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• W2023651881 abstract "The circadian clock facilitates a temporal coordination of most homeostatic activities and their synchronization with the environmental cycles of day and night. The core oscillating activity of the circadian clock is formed by a heterodimer of the transcription factors CLOCK (CLK) and CYCLE (CYC). Post-translational regulation of CLK/CYC has previously been shown to be crucial for clock function and accurate timing of circadian transcription. Here we report that a sequential and compartment-specific phosphorylation of the Drosophila CLK protein assigns specific localization and activity patterns. Total and nuclear amounts of CLK protein were found to oscillate over the course of a day in circadian neurons. Detailed analysis of the cellular distribution and phosphorylation of CLK revealed that newly synthesized CLK is hypophosphorylated in the cytoplasm prior to nuclear import. In the nucleus, CLK is converted into an intermediate phosphorylation state that correlates with trans-activation of circadian transcription. Hyperphosphorylation and degradation are promoted by nuclear export of the CLK protein. Surprisingly, CLK localized to discrete nuclear foci in cell culture as well as in circadian neurons of the larval brain. These subnuclear sites likely contain a storage form of the transcription factor, while homogeneously distributed nuclear CLK appears to be the transcriptionally active form. These results show that sequential post-translational modifications and subcellular distribution regulate the activity of the CLK protein, indicating a core post-translational timing mechanism of the circadian clock. The circadian clock facilitates a temporal coordination of most homeostatic activities and their synchronization with the environmental cycles of day and night. The core oscillating activity of the circadian clock is formed by a heterodimer of the transcription factors CLOCK (CLK) and CYCLE (CYC). Post-translational regulation of CLK/CYC has previously been shown to be crucial for clock function and accurate timing of circadian transcription. Here we report that a sequential and compartment-specific phosphorylation of the Drosophila CLK protein assigns specific localization and activity patterns. Total and nuclear amounts of CLK protein were found to oscillate over the course of a day in circadian neurons. Detailed analysis of the cellular distribution and phosphorylation of CLK revealed that newly synthesized CLK is hypophosphorylated in the cytoplasm prior to nuclear import. In the nucleus, CLK is converted into an intermediate phosphorylation state that correlates with trans-activation of circadian transcription. Hyperphosphorylation and degradation are promoted by nuclear export of the CLK protein. Surprisingly, CLK localized to discrete nuclear foci in cell culture as well as in circadian neurons of the larval brain. These subnuclear sites likely contain a storage form of the transcription factor, while homogeneously distributed nuclear CLK appears to be the transcriptionally active form. These results show that sequential post-translational modifications and subcellular distribution regulate the activity of the CLK protein, indicating a core post-translational timing mechanism of the circadian clock. 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.Yu W. Hardin P.E. J. Cell Sci. 2006; 119: 4793-4795Crossref PubMed Scopus (95) Google Scholar, 2.Weber F. Naturwissenschaften. 2009; 96: 321-337Crossref PubMed Scopus (18) Google Scholar, 3.Hung H.C. Kay S.A. Weber F. J. Biol. Rhythms. 2009; 24: 183-192Crossref PubMed Scopus (18) Google Scholar). The central oscillating activity of the Drosophila and mammalian circadian clock is formed by the heterodimeric complex of the transcription factors CLOCK (CLK) 2The abbreviations used are: CLKCLOCK transcription factorCYCCYCLE transcription factorNGSnormal goat serumTRITCtetramethylrhodamine isothiocyanateLNlateral neuronZTZeitgeber TimeLDlight and darknessS2Schneider 2NLSnuclear localization signalbHLHbasic helix-loop-helixNESnuclear export signalPDFpigment dispersing factorPAPPDF-associated peptide.2The abbreviations used are: CLKCLOCK transcription factorCYCCYCLE transcription factorNGSnormal goat serumTRITCtetramethylrhodamine isothiocyanateLNlateral neuronZTZeitgeber TimeLDlight and darknessS2Schneider 2NLSnuclear localization signalbHLHbasic helix-loop-helixNESnuclear export signalPDFpigment dispersing factorPAPPDF-associated peptide. and CYCLE (CYC) that ultimately controls genome-wide transcription and activity states of key regulatory components (4.McDonald M.J. Rosbash M. Cell. 2001; 107: 567-578Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 5.Claridge-Chang A. Wijnen H. Naef F. Boothroyd C. Rajewsky N. Young M.W. Neuron. 2001; 32: 657-671Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar, 6.Akhtar R.A. Reddy A.B. Maywood E.S. Clayton J.D. King V.M. Smith A.G. Gant T.W. Hastings M.H. Kyriacou C.P. Curr. Biol. 2002; 12: 540-550Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar, 7.Panda S. Antoch M.P. Miller B.H. Su A.I. Schook A.B. Straume M. Schultz P.G. Kay S.A. Takahashi J.S. Hogenesch J.B. Cell. 2002; 109: 307-320Abstract Full Text Full Text PDF PubMed Scopus (1872) Google Scholar). Importantly, the circadian oscillator is synchronized by environmental cycles, primarily light/dark and temperature cycles (8.Stanewsky R. Kaneko M. Emery P. Beretta B. Wager-Smith K. Kay S.A. Rosbash M. Hall J.C. Cell. 1998; 95: 681-692Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar, 9.Glaser F.T. Stanewsky R. Curr. Biol. 2005; 15: 1352-1363Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), but keeps time on its own in the absence of environmental cues, therefore representing a true molecular clock. CLOCK transcription factor CYCLE transcription factor normal goat serum tetramethylrhodamine isothiocyanate lateral neuron Zeitgeber Time light and darkness Schneider 2 nuclear localization signal basic helix-loop-helix nuclear export signal pigment dispersing factor PDF-associated peptide. CLOCK transcription factor CYCLE transcription factor normal goat serum tetramethylrhodamine isothiocyanate lateral neuron Zeitgeber Time light and darkness Schneider 2 nuclear localization signal basic helix-loop-helix nuclear export signal pigment dispersing factor PDF-associated peptide. 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.Weber F. Naturwissenschaften. 2009; 96: 321-337Crossref PubMed Scopus (18) Google Scholar, 10.Gallego M. Virshup D.M. Nat. Rev. Mol. Cell Biol. 2007; 8: 139-148Crossref PubMed Scopus (623) Google Scholar, 11.Bae K. Edery I. J. Biochem. 2006; 140: 609-617Crossref PubMed Scopus (57) Google Scholar). 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.Cyran S.A. Buchsbaum A.M. Reddy K.L. Lin M.C. Glossop N.R. Hardin P.E. Young M.W. Storti R.V. Blau J. Cell. 2003; 112: 329-341Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar, 13.Glossop N.R. Houl J.H. Zheng H. Ng F.S. Dudek S.M. Hardin P.E. Neuron. 2003; 37: 249-261Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 14.Blau J. Young M.W. Cell. 1999; 99: 661-671Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). Rhythmic Clk transcription appears however not essential for self-sustained molecular oscillations, because expression of CLK from a per-promoter in anti-phase to its endogenous rhythm was found to support normal clock function (15.Kim E.Y. Bae K. Ng F.S. Glossop N.R. Hardin P.E. Edery I. Neuron. 2002; 34: 69-81Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The post-translational regulation of CLK/CYC is however crucial for constitution of a circadian oscillator. Two CLK/CYC-activated genes period (per) and timeless (tim) feed back onto CLK/CYC activity, by binding and inhibition of CLK/CYC (16.Darlington T.K. Wager-Smith K. Ceriani M.F. Staknis D. Gekakis N. Steeves T.D. Weitz C.J. Takahashi J.S. Kay S.A. Science. 1998; 280: 1599-1603Crossref PubMed Scopus (693) Google Scholar). Nucleocytoplasmic shuttling of TIM (17.Ashmore L.J. Sathyanarayanan S. Silvestre D.W. Emerson M.M. Schotland P. Sehgal A. J. Neurosci. 2003; 23: 7810-7819Crossref PubMed Google Scholar) allows a temporal control of nuclear import and inhibitor activity of PER (18.Sathyanarayanan S. Zheng X. Xiao R. Sehgal A. Cell. 2004; 116: 603-615Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 19.Fang Y. Sathyanarayanan S. Sehgal A. Genes Dev. 2007; 21: 1506-1518Crossref PubMed Scopus (106) Google Scholar, 20.Saez L. Young M.W. Neuron. 1996; 17: 911-920Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 21.Meyer P. Saez L. Young M.W. Science. 2006; 311: 226-229Crossref PubMed Scopus (143) Google Scholar). PER-mediated inhibition of CLK/CYC (22.Weber F. Kay S.A. FEBS Lett. 2003; 555: 341-345Crossref PubMed Scopus (18) Google Scholar, 23.Rothenfluh A. Young M.W. Saez L. Neuron. 2000; 26: 505-514Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) involves the recruitment of the casein kinase Iϵ homolog DOUBLE-TIME, which contributes to phosphorylation-induced inhibition and degradation of the CLK protein (24.Yu W. Zheng H. Houl J.H. Dauwalder B. Hardin P.E. Genes Dev. 2006; 20: 723-733Crossref PubMed Scopus (177) Google Scholar, 25.Kim E.Y. Edery I. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 6178-6183Crossref PubMed Scopus (107) Google Scholar). Activation of CLK/CYC is affected by calcium/calmodulin-dependent kinase II and mitogen-activated protein kinase, which both phosphorylate CLK in vitro (26.Weber F. Hung H.C. Maurer C. Kay S.A. J. Neurochem. 2006; 98: 248-257Crossref PubMed Scopus (46) Google Scholar). Phosphorylation of CLK may also control recruitment of the transcription co-activator CREB-binding protein to the CLK/CYC complex, which is essential for trans-activation by CLK/CYC (27.Hung H.C. Maurer C. Kay S.A. Weber F. J. Biol. Chem. 2007; 282: 31349-31357Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 28.Etchegaray J.P. Lee C. Wade P.A. Reppert S.M. Nature. 2003; 421: 177-182Crossref PubMed Scopus (540) Google Scholar). These studies suggest a fundamental role of post-translational modifications for the temporal regulation of CLK/CYC-dependent transcription. Here we show that sequential and compartment-specific phosphorylation controls the life cycle of the CLK protein. Newly synthesized CLK accumulates in the cytoplasm in a hypophosphorylated state. Subsequent nuclear translocation converts the CLK protein into an intermediately phosphorylated form. Interestingly, we found that CLK is stored in subnuclear compartments, and homogeneously distributed CLK protein represents a transcriptionally active species. Hyperphosphorylation as well as degradation of CLK are promoted by nuclear export of the transcription factor. The data reveal specific localization and activity patterns for the phosphorylation states of the CLK protein that oscillate over the course of a day (29.Lee C. Bae K. Edery I. Neuron. 1998; 21: 857-867Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). CLK proteins were expressed from a pAc5.1/V5-HisA vector (Invitrogen) either without any tag (pAc-Clk) for Western blot analysis and transcription activation assays as described in a previous study (26.Weber F. Hung H.C. Maurer C. Kay S.A. J. Neurochem. 2006; 98: 248-257Crossref PubMed Scopus (46) Google Scholar, 27.Hung H.C. Maurer C. Kay S.A. Weber F. J. Biol. Chem. 2007; 282: 31349-31357Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), or as fusion proteins with a N-terminal DsRed1 red fluorescent protein tag (pDsRed-Clk) for fluorescence microscopy (30.Maurer C. Hung H.C. Weber F. FEBS Lett. 2009; 583: 1561-1566Crossref PubMed Scopus (7) Google Scholar). Mutations and deletions were inserted into these constructs with the QuikChange II XL system (Stratagene, La Jolla, CA) according to the instructions by the manufacturer and using the following primers: CLKNLS (substitution of Pro-484 to Leu, Lys-485 to Glu, and Lys-487 to Glu; fwd, CCA CAG GAA TAT CGC TCG AGG CCG AAC GAA AGT GC; rev, GCA CTT TCG TTC GGC CTC GAG CGA TAT TCC TGT GG), CLKΔNES (deletion of 25 amino acids from Gln-836 to Leu-860; fwd, GGC TCG CAA TCC ACC ATT AAT C; rev, TTG TTG CAG CTG CAA CTG TTG CTG). A 25tag-CLKΔNES construct carries an N- terminal 25-amino acid tag (MDYKDDDDKDYKDDDDKDYPRYFQS) that compensates for the change in protein size caused by the NES deletion. For cellular localization studies, DsRed-CLK constructs were expressed in Drosophila S2 cells or S2R+ cells as specified in the figure legends after transient transfection with Lipofectin (Invitrogen) as instructed by the manufacturer. 5 × 105 cells were transfected with 1 or 2 μg of plasmid DNA and 10 μl of Lipofectin, as described previously (16.Darlington T.K. Wager-Smith K. Ceriani M.F. Staknis D. Gekakis N. Steeves T.D. Weitz C.J. Takahashi J.S. Kay S.A. Science. 1998; 280: 1599-1603Crossref PubMed Scopus (693) Google Scholar, 22.Weber F. Kay S.A. FEBS Lett. 2003; 555: 341-345Crossref PubMed Scopus (18) Google Scholar, 26.Weber F. Hung H.C. Maurer C. Kay S.A. J. Neurochem. 2006; 98: 248-257Crossref PubMed Scopus (46) Google Scholar). Cells were cultured in poly-l-lysine (Sigma)-coated cover slide chambers (Nalgene, Rochester, NY). Fluorescence microscopy was performed using an Axiovert 200M (Zeiss, Jena, Germany) fluorescence microscope or a Nikon C1Si-CLEM confocal microscope (Fig. 6) after staining of nuclei with Hoechst 33342 (Invitrogen). At least 60 transfected cells from at least two independent experiments were analyzed per construct. Images were collected with a laser wavelength of 563 nm for DsRed-CLK and 352 nm for Hoechst 33342-stained nuclei. Third instar larvae were raised and collected at time points as indicated in the figure in cycles of 12-h light and 12-h darkness. Larvae were dissected and brains were fixed in 4% formaldehyde in PEM buffer (100 mm Pipes, pH 6.9, 1 mm EGTA, 2 mm magnesium sulfate) for 2 h, blocked with 10% normal goat serum (NGS) in PT buffer (phosphate-buffered saline plus 0.3% Triton X-100) for 2 h at room temperature and subsequently incubated for 48 h at 4 °C in 50 μl of primary antiserum solution containing 1:500-diluted rabbit anti-CLK (27.Hung H.C. Maurer C. Kay S.A. Weber F. J. Biol. Chem. 2007; 282: 31349-31357Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) and 1:200-diluted guinea pig anti-PAP (31.Renn S.C. Park J.H. Rosbash M. Hall J.C. Taghert P.H. Cell. 1999; 99: 791-802Abstract Full Text Full Text PDF PubMed Scopus (863) Google Scholar) in PT buffer with 10% NGS. The rabbit anti-CLK antibody was incubated with ClkJrk protein extract to remove unspecific interactions prior to the application in immunohistochemistry. Brains were rinsed with PT buffer and PT buffer plus 5% NGS for 20 min each and then incubated at room temperature for 2 h in secondary antiserum solution containing 1:200-diluted TRITC-conjugated donkey anti-guinea pig (Jackson Immuno Research, West Grove, PA) and 1:200-diluted Cy2-conjugated goat anti-rabbit (Jackson ImmunoResearch) in PT buffer with 10% NGS. After incubation, brains were rinsed with PT buffer and PT buffer plus 5% NGS for 20 min each and mounted in the mounting medium (50 mm Tris-Cl, pH 8, 90% glycerol, 2.5% DABCO (Sigma)). Optical sections of larval lateral neurons (LNs) were imaged on a Nikon C1Si-CLEM confocal microscope. For each Zeitgeber Time (ZT), LNs from at least 10 brain hemispheres were scanned. For each LN sample, PAP staining was used to identify and select an optical section, which was then scanned for CLK immunoreactivity. A 0.31-μm Z-stack of images was taken per LN with single laser at 488 nm, 543 nm, and with both lasers together. Images were imported to NIH ImageJ 1.34S, and the localization of neuronal PAP and CLK staining was determined. Total pixel intensity of CLK staining was measured for the whole cell or nucleus only and the means ± S.E. from all brains at a particular ZT are reported in the graphs. Transcriptional activation assays in Drosophila S2 and S2R+ cells were performed as described previously (16.Darlington T.K. Wager-Smith K. Ceriani M.F. Staknis D. Gekakis N. Steeves T.D. Weitz C.J. Takahashi J.S. Kay S.A. Science. 1998; 280: 1599-1603Crossref PubMed Scopus (693) Google Scholar, 22.Weber F. Kay S.A. FEBS Lett. 2003; 555: 341-345Crossref PubMed Scopus (18) Google Scholar, 26.Weber F. Hung H.C. Maurer C. Kay S.A. J. Neurochem. 2006; 98: 248-257Crossref PubMed Scopus (46) Google Scholar). In brief, cells from 500 μl of culture (106 cells/ml) were transfected after 12-h incubation at 25 °C with 200 μl of serum-free Schneider's insect medium (Sigma) containing 1.5% Lipofectin (Invitrogen), 25 ng of pRLcopia, 10 ng of pGL3-(4-per-E-box)hs::luc+, 200 ng of pHT control vector, and 0.25 ng of pAc-Clk construct. Identical controls were performed for each condition without pAc-Clk. 200 μl of 20% fetal bovine serum (Invitrogen) in Schneider's insect medium was added 4–6 h after transfection. After 40–48 h luciferase activities were determined using the dual-luciferase reporter assay system (Promega) according to the instructions by the manufacturer. Firefly luciferase activity was normalized toward Renilla luciferase activity to control for transfection efficiency and lysate concentration (16.Darlington T.K. Wager-Smith K. Ceriani M.F. Staknis D. Gekakis N. Steeves T.D. Weitz C.J. Takahashi J.S. Kay S.A. Science. 1998; 280: 1599-1603Crossref PubMed Scopus (693) Google Scholar, 26.Weber F. Hung H.C. Maurer C. Kay S.A. J. Neurochem. 2006; 98: 248-257Crossref PubMed Scopus (46) Google Scholar). w1118 flies were entrained during eclosion in LD cycles of 12-h light and 12-h darkness. Flies, 1–7 days old, were harvested in LD 2 h after lights were on (ZT 2), and 2 h after lights were off (ZT 14). 400–500 μl of fly heads were subjected to subcellular fractionation according to a protocol that was adapted from a previous study (32.Luo C. Loros J.J. Dunlap J.C. EMBO J. 1998; 17: 1228-1235Crossref PubMed Scopus (111) Google Scholar). Fly heads were gently homogenized in liquid nitrogen and transferred to 800 μl of buffer A (1 m sorbitol, 7% Ficoll, 20% glycerol, 50 mm Tris, pH 7.5, 5 mm magnesium acetate, 5 mm EGTA, 3 mm CaCl2, 3 mm dithiothreitol, 1× complete protease inhibitor (Roche Applied Science)). Coarse debris was filtered by passing the homogenate through a nylon mesh. Subsequently, Nonidet P-40 was added to a final concentration of 0.1%, and the mixture was passed through a 20-gauge needle. 1200 μl of ice-cold buffer B (10% glycerol, 25 mm Tris, pH 7.5, 5 mm magnesium acetate, 5 mm EGTA, 1× complete protease inhibitor (Roche Applied Science)) were added during gentle mixing. The mixture was carefully added on top of 300 μl of buffer AB (at a ratio of 1:1.7) and centrifuged at 700 × g for 3 min. 120 μl of the supernatant was collected as total protein fraction, and the rest was layered on top of a sucrose step-gradient (1 m sucrose, 10% glycerol, 25 mm Tris, pH 7.5, 5 mm magnesium acetate, 1 mm dithiothreitol, 1× complete protease inhibitor (Roche Applied Science)). After centrifugation at 9000 × g for 8 min, the nuclear pellet was resuspended in 50 μl of SDS-loading buffer. The supernatant was collected as cytosolic fraction, and proteins were precipitated with trichloroacetic acid (Sigma) and re-dissolved in HU buffer (8 m urea, 200 mm Tris, pH 6.8, 5% SDS, 1 mm EDTA, 100 mm dithiothreitol). CLK proteins were analyzed from fly head extracts or after expression in S2 or S2R+ cells that were transiently transfected as described for fluorescence microscopy with 2 μg of pAc-Clk constructs. After 40–48 h cells were harvested and lysed in 4× SDS loading buffer (240 mm Tris/HCl, pH 6.8, 8% SDS, 20% glycerol) and boiled at 95 °C, and lysates were subjected to SDS-PAGE on 6% gels. CLK protein was analyzed by Western blot using an antibody raised in rabbits against the C terminus of CLK (27.Hung H.C. Maurer C. Kay S.A. Weber F. J. Biol. Chem. 2007; 282: 31349-31357Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). For phosphatase treatment, CLK proteins were similarly expressed in S2R+ cells. Cells were lysed in RBS buffer (10 mm HEPES, pH 7.5, 5 mm Tris, pH 7.5, 50 mm KCl, 10% glycerol, 2 mm EDTA, 1 mm dithiothreitol, 1% Triton X-100, 0.4% Nonidet P-40) (24.Yu W. Zheng H. Houl J.H. Dauwalder B. Hardin P.E. Genes Dev. 2006; 20: 723-733Crossref PubMed Scopus (177) Google Scholar) to which complete protease inhibitor (Roche Applied Science) was added. Proteins were subsequently de-phosphorylated by addition of 25 units of λ-phosphatase (New England Biolabs, Ipswich, MA) to a total of a 50-μl reaction, which was incubated for 1 h at 37 °C. After denaturation by addition of SDS loading buffer and boiling at 95 °C, proteins were analyzed by 6% SDS-PAGE and Western blot. To determine degradation kinetics, S2 cells (2 ml of 106 cells/ml) were transfected as described for fluorescence microscopy with 2 μg of pAc-Clk expression constructs. One day after transfection, cells were split in 24-well plates and incubated for a further 24 h. Subsequently, new protein synthesis was inhibited by the addition of cycloheximide at a final concentration of 260 μm. Cells were harvested over a time course after the addition of cycloheximide, and CLK protein levels were determined by Western blot analysis on 6% SDS-PAGE. CLK protein levels were quantified for different time points by densitometry and normalized toward an unspecific control band, which was detected by the anti-CLK antibody. Amounts of CLK proteins are shown relative to the amount of CLK prior to the addition of cycloheximide (time point 0, set to 100). We first investigated the cellular distribution of the CLK protein in circadian neurons of the larval brain (Fig. 1A). We found that the majority of the CLK protein localizes inside the nucleus (33.Houl J.H. Yu W. Dudek S.M. Hardin P.E. J. Biol. Rhythms. 2006; 21: 93-103Crossref PubMed Scopus (55) Google Scholar). Interestingly, nuclear and total amounts of CLK revealed robust oscillations over the course of a day, with a maximum in the morning and trough levels in the evening (Fig. 1, A and B). These oscillations correlated with the circadian profile of Clk transcription that reaches a maximum toward the end of the night (16.Darlington T.K. Wager-Smith K. Ceriani M.F. Staknis D. Gekakis N. Steeves T.D. Weitz C.J. Takahashi J.S. Kay S.A. Science. 1998; 280: 1599-1603Crossref PubMed Scopus (693) Google Scholar). In addition, phosphorylation states of the CLK protein oscillate in abundance over the course of a day (Fig. 1C) in accord with previous reports (25.Kim E.Y. Edery I. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 6178-6183Crossref PubMed Scopus (107) Google Scholar, 29.Lee C. Bae K. Edery I. Neuron. 1998; 21: 857-867Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 33.Houl J.H. Yu W. Dudek S.M. Hardin P.E. J. Biol. Rhythms. 2006; 21: 93-103Crossref PubMed Scopus (55) Google Scholar). Hypo- and hyperphosphorylated forms of CLK were mainly observed during late night and in the morning, whereas intermediate phosphorylation states were the predominant species in the evening, when CLK/CYC is transcriptionally active. To investigate the cellular localization of CLK in more detail, we expressed CLK with an N-terminal red fluorescent protein (DsRed) fusion tag in Drosophila Schneider 2 (S2) cells and analyzed the localization by fluorescence microscopy. The Drosophila CLK sequence reveals two consensus nuclear localization signals (NLSs) (Fig. 2A). The first consensus NLS is located in the N terminus at the beginning of the basic helix-loop-helix (bHLH) DNA-binding domain. Although this region is conserved in mammalian CLK orthologs, mutagenesis of this site did not affect the subcellular localization of the CLK protein, indicating that the consensus NLS in the bHLH domain is not involved in nuclear translocation (data not shown). A second consensus NLS located C-terminal of the PAS domains is unique to the Drosophila CLK protein. Rendering this site non-functional by mutagenesis (Fig. 2A) caused a cytoplasmic localization of the CLKNLS mutant protein in S2 cells (Fig. 2B). These results show that the consensus NLS located C-terminal of the PAS domains is a functional nuclear translocation site and essential for nuclear import of the CLK protein. Interestingly, nuclear import-deficient CLKNLS localized largely in cytoplasmic speckles (Fig. 2B). These speckles were also observed for non-tagged CLKNLS mutants, indicating that the localization pattern is not due to the fluorescent protein fusion tag (data not shown). Similarly, PER/TIM complexes were found to localize in cytoplasmic foci shortly before they translocate into the nucleus (21.Meyer P. Saez L. Young M.W. Science. 2006; 311: 226-229Crossref PubMed Scopus (143) Google Scholar). The nuclear import incompetent mutant of CLK may therefore be stalled in pre-import cytoplasmic foci. Cellular fractionation experiments of mammalian CLK from mouse liver (34.Lee C. Etchegaray J.P. Cagampang F.R. Loudon A.S. Reppert S.M. Cell. 2001; 107: 855-867Abstract Full Text Full Text PDF PubMed Scopus (919) Google Scholar) revealed a hypophosphorylation of cytoplasmic mCLK, whereas nuclear mCLK was hyperphosphorylated. We therefore analyzed the phosphorylation state of nuclear and cytoplasmic CLK proteins (Fig. 2, B and C) in Drosophila S2 cells. Different hypo-, intermediate, and hyperphosphorylated forms of CLK could be resolved by one-dimensional gel electrophoresis. Phosphatase treatment converted these phosphorylated forms into two bands with increased electrophoretic mobility that likely represent a non- and a hypophosphorylated species (supplemental Fig. S1). Wild-type CLK showed predominantly hyperphosphorylated, low electrophoretic mobility forms (Fig. 2C). In contrast, cytoplasmic CLKNLS accumulated mainly in a hypophosphorylated state, indicating that newly synthesized CLK protein is converted to a low phosphorylated species in the cytoplasm, whereas hyperphosphorylation requires nuclear import (Fig. 2C). The nuclear localization of wild-type CLK in S2 cells (Fig. 2) mimics the predominant nuclear localization of CLK in circadian neurons of the larval brain (Fig. 1). When we investigated the cellular localization of CLK in S2R+ cells, we made the surprising observation that CLK showed a strong cytoplasmic localization in most cells, in addition to nuclear staining (Fig. 3A). Co-expression of CYC facilitated, however, an efficient nuclear localization of CLK in S2R+ cells, suggesting that CYC promotes the nuclear localization of CLK. Such a role of CYC is consistent with the finding of reduced endogenous cyc levels in S2R+ cells that reached only 50% of the transcript levels found in S2 cells, as determined by quantitative real-time PCR. In addition, pharmacological inhibition of CRM1-dependent nuclear export by leptomycin B significantly increased the amount of nuclear CLK, suggesting that rapid nuclear export causes an inefficient nuclear localization of CLK in S2R+ cells (Fig. 3A). Interestingly, deletion of a C-terminal consensus nuclear export signal (NES) resulted in a complete nuclear localization of the mutant CLKΔNES protein in most cells, indicating that this sequence is important for nuclear export of CLK (Fig. 3A). The CLKΔNES mutant activated transcription of a luciferase reporter gene in S2R+ cells similar as wild type (Fig. 3B) demonstrating that deletion of the NES did not compromise CLK function and allowed a native fold of the mutant protein. The results are consistent with a nucleocytoplasmic shuttling of the CLK protein, which, in the absence of CYC, results in a cytoplasmic localization of CLK, due to inefficient nuclear retention. When the phosphorylation states of cytoplasmic and nuclear CLK proteins were investigated after expression in S2R+ cells, we observed that cytoplasmic CLKNLS/ΔNES was hypophosphorylated (Fig. 3C and data not shown), whereas wild-type CLK showed predominantly hyperphosphorylated forms (Fig. 3C). Interestingly, the nuclear export-deficient CLKΔNES protein accumulated in an intermediate and a hyperphosphorylated state. The phosphorylation level of CLKΔNES was increased compared with nuclear import-deficient CLKNLS/ΔNES, indicating that nuclear translocation is important for hyperphosphorylation of CLK. On the other hand, CLKΔNES showed a stronger accumulation in the intermediate phosphorylation state compared with shuttling wild-type CLK (Fig. 3C), which was mainly hyperphosphorylated. These findings suggest that nuclear export promotes phosphorylation of the CLK protein and further phosphorylation events may take place in the cytoplasm after nuclear export. Because the CLKΔNES construct carries a 25-amino acid deletion of the NES, we validated these findings using a CLKΔNES construct that carries a 25-amino acid tag (25tag-CLKΔNES) (supplemental Fig. S2). No significant differences were however observed in the phosphorylation patterns of CLKΔNES and 25tag-CLKΔNES (supplemental Fig. S2A). Similarly, 25tag-CLKΔNES accumulated in a phosphorylation state that was intermediate compared with mainly hyperphosphorylated wild-type CLK and hypophosphorylated cytoplasmic CLKNLS (supplemental Fig. S2B). The above observations rev" @default.
• W2023651881 created "2016-06-24" @default.
• W2023651881 creator A5019503490 @default.
• W2023651881 creator A5032357342 @default.
• W2023651881 creator A5062189732 @default.
• W2023651881 creator A5069761433 @default.
• W2023651881 creator A5080757112 @default.
• W2023651881 date "2009-08-01" @default.
• W2023651881 modified "2023-10-14" @default.
• W2023651881 title "Sequential and Compartment-specific Phosphorylation Controls the Life Cycle of the Circadian CLOCK Protein" @default.
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