neurons/physiology , Animals , Astrocytes/metabolism , Behavior, Animal , Brain/metabolism , Brain/physiology , CLOCK Proteins/metabolism , CLOCK Proteins/physiology , Calcium/metabolism , Drosophila/genetics , Drosophila/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila Proteins/physiology , Motor Activity , Neurons/metabolism , Signal Transduction , Sodium Channels/metabolismABSTRACT
There is a universal requirement for post-translational regulatory mechanisms in circadian clock systems. Previous work in Drosophila has identified several kinases, phosphatases, and an E3 ligase that are critical for determining the nuclear translocation and/or stability of clock proteins. The present study evaluated the function of p90 ribosomal S6 kinase (RSK) in the Drosophila circadian system. In mammals, RSK1 is a light- and clock-regulated kinase known to be activated by the mitogen-activated protein kinase pathway, but there is no direct evidence that it functions as a component of the circadian system. Here, we show that Drosophila S6KII RNA displays rhythms in abundance, indicative of circadian control. Importantly, an S6KII null mutant exhibits a short-period circadian phenotype that can be rescued by expression of the wild-type gene in clock neurons, indicating a role for S6KII in the molecular oscillator. Peak PER clock protein expression is elevated in the mutant, indicative of enhanced stability, whereas per mRNA level is decreased, consistent with enhanced feedback repression. Gene reporter assays show that decreased S6KII is associated with increased PER repression. Surprisingly, we demonstrate a physical interaction between S6KII and the casein kinase 2 regulatory subunit (CK2beta), suggesting a functional relationship between the two kinases. In support of such a relationship, there are genetic interactions between S6KII and CK2 mutations, in vivo, which indicate that CK2 activity is required for S6KII action. We propose that the two kinases cooperate within clock neurons to fine-tune circadian period, improving the precision of the clock mechanism.