ABSTRACT
The outgrowth of many neurons within the central nervous system is initially directed towards or away from the cells lying at the midline. Recent genetic evidence suggests that a simple model of differential sensitivity to the conserved Netrin attractants and Slit repellents is insufficient to explain the guidance of all axons at the midline. In the Drosophila embryonic ventral nerve cord, many axons still cross the midline in the absence of the Netrin genes (NetA and NetB) or their receptor frazzled. Here we show that mutation of mushroom body defect (mud) dramatically enhances the phenotype of Netrin or frazzled mutants, resulting in many more axons failing to cross the midline, although mutations in mud alone have little effect. This suggests that mud, which encodes a microtubule-binding coiled-coil protein homologous to NuMA and LIN-5, is an essential component of a Netrin-independent pathway that acts in parallel to promote midline crossing. We demonstrate that this novel role of Mud in axon guidance is independent of its previously described role in neural precursor development. These studies identify a parallel pathway controlling midline guidance in Drosophila and highlight a novel role for Mud potentially acting downstream of Frizzled to aid axon guidance.
Subject(s)
Axons/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Membrane Proteins/metabolism , Mushroom Bodies/metabolism , Nerve Growth Factors/metabolism , Nerve Tissue Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Cell Cycle Proteins , Central Nervous System/embryology , Drosophila Proteins/deficiency , Drosophila melanogaster/cytology , Embryo, Nonmammalian/metabolism , Guanine Nucleotide Dissociation Inhibitors/metabolism , Mitosis , Mutation/genetics , Nerve Growth Factors/deficiency , Netrin-1 , Netrins , Phenotype , Tumor Suppressor Proteins/deficiencyABSTRACT
All organisms have to adapt to acute as well as to regularly occurring changes in the environment. To deal with these major challenges organisms evolved two fundamental mechanisms: the p38 mitogen-activated protein kinase (MAPK) pathway, a major stress pathway for signaling stressful events, and circadian clocks to prepare for the daily environmental changes. Both systems respond sensitively to light. Recent studies in vertebrates and fungi indicate that p38 is involved in light-signaling to the circadian clock providing an interesting link between stress-induced and regularly rhythmic adaptations of animals to the environment, but the molecular and cellular mechanisms remained largely unknown. Here, we demonstrate by immunocytochemical means that p38 is expressed in Drosophila melanogaster's clock neurons and that it is activated in a clock-dependent manner. Surprisingly, we found that p38 is most active under darkness and, besides its circadian activation, additionally gets inactivated by light. Moreover, locomotor activity recordings revealed that p38 is essential for a wild-type timing of evening activity and for maintaining â¼ 24 h behavioral rhythms under constant darkness: flies with reduced p38 activity in clock neurons, delayed evening activity and lengthened the period of their free-running rhythms. Furthermore, nuclear translocation of the clock protein Period was significantly delayed on the expression of a dominant-negative form of p38b in Drosophila's most important clock neurons. Western Blots revealed that p38 affects the phosphorylation degree of Period, what is likely the reason for its effects on nuclear entry of Period. In vitro kinase assays confirmed our Western Blot results and point to p38 as a potential "clock kinase" phosphorylating Period. Taken together, our findings indicate that the p38 MAP Kinase is an integral component of the core circadian clock of Drosophila in addition to playing a role in stress-input pathways.
Subject(s)
Circadian Clocks/genetics , Drosophila melanogaster/physiology , Motor Activity/genetics , p38 Mitogen-Activated Protein Kinases/genetics , Animals , Circadian Clocks/physiology , Circadian Rhythm/physiology , Darkness , Drosophila melanogaster/genetics , Light , Motor Activity/physiology , Neurons/metabolism , Neurons/physiology , Phosphorylation , Stress, Physiological/geneticsABSTRACT
The final size of the central nervous system is determined by precisely controlled generation, proliferation and death of neural stem cells. We show here that the Drosophila PAK protein Mushroom bodies tiny (Mbt) is expressed in central brain progenitor cells (neuroblasts) and becomes enriched to the apical cortex of neuroblasts in a cell cycle- and Cdc42-dependent manner. Using mushroom body neuroblasts as a model system, we demonstrate that in the absence of Mbt function, neuroblasts and their progeny are correctly specified and are able to generate different neuron subclasses as in the wild type, but are impaired in their proliferation activity throughout development. In general, loss of Mbt function does not interfere with establishment or maintenance of cell polarity, orientation of the mitotic spindle and organization of the actin or tubulin cytoskeleton in central brain neuroblasts. However, we show that mbt mutant neuroblasts are significantly reduced in cell size during different stages of development, which is most pronounced for mushroom body neuroblasts. This phenotype correlates with reduced mitotic activity throughout development. Additionally, postembryonic neuroblasts are lost prematurely owing to apoptosis. Yet, preventing apoptosis did not rescue the loss of neurons seen in the adult mushroom body of mbt mutants. From these results, we conclude that Mbt is part of a regulatory network that is required for neuroblast growth and thereby allows proper proliferation of neuroblasts throughout development.
Subject(s)
Cell Proliferation , Drosophila Proteins/metabolism , Drosophila/cytology , Gene Expression Regulation, Developmental , Neural Stem Cells/enzymology , Protein Kinases/metabolism , Actin Cytoskeleton/genetics , Actin Cytoskeleton/metabolism , Animals , Apoptosis , Asymmetric Cell Division , Binding Sites , Brain/cytology , Brain/enzymology , Cell Count , Cell Polarity , Cell Size , Drosophila/embryology , Drosophila/enzymology , Drosophila Proteins/genetics , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/enzymology , Enzyme Activation , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Larva/cytology , Larva/enzymology , Mitosis , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Neural Stem Cells/cytology , Phenotype , Protein Kinases/genetics , Spindle Apparatus/genetics , Spindle Apparatus/metabolism , Tubulin/genetics , Tubulin/metabolismABSTRACT
Phosphorylation is a pivotal regulatory mechanism for protein stability and activity in circadian clocks regardless of their evolutionary origin. It determines the speed and strength of molecular oscillations by acting on transcriptional activators and their repressors, which form negative feedback loops. In Drosophila, the CK2 kinase phosphorylates and destabilizes the PERIOD (PER) and TIMELESS (TIM) proteins, which inhibit CLOCK (CLK) transcriptional activity. Here we show that CK2 also targets the CLK activator directly. Downregulating the activity of the catalytic α subunit of CK2 induces CLK degradation, even in the absence of PER and TIM. Unexpectedly, the regulatory ß subunit of the CK2 holoenzyme is not required for the regulation of CLK stability. In addition, downregulation of CK2α activity decreases CLK phosphorylation and increases per and tim transcription. These results indicate that CK2 inhibits CLK degradation while reducing its activity. Since the CK1 kinase promotes CLK degradation, we suggest that CLK stability and transcriptional activity result from counteracting effects of CK1 and CK2.
Subject(s)
CLOCK Proteins/metabolism , Circadian Rhythm/physiology , Drosophila Proteins/metabolism , Animals , CLOCK Proteins/genetics , Circadian Rhythm/genetics , Drosophila , Drosophila Proteins/genetics , PhosphorylationABSTRACT
BACKGROUND: Myc proteins are essential regulators of animal growth during normal development, and their deregulation is one of the main driving factors of human malignancies. They function as transcription factors that (in vertebrates) control many growth- and proliferation-associated genes, and in some contexts contribute to global gene regulation. RESULTS: We combine chromatin immunoprecipitation-sequencing (ChIPseq) and RNAseq approaches in Drosophila tissue culture cells to identify a core set of less than 500 Myc target genes, whose salient function resides in the control of ribosome biogenesis. Among these genes we find the non-coding snoRNA genes as a large novel class of Myc targets. All assayed snoRNAs are affected by Myc, and many of them are subject to direct transcriptional activation by Myc, both in Drosophila and in vertebrates. The loss of snoRNAs impairs growth during normal development, whereas their overexpression increases tumor mass in a model for neuronal tumors. CONCLUSIONS: This work shows that Myc acts as a master regulator of snoRNP biogenesis. In addition, in combination with recent observations of snoRNA involvement in human cancer, it raises the possibility that Myc's transforming effects are partially mediated by this class of non-coding transcripts.
Subject(s)
Drosophila melanogaster/genetics , Proto-Oncogene Proteins c-myc/metabolism , RNA, Small Nucleolar/metabolism , Animals , Binding Sites/genetics , Cell Line, Tumor , Drosophila melanogaster/growth & development , Gene Expression Regulation, Developmental , Genes, Insect , Humans , Models, Biological , Promoter Regions, Genetic/genetics , Protein Binding/genetics , RNA, Small Nucleolar/genetics , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Ribosomes/metabolism , Vertebrates/geneticsABSTRACT
The transcription factor SPT5 physically interacts with MYC oncoproteins and is essential for efficient transcriptional activation of MYC targets in cultured cells. Here, we use Drosophila to address the relevance of this interaction in a living organism. Spt5 displays moderate synergy with Myc in fast proliferating young imaginal disc cells. During later development, Spt5-knockdown has no detectable consequences on its own, but strongly enhances eye defects caused by Myc overexpression. Similarly, Spt5-knockdown in larval type 2 neuroblasts has only mild effects on brain development and survival of control flies, but dramatically shrinks the volumes of experimentally induced neuroblast tumors and significantly extends the lifespan of tumor-bearing animals. This beneficial effect is still observed when Spt5 is knocked down systemically and after tumor initiation, highlighting SPT5 as a potential drug target in human oncology.
Subject(s)
Brain Neoplasms , Drosophila , Animals , Humans , Brain/metabolism , Brain Neoplasms/genetics , Drosophila/genetics , Drosophila/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Transcriptional Elongation Factors/metabolismABSTRACT
The orientation of the mitotic spindle relative to the cell axis determines whether polarized cells undergo symmetric or asymmetric divisions. Drosophila epithelial cells and neuroblasts provide an ideal pair of cells to study the regulatory mechanisms involved. Epithelial cells divide symmetrically, perpendicular to the apical-basal axis. In the asymmetric divisions of neuroblasts, by contrast, the spindle reorients parallel to that axis, leading to the unequal distribution of cell-fate determinants to one daughter cell. Receptor-independent G-protein signalling involving the GoLoco protein Pins is essential for spindle orientation in both cell types. Here, we identify Mushroom body defect (Mud) as a downstream effector in this pathway. Mud directly associates and colocalizes with Pins at the cell cortex overlying the spindle pole(s) in both neuroblasts and epithelial cells. The cortical Mud protein is essential for proper spindle orientation in the two different division modes. Moreover, Mud localizes to centrosomes during mitosis independently of Pins to regulate centrosomal organization. We propose that Drosophila Mud, vertebrate NuMA and Caenorhabditis elegans Lin-5 (refs 5, 6) have conserved roles in the mechanism by which G-proteins regulate the mitotic spindle.
Subject(s)
Centrosome , Drosophila Proteins/metabolism , Drosophila Proteins/physiology , Guanine Nucleotide Dissociation Inhibitors/metabolism , Membrane Proteins/physiology , Nerve Tissue Proteins/physiology , Spindle Apparatus , Animals , Cell Cycle Proteins , Cell Polarity , Drosophila , Epithelial Cells/cytology , GTP-Binding Proteins/physiology , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Neurons/cytology , Protein BindingABSTRACT
Endogenous clocks enable organisms to adapt cellular processes, physiology, and behavior to daily variation in environmental conditions. Metabolic processes in cyanobacteria to humans are under the influence of the circadian clock, and dysregulation of the circadian clock causes metabolic disorders. In mouse and Drosophila, the circadian clock influences translation of factors involved in ribosome biogenesis and synchronizes protein synthesis. Notably, nutrition signals are mediated by the insulin receptor/target of rapamycin (InR/TOR) pathways to regulate cellular metabolism and growth. However, the role of the circadian clock in Drosophila brain development and the potential impact of clock impairment on neural circuit formation and function is less understood. Here we demonstrate that changes in light stimuli or disruption of the molecular circadian clock cause a defect in neural stem cell growth and proliferation. Moreover, we show that disturbed cell growth and proliferation are accompanied by reduced nucleolar size indicative of impaired ribosomal biogenesis. Further, we define that light and clock independently affect the InR/TOR growth regulatory pathway due to the effect on regulators of protein biosynthesis. Altogether, these data suggest that alterations in InR/TOR signaling induced by changes in light conditions or disruption of the molecular clock have an impact on growth and proliferation properties of neural stem cells in the developing Drosophila brain.
ABSTRACT
Vocalization is an important part of social communication, not only for humans but also for mice. Here, we show in a mouse model that functional deficiency of Sprouty-related EVH1 domain-containing 2 (SPRED2), a protein ubiquitously expressed in the brain, causes differences in social ultrasound vocalizations (USVs), using an uncomplicated and reliable experimental setting of a short meeting of two individuals. SPRED2 mutant mice show an OCD-like behaviour, accompanied by an increased release of stress hormones from the hypothalamic-pituitary-adrenal axis, both factors probably influencing USV usage. To determine genotype-related differences in USV usage, we analyzed call rate, subtype profile, and acoustic parameters (i.e., duration, bandwidth, and mean peak frequency) in young and old SPRED2-KO mice. We recorded USVs of interacting male and female mice, and analyzed the calls with the deep-learning DeepSqueak software, which was trained to recognize and categorize the emitted USVs. Our findings provide the first classification of SPRED2-KO vs. wild-type mouse USVs using neural networks and reveal significant differences in their development and use of calls. Our results show, first, that simple experimental settings in combination with deep learning are successful at identifying genotype-dependent USV usage and, second, that SPRED2 deficiency negatively affects the vocalization usage and social communication of mice.
ABSTRACT
Parkinson's disease (PD) provokes bradykinesia, resting tremor, rigidity and postural instability, and also non-motor symptoms such as depression, anxiety, sleep and cognitive impairments. Similar phenotypes can be induced in Drosophila melanogaster through modification of PD-relevant genes or the administration of PD-inducing toxins. Recent studies correlated deregulation of human p21-activated kinase 4 (PAK4) with PD, leaving open the question of a causative relationship of mutations in this gene for manifestation of PD symptoms. To determine whether flies lacking the PAK4 homolog Mushroom bodies tiny (Mbt) show PD-like phenotypes, we tested for a variety of PD criteria. Here, we demonstrate that mbt mutant flies show PD-like phenotypes including age-dependent movement deficits, reduced life expectancy and fragmented sleep. They also react to a stressful situation with higher immobility, indicating an influence of Mbt on emotional behavior. Loss of Mbt function has a negative effect on the number of dopaminergic protocerebral anterior medial (PAM) neurons, most likely caused by a proliferation defect of neural progenitors. The age-dependent movement deficits are not accompanied by a corresponding further loss of PAM neurons. Previous studies highlighted the importance of a small PAM subgroup for age-dependent PD motor impairments. We show that impaired motor skills are caused by a lack of Mbt in this PAM subgroup. In addition, a broader re-expression of Mbt in PAM neurons improves life expectancy. Conversely, selective Mbt knockout in the same cells shortens lifespan. We conclude that mutations in Mbt/PAK4 can play a causative role in the development of PD phenotypes.
Subject(s)
Parkinson Disease/genetics , Phenotype , p21-Activated Kinases/genetics , Animals , Drosophila/physiology , Gene Knockdown Techniques , Life Expectancy , Motor Activity/genetics , Neurons/physiology , Sleep/geneticsABSTRACT
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.
Subject(s)
Casein Kinase II/metabolism , Circadian Rhythm/physiology , Gene Expression Regulation/physiology , Periodicity , Ribosomal Protein S6 Kinases/metabolism , Animals , Animals, Genetically Modified , Casein Kinase II/genetics , Cell Line, Transformed , Circadian Rhythm/genetics , Drosophila , Drosophila Proteins/genetics , Gene Expression Regulation/genetics , Humans , Motor Activity/genetics , Mutation/genetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Period Circadian Proteins , RNA Interference/physiology , RNA, Messenger/metabolism , Ribosomal Protein S6 Kinases/genetics , TransfectionABSTRACT
The molecular heterogeneity of human cancer cells at the level of signaling protein activities remains poorly understood. Using a panel of 64 colorectal (CRC) cancer cell lines the activity status of the MAP kinases Erk1 and Erk2 was investigated. Erk1/2 activity varied greatly within the CRC cell line panel and was not detectably associated with the speed of cell growth in 10 CRC lines analyzed. As expected, mutations in K-Ras or B-Raf were often, albeit not always, linked to high Erk1/2 activity. The phosphorylation of several known Erk1/2 targets investigated did not generally reflect Erk1/2 activity in the 10 CRC lines analyzed. However, the reduction of Erk1/2 activity with MEK inhibitors generally abolished cell growth but only led to an increase of cellular p27Kip1 levels in CRC cells with high Erk1/2 activity levels. The results indicate that high Erk1/2 activation is utilized by some CRC lines to override the cell cycle brake p27Kip1, while others presumably rely on different mechanisms in order to inactivate this important cell cycle brake. Such detailed knowledge of the molecular diversity of cancer cell signaling mechanisms may eventually help to develop molecularly targeted, patient-specific therapeutic strategies and treatments.
ABSTRACT
The role of CK2beta has been defined as the regulatory subunit of protein kinase CK2, which is a heterotetrameric complex composed of two CK2beta and two catalytic active CK2alpha subunits. The identification of other serine/threonine kinases such as A-Raf, Chk1, and c-Mos that interact with and are regulated by CK2beta has challenged this view and provided evidence for functions of CK2beta outside the CK2 holoenzyme. In this report we describe the first interaction of Drosophila CK2beta outside the CK2 holoenzyme with p21-activated kinase (PAK) proteins. This interaction is seen for distinct PAK and CK2beta isoforms. In contrast to the CK2alpha-CK2beta interaction, dimer formation of the CK2beta subunits is not a prerequisite for binding of PAK proteins. Our results support the idea that CK2beta can bind to PAK proteins in a CK2alpha independent manner and negatively regulates PAK kinase activity.
Subject(s)
Casein Kinase II/metabolism , Drosophila melanogaster/enzymology , p21-Activated Kinases/metabolism , Animals , Casein Kinase II/genetics , Protein Interaction MappingABSTRACT
Single-gene mutants of Drosophila have not only increased our understanding of the biochemical processes underlying learning and memory processes, but also established structure-function relationships. The first relevant mutants were identified by Martin Heisenberg nearly 30 years ago in a screen for altered adult brain structure and were used to link the mushroom bodies in the central brain with olfactory learning and memory processes. Because the observed structural defects in the adult are the consequence of deregulated developmental processes, the characterization of these mutants can also provide insight into the genetic programs underlying the establishment, maintenance, and remodeling of functional neuronal circuits. As an example for the value of this approach, we trace the history of mushroom body defect (mud), from the original anatomical description of the mutation to most recent insights of the function of the protein as a regulator of neuronal progenitor cell division.
Subject(s)
Brain/embryology , Drosophila Proteins/genetics , Drosophila/genetics , Membrane Proteins/genetics , Mushroom Bodies/embryology , Mutation , Nerve Tissue Proteins/genetics , Animals , Brain/cytology , Cell Division/genetics , Drosophila Proteins/physiology , Genes, Insect , Membrane Proteins/physiology , Mushroom Bodies/cytology , Nerve Tissue Proteins/physiology , Neurogenesis/genetics , PhenotypeABSTRACT
Phosphorylation by tyrosine and serine/threonine kinases regulate the interactions between components of the cadherin-catenin cell-adhesion complex and thus can influence the dynamic modulation of cell adhesion under normal and disease conditions. Previous mutational analysis and localization experiments suggested an involvement of single members of the family of PAKs (p21-activated kinases) in the regulation of cadherin-mediated cell adhesion, but the molecular mechanism remained elusive. In the present study, we address this question using the Drosophila PAK protein Mbt, which is most similar to vertebrate PAK4. Previous phenotypic analysis showed that Mbt has a function to maintain adherens junctions during eye development and indicated a requirement of the protein in regulation of the actin cytoskeleton and the cadherin-catenin complex. Here we show that activation of Mbt leads to destabilization of the interaction of the Drosophila beta-catenin homologue Armadillo with DE-cadherin resulting in a decrease in DE-cadherin-mediated adhesion. Two conserved phosphorylation sites in Armadillo were identified that mediate this effect. The findings of the present study support the previous observation that activation of the human Mbt homologue PAK4 leads to anchorage-independent growth and provide a functional link between a PAK protein and the cadherin-catenin complex.
Subject(s)
Armadillo Domain Proteins/physiology , Drosophila Proteins/metabolism , Drosophila Proteins/physiology , Drosophila melanogaster/physiology , Protein Kinases/metabolism , Transcription Factors/physiology , p21-Activated Kinases/metabolism , Animals , Cadherins/genetics , Cell Line , Cloning, Molecular , Humans , Kidney , Phosphorylation , p21-Activated Kinases/physiologyABSTRACT
The p21 activated kinase (Pak) family of protein kinases are involved in many cellular functions like re-organisation of the cytoskeleton, transcriptional control, cell division, and survival. These pleiotropic actions are reflected in a plethora of known interacting proteins and phosphorylation substrates. Yet, the integration of a single Pak protein into signalling pathways controlling a particular developmental process are less well studied. For two of the three known Pak proteins in Drosophila melanogaster, D-Pak and Mbt, distinct functions during eye development have been established. In this study we undertook a genetic approach to identify proteins acting in the Mbt signalling pathway during photoreceptor cell morphogenesis. The genetic screen identified the actin depolymerisation factor Twinstar/Cofilin as one target of Mbt signalling. Twinstar/Cofilin becomes phosphorylated upon activation of Mbt. However, biochemical and genetic experiments question the role of the LIM domain protein kinase (Limk) as a major link between Mbt and Twinstar/Cofilin as it has been suggested for other PAK proteins. Constitutive activation of Mbt not only disturbs the actin cytoskeleton but also affects adherens junction organisation indicating a requirement of the protein in cell adhesion dependent processes during photoreceptor cell differentiation.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/growth & development , Drosophila melanogaster/metabolism , Photoreceptor Cells, Invertebrate/metabolism , Protein Kinases/metabolism , Actins/metabolism , Adherens Junctions/metabolism , Animals , Animals, Genetically Modified , Base Sequence , Cell Differentiation , Cell Line , Cytoskeleton/metabolism , DNA/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/cytology , Drosophila melanogaster/genetics , Eye/cytology , Eye/growth & development , Eye/metabolism , Genes, Insect , Humans , Lim Kinases , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Microscopy, Electron, Scanning , Mutagenesis, Site-Directed , Mutation , Phenotype , Phosphorylation , Photoreceptor Cells, Invertebrate/cytology , Photoreceptor Cells, Invertebrate/growth & development , Protein Kinases/genetics , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction , Transfection , p21-Activated KinasesABSTRACT
The posttranslational modification of clock proteins is critical for the function of circadian oscillators. By genetic analysis of a Drosophila melanogaster circadian clock mutant known as Andante, which has abnormally long circadian periods, we show that casein kinase 2 (CK2) has a role in determining period length. Andante is a mutation of the gene encoding the beta subunit of CK2 and is predicted to perturb CK2beta subunit dimerization. It is associated with reduced beta subunit levels, indicative of a defect in alpha:beta association and production of the tetrameric alpha2:beta2 holoenzyme. Consistent with a direct action on the clock mechanism, we show that CK2beta is localized within clock neurons and that the clock proteins Period (Per) and Timeless (Tim) accumulate to abnormally high levels in the Andante mutant. Furthermore, the nuclear translocation of Per and Tim is delayed in Andante, and this defect accounts for the long-period phenotype of the mutant. These results suggest a function for CK2-dependent phosphorylation in the molecular oscillator.
Subject(s)
Biological Clocks/physiology , Circadian Rhythm/physiology , Drosophila Proteins , Drosophila melanogaster/physiology , Protein Serine-Threonine Kinases/physiology , Active Transport, Cell Nucleus/physiology , Animals , Casein Kinase II , Dimerization , Insect Proteins/metabolism , Motor Activity/physiology , Mutation , Neurons/cytology , Neurons/metabolism , Nuclear Proteins/metabolism , Period Circadian Proteins , Phenotype , Protein Binding/physiology , Protein Serine-Threonine Kinases/genetics , Protein Subunits/genetics , Protein Subunits/physiologyABSTRACT
Loss of function mutations in the rsk2 gene cause Coffin-Lowry syndrome (CLS), which is associated with multiple symptoms including severe mental disabilities. Despite the characterization of ribosomal S6 kinase 2 (RSK2) as a protein kinase acting as a downstream effector of the well characterized ERK MAP-kinase signaling pathway, it turns out to be a challenging task to link RSK2 to specific neuronal processes dysregulated in case of mutation. Animal models such as mouse and Drosophila combine advanced genetic manipulation tools with in vivo imaging techniques, high-resolution connectome analysis and a variety of behavioral assays, thereby allowing for an in-depth analysis for gene functions in the nervous system. Although modeling mental disability in animal systems has limitations because of the complexity of phenotypes, the influence of genetic variation and species-specific characteristics at the neural circuit and behavioral level, some common aspects of RSK2 function in the nervous system have emerged, which will be presented. Only with this knowledge our understanding of the pathophysiology of CLS can be improved, which might open the door for development of potential intervention strategies.
ABSTRACT
Endogenous molecular circadian clocks drive daily rhythmic changes at the cellular, physiological, and behavioral level for adaptation to and anticipation of environmental signals. The core molecular system consists of autoregulatory feedback loops, where clock proteins inhibit their own transcription. A complex and not fully understood interplay of regulatory proteins influences activity, localization and stability of clock proteins to set the pace of the clock. This study focuses on the molecular function of Ribosomal S6 Kinase (RSK) in the Drosophila melanogaster circadian clock. Mutations in the human rsk2 gene cause Coffin-Lowry syndrome, which is associated with severe mental disabilities. Knock-out studies with Drosophila ortholog rsk uncovered functions in synaptic processes, axonal transport and adult behavior including associative learning and circadian activity. However, the molecular targets of RSK remain elusive. Our experiments provide evidence that RSK acts in the key pace maker neurons as a negative regulator of Shaggy (SGG) kinase activity, which in turn determines timely nuclear entry of the clock proteins Period and Timeless to close the negative feedback loop. Phosphorylation of serine 9 in SGG is mediated by the C-terminal kinase domain of RSK, which is in agreement with previous genetic studies of RSK in the circadian clock but argues against the prevailing view that only the N-terminal kinase domain of RSK proteins carries the effector function. Our data provide a mechanistic explanation how RSK influences the molecular clock and imply SGG S9 phosphorylation by RSK and other kinases as a convergence point for diverse cellular and external stimuli.
ABSTRACT
Genes encoding for the two evolutionary highly conserved subunits of a heterotetrameric protein kinase CK2 holoenzyme are present in all examined eukaryotic genomes. Depending on the organism, multiple transcription units encoding for a catalytically active CK2alpha subunit and/or a regulatory CK2beta subunit may exist. The phosphotransferase activity of members of the protein kinase CK2alpha family is thought to be independent of second messengers but is modulated by interaction with CK2beta-like proteins. In the genome of Drosophila melanogaster, one gene encoding for a CK2alpha subunit and three genes encoding for CK2beta-like proteins are present. The X-linked DmCK2beta transcription unit encodes for several CK2beta protein isoforms due to alternative splicing of its primary transcript. We addressed the question whether CK2beta-like proteins are redundant in function. Our in vivo experiments show that variations of the very C-terminal tail of CK2beta isoforms encoded by the X-linked DmCK2beta transcription unit influence their functional properties. In addition, we find that CK2beta-like proteins encoded by the autosomal D. melanogaster genes CK2betates and CK2beta' cannot fully substitute for a loss of CK2beta isoforms encoded by DmCK2beta.