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• W1540772015 abstract "•Different PP2A complexes control the activity of the Drosophila CLOCK (CLK) protein•STRIP-containing PP2A/CKA (STRIPAK) complexes promote daytime CLK dephosphorylation•PP2A/WDB complexes stabilize CLK In the Drosophila circadian oscillator, the CLOCK/CYCLE complex activates transcription of period (per) and timeless (tim) in the evening. PER and TIM proteins then repress CLOCK (CLK) activity during the night. The pace of the oscillator depends upon post-translational regulation that affects both positive and negative components of the transcriptional loop. CLK protein is highly phosphorylated and inactive in the morning, whereas hypophosphorylated active forms are present in the evening. How this critical dephosphorylation step is mediated is unclear. We show here that two components of the STRIPAK complex, the CKA regulatory subunit of the PP2A phosphatase and its interacting protein STRIP, promote CLK dephosphorylation during the daytime. In contrast, the WDB regulatory PP2A subunit stabilizes CLK without affecting its phosphorylation state. Inhibition of the PP2A catalytic subunit and CKA downregulation affect daytime CLK similarly, suggesting that STRIPAK complexes are the main PP2A players in producing transcriptionally active hypophosphorylated CLK. In the Drosophila circadian oscillator, the CLOCK/CYCLE complex activates transcription of period (per) and timeless (tim) in the evening. PER and TIM proteins then repress CLOCK (CLK) activity during the night. The pace of the oscillator depends upon post-translational regulation that affects both positive and negative components of the transcriptional loop. CLK protein is highly phosphorylated and inactive in the morning, whereas hypophosphorylated active forms are present in the evening. How this critical dephosphorylation step is mediated is unclear. We show here that two components of the STRIPAK complex, the CKA regulatory subunit of the PP2A phosphatase and its interacting protein STRIP, promote CLK dephosphorylation during the daytime. In contrast, the WDB regulatory PP2A subunit stabilizes CLK without affecting its phosphorylation state. Inhibition of the PP2A catalytic subunit and CKA downregulation affect daytime CLK similarly, suggesting that STRIPAK complexes are the main PP2A players in producing transcriptionally active hypophosphorylated CLK. Circadian clocks govern a wide range of physiological and behavioral rhythms to adapt organisms to the 24-hr day-night cycles generated by the rotation of the Earth on its axis. They stem upon feedback loops where transcription factors activate the expression of their own inhibitors, which repress transcription with a timely regulated delay. In the Drosophila circadian oscillator, the CLOCK (CLK) and CYCLE (CYC) bHLH-PAS domain transcription factors activate expression of the period (per) and timeless (tim) genes at the end of the day (Hardin, 2011Hardin P.E. Molecular genetic analysis of circadian timekeeping in Drosophila.Adv. Genet. 2011; 74: 141-173Crossref PubMed Scopus (248) Google Scholar, Ozkaya and Rosato, 2012Ozkaya O. Rosato E. The circadian clock of the fly: a neurogenetics journey through time.Adv. Genet. 2012; 77: 79-123Crossref PubMed Scopus (75) Google Scholar). In a second loop, CLK/CYC activate the vrille (vri) and pdp1ε genes, whose protein products repress (VRI) or activate (PDP1ϵ) the transcription of the Clk gene. The delayed accumulation of PER and TIM leads to transcriptional repression of CLK/CYC during the late night, and their subsequent morning degradation allows transcription to resume in the second part of the day. PER and TIM stability is largely controlled by phosphorylation. PER is phosphorylated by the DOUBLETIME (DBT, CK1δ/ε), CK2, and NEMO kinases and polyubiquitylated by the SCFSlimb ubiquitin ligase complex (Kloss et al., 1998Kloss B. Price J.L. Saez L. Blau J. Rothenfluh A. Wesley C.S. Young M.W. The Drosophila clock gene double-time encodes a protein closely related to human casein kinase Iepsilon.Cell. 1998; 94: 97-107Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar, Price et al., 1998Price J.L. Blau J. Rothenfluh A. Abodeely M. Kloss B. Young M.W. double-time is a novel Drosophila clock gene that regulates PERIOD protein accumulation.Cell. 1998; 94: 83-95Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar, Ko et al., 2002Ko H.W. Jiang J. Edery I. Role for Slimb in the degradation of Drosophila Period protein phosphorylated by Doubletime.Nature. 2002; 420: 673-678Crossref PubMed Scopus (265) Google Scholar, Grima et al., 2002Grima B. Lamouroux A. Chélot E. Papin C. Limbourg-Bouchon B. Rouyer F. The F-box protein slimb controls the levels of clock proteins period and timeless.Nature. 2002; 420: 178-182Crossref PubMed Scopus (226) Google Scholar, Lin et al., 2002Lin J.-M. Kilman V.L. Keegan K. Paddock B. Emery-Le M. Rosbash M. Allada R. A role for casein kinase 2alpha in the Drosophila circadian clock.Nature. 2002; 420: 816-820Crossref PubMed Scopus (297) Google Scholar, Akten et al., 2003Akten B. Jauch E. Genova G.K. Kim E.Y. Edery I. Raabe T. Jackson F.R. A role for CK2 in the Drosophila circadian oscillator.Nat. Neurosci. 2003; 6: 251-257Crossref PubMed Scopus (234) Google Scholar, Nawathean and Rosbash, 2004Nawathean P. Rosbash M. The doubletime and CKII kinases collaborate to potentiate Drosophila PER transcriptional repressor activity.Mol. Cell. 2004; 13: 213-223Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, Chiu et al., 2008Chiu J.C. Vanselow J.T. Kramer A. Edery I. The phospho-occupancy of an atypical SLIMB-binding site on PERIOD that is phosphorylated by DOUBLETIME controls the pace of the clock.Genes Dev. 2008; 22: 1758-1772Crossref PubMed Scopus (121) Google Scholar, Chiu et al., 2011Chiu J.C. Ko H.W. Edery I. NEMO/NLK phosphorylates PERIOD to initiate a time-delay phosphorylation circuit that sets circadian clock speed.Cell. 2011; 145: 357-370Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). TIM phosphorylation involves the CK2 and SHAGGY (SGG, GSK-3) kinases, and TIM degradation also depends on SCFSlimb and a Cullin-3-based ubiquitin ligase complex (Martinek et al., 2001Martinek S. Inonog S. Manoukian A.S. Young M.W. A role for the segment polarity gene shaggy/GSK-3 in the Drosophila circadian clock.Cell. 2001; 105: 769-779Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, Grima et al., 2002Grima B. Lamouroux A. Chélot E. Papin C. Limbourg-Bouchon B. Rouyer F. The F-box protein slimb controls the levels of clock proteins period and timeless.Nature. 2002; 420: 178-182Crossref PubMed Scopus (226) Google Scholar, Grima et al., 2012Grima B. Dognon A. Lamouroux A. Chélot E. Rouyer F. CULLIN-3 controls TIMELESS oscillations in the Drosophila circadian clock.PLoS Biol. 2012; 10: e1001367Crossref PubMed Scopus (41) Google Scholar, Lin et al., 2002Lin J.-M. Kilman V.L. Keegan K. Paddock B. Emery-Le M. Rosbash M. Allada R. A role for casein kinase 2alpha in the Drosophila circadian clock.Nature. 2002; 420: 816-820Crossref PubMed Scopus (297) Google Scholar, Meissner et al., 2008Meissner R.A. Kilman V.L. Lin J.M. Allada R. TIMELESS is an important mediator of CK2 effects on circadian clock function in vivo.J. Neurosci. 2008; 28: 9732-9740Crossref PubMed Scopus (32) Google Scholar). In addition, PER and TIM are controlled by phosphatase activities: PP2A regulates PER abundance and nuclear entry (Sathyanarayanan et al., 2004Sathyanarayanan S. Zheng X. Xiao R. Sehgal A. Posttranslational regulation of Drosophila PERIOD protein by protein phosphatase 2A.Cell. 2004; 116: 603-615Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), while PP1 dephosphorylates TIM (Fang et al., 2007Fang Y. Sathyanarayanan S. Sehgal A. Post-translational regulation of the Drosophila circadian clock requires protein phosphatase 1 (PP1).Genes Dev. 2007; 21: 1506-1518Crossref PubMed Scopus (107) Google Scholar). CLK phosphorylation cycles with a peak in the morning and requires both PER and DBT (Lee et al., 1998Lee C. Bae K. Edery I. The Drosophila CLOCK protein undergoes daily rhythms in abundance, phosphorylation, and interactions with the PER-TIM complex.Neuron. 1998; 21: 857-867Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, Kim and Edery, 2006Kim E.Y. Edery I. Balance between DBT/CKIepsilon kinase and protein phosphatase activities regulate phosphorylation and stability of Drosophila CLOCK protein.Proc. Natl. Acad. Sci. USA. 2006; 103: 6178-6183Crossref PubMed Scopus (108) Google Scholar, Yu et al., 2006Yu W. Zheng H. Houl J.H. Dauwalder B. Hardin P.E. PER-dependent rhythms in CLK phosphorylation and E-box binding regulate circadian transcription.Genes Dev. 2006; 20: 723-733Crossref PubMed Scopus (178) Google Scholar, Hung et al., 2009Hung H.C. Maurer C. Zorn D. Chang W.L. Weber F. Sequential and compartment-specific phosphorylation controls the life cycle of the circadian CLOCK protein.J. Biol. Chem. 2009; 284: 23734-23742Crossref PubMed Scopus (23) Google Scholar). However, DBT kinase activity does not seem to be required, and it was proposed that DBT function is to recruit other kinases (Yu et al., 2009Yu W. Zheng H. Price J.L. Hardin P.E. DOUBLETIME plays a noncatalytic role to mediate CLOCK phosphorylation and repress CLOCK-dependent transcription within the Drosophila circadian clock.Mol. Cell. Biol. 2009; 29: 1452-1458Crossref PubMed Scopus (61) Google Scholar). A similar cycling was reported for CLK immunoreactivity (Hung et al., 2009Hung H.C. Maurer C. Zorn D. Chang W.L. Weber F. Sequential and compartment-specific phosphorylation controls the life cycle of the circadian CLOCK protein.J. Biol. Chem. 2009; 284: 23734-23742Crossref PubMed Scopus (23) Google Scholar, Menet et al., 2010Menet J.S. Abruzzi K.C. Desrochers J. Rodriguez J. Rosbash M. Dynamic PER repression mechanisms in the Drosophila circadian clock: from on-DNA to off-DNA.Genes Dev. 2010; 24: 358-367Crossref PubMed Scopus (108) Google Scholar, Lamaze et al., 2011Lamaze A. Lamouroux A. Vias C. Hung H.C. Weber F. Rouyer F. The E3 ubiquitin ligase CTRIP controls CLOCK levels and PERIOD oscillations in Drosophila.EMBO Rep. 2011; 12: 549-557Crossref PubMed Scopus (28) Google Scholar), but relatively constant CLK levels have been observed with harsh extraction conditions (Yu et al., 2006Yu W. Zheng H. Houl J.H. Dauwalder B. Hardin P.E. PER-dependent rhythms in CLK phosphorylation and E-box binding regulate circadian transcription.Genes Dev. 2006; 20: 723-733Crossref PubMed Scopus (178) Google Scholar, Sun et al., 2010Sun W.C. Jeong E.H. Jeong H.J. Ko H.W. Edery I. Kim E.Y. Two distinct modes of PERIOD recruitment onto dCLOCK reveal a novel role for TIMELESS in circadian transcription.J. Neurosci. 2010; 30: 14458-14469Crossref PubMed Scopus (17) Google Scholar, Luo et al., 2012Luo W. Li Y. Tang C.H. Abruzzi K.C. Rodriguez J. Pescatore S. Rosbash M. CLOCK deubiquitylation by USP8 inhibits CLK/CYC transcription in Drosophila.Genes Dev. 2012; 26: 2536-2549Crossref PubMed Scopus (31) Google Scholar). CLK DNA-binding and transcriptional activity are maximal in the evening when CLK phosphorylation is minimal (Yu et al., 2006Yu W. Zheng H. Houl J.H. Dauwalder B. Hardin P.E. PER-dependent rhythms in CLK phosphorylation and E-box binding regulate circadian transcription.Genes Dev. 2006; 20: 723-733Crossref PubMed Scopus (178) Google Scholar, Menet et al., 2010Menet J.S. Abruzzi K.C. Desrochers J. Rodriguez J. Rosbash M. Dynamic PER repression mechanisms in the Drosophila circadian clock: from on-DNA to off-DNA.Genes Dev. 2010; 24: 358-367Crossref PubMed Scopus (108) Google Scholar, Abruzzi et al., 2011Abruzzi K.C. Rodriguez J. Menet J.S. Desrochers J. Zadina A. Luo W. Tkachev S. Rosbash M. Drosophila CLOCK target gene characterization: implications for circadian tissue-specific gene expression.Genes Dev. 2011; 25: 2374-2386Crossref PubMed Scopus (124) Google Scholar). The release of CLK from DNA correlates with its PER/DBT-dependent hyperphosphorylation (Yu et al., 2006Yu W. Zheng H. Houl J.H. Dauwalder B. Hardin P.E. PER-dependent rhythms in CLK phosphorylation and E-box binding regulate circadian transcription.Genes Dev. 2006; 20: 723-733Crossref PubMed Scopus (178) Google Scholar, Kim and Edery, 2006Kim E.Y. Edery I. Balance between DBT/CKIepsilon kinase and protein phosphatase activities regulate phosphorylation and stability of Drosophila CLOCK protein.Proc. Natl. Acad. Sci. USA. 2006; 103: 6178-6183Crossref PubMed Scopus (108) Google Scholar, Menet et al., 2010Menet J.S. Abruzzi K.C. Desrochers J. Rodriguez J. Rosbash M. Dynamic PER repression mechanisms in the Drosophila circadian clock: from on-DNA to off-DNA.Genes Dev. 2010; 24: 358-367Crossref PubMed Scopus (108) Google Scholar). In addition to DBT, kinases known to be involved in CLK phosphorylation include NEMO (NMO), which destabilizes CLK (Yu et al., 2011Yu W. Houl J.H. Hardin P.E. NEMO kinase contributes to core period determination by slowing the pace of the Drosophila circadian oscillator.Curr. Biol. 2011; 21: 756-761Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), and CK2α that stabilizes CLK and inhibits its transcriptional activity (Szabó et al., 2013Szabó A. Papin C. Zorn D. Ponien P. Weber F. Raabe T. Rouyer F. The CK2 kinase stabilizes CLOCK and represses its activity in the Drosophila circadian oscillator.PLoS Biol. 2013; 11: e1001645Crossref PubMed Scopus (25) Google Scholar). CLK is also regulated by ubiquitylation with the HECT-domain ubiquitin ligase CTRIP destabilizing CLK (Lamaze et al., 2011Lamaze A. Lamouroux A. Vias C. Hung H.C. Weber F. Rouyer F. The E3 ubiquitin ligase CTRIP controls CLOCK levels and PERIOD oscillations in Drosophila.EMBO Rep. 2011; 12: 549-557Crossref PubMed Scopus (28) Google Scholar), and the USP8 ubiquitin protease decreasing its transcriptional activity (Luo et al., 2012Luo W. Li Y. Tang C.H. Abruzzi K.C. Rodriguez J. Pescatore S. Rosbash M. CLOCK deubiquitylation by USP8 inhibits CLK/CYC transcription in Drosophila.Genes Dev. 2012; 26: 2536-2549Crossref PubMed Scopus (31) Google Scholar). Finally, CLK activity is controlled by repressors/activators such as CLOCKWORK ORANGE (CWO) (Matsumoto et al., 2007Matsumoto A. Ukai-Tadenuma M. Yamada R.G. Houl J. Uno K.D. Kasukawa T. Dauwalder B. Itoh T.Q. Takahashi K. Ueda R. et al.A functional genomics strategy reveals clockwork orange as a transcriptional regulator in the Drosophila circadian clock.Genes Dev. 2007; 21: 1687-1700Crossref PubMed Scopus (135) Google Scholar, Kadener et al., 2007Kadener S. Stoleru D. McDonald M. Nawathean P. Rosbash M. Clockwork Orange is a transcriptional repressor and a new Drosophila circadian pacemaker component.Genes Dev. 2007; 21: 1675-1686Crossref PubMed Scopus (146) Google Scholar, Lim et al., 2007Lim C. Chung B.Y. Pitman J.L. McGill J.J. Pradhan S. Lee J. Keegan K.P. Choe J. Allada R. Clockwork orange encodes a transcriptional repressor important for circadian-clock amplitude in Drosophila.Curr. Biol. 2007; 17: 1082-1089Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, Richier et al., 2008Richier B. Michard-Vanhée C. Lamouroux A. Papin C. Rouyer F. The clockwork orange Drosophila protein functions as both an activator and a repressor of clock gene expression.J. Biol. Rhythms. 2008; 23: 103-116Crossref PubMed Scopus (67) Google Scholar) and the FOS ortholog KAYAK (KAY) (Ling et al., 2012Ling J. Dubruille R. Emery P. KAYAK-α modulates circadian transcriptional feedback loops in Drosophila pacemaker neurons.J. Neurosci. 2012; 32: 16959-16970Crossref PubMed Scopus (16) Google Scholar). PP2A is the main source of serine/threonine phosphatase activity in the cell (Virshup and Shenolikar, 2009Virshup D.M. Shenolikar S. From promiscuity to precision: protein phosphatases get a makeover.Mol. Cell. 2009; 33: 537-545Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar). PP2A holoenzymes are predominantly heterotrimers in which the scaffold A and catalytic C subunits associate with a single variable regulatory B subunit, which defines substrate specificity and holoenzyme localization (Shi, 2009Shi Y. Serine/threonine phosphatases: mechanism through structure.Cell. 2009; 139: 468-484Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar, Virshup and Shenolikar, 2009Virshup D.M. Shenolikar S. From promiscuity to precision: protein phosphatases get a makeover.Mol. Cell. 2009; 33: 537-545Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar). In Drosophila, the scaffold and catalytic subunits are encoded by the pp2a-29b and microtubule star (mts) genes, respectively. Regulatory subunits are encoded by twins (tws) (B type), widerborst (wdb) and PP2A-B′ (B′ type), CG4733 (B′′ type), and Connector of kinase to AP-1 (Cka) (B′′′ type). CKA is the ortholog of the mammalian striatin proteins (Ribeiro et al., 2010Ribeiro P.S. Josué F. Wepf A. Wehr M.C. Rinner O. Kelly G. Tapon N. Gstaiger M. Combined functional genomic and proteomic approaches identify a PP2A complex as a negative regulator of Hippo signaling.Mol. Cell. 2010; 39: 521-534Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). B′′′-containing PP2A holoenzymes are associated with other proteins including the striatin-interacting protein (STRIP) and kinases in STRIPAK (STRiatin-Interacting Phosphatase And Kinase) complexes (Glatter et al., 2009Glatter T. Wepf A. Aebersold R. Gstaiger M. An integrated workflow for charting the human interaction proteome: insights into the PP2A system.Mol. Syst. Biol. 2009; 5: 237Crossref PubMed Scopus (215) Google Scholar, Goudreault et al., 2009Goudreault M. D’Ambrosio L.M. Kean M.J. Mullin M.J. Larsen B.G. Sanchez A. Chaudhry S. Chen G.I. Sicheri F. Nesvizhskii A.I. et al.A PP2A phosphatase high density interaction network identifies a novel striatin-interacting phosphatase and kinase complex linked to the cerebral cavernous malformation 3 (CCM3) protein.Mol. Cell. Proteomics. 2009; 8: 157-171Crossref PubMed Scopus (273) Google Scholar, Herzog et al., 2012Herzog F. Kahraman A. Boehringer D. Mak R. Bracher A. Walzthoeni T. Leitner A. Beck M. Hartl F.U. Ban N. et al.Structural probing of a protein phosphatase 2A network by chemical cross-linking and mass spectrometry.Science. 2012; 337: 1348-1352Crossref PubMed Scopus (312) Google Scholar, Ribeiro et al., 2010Ribeiro P.S. Josué F. Wepf A. Wehr M.C. Rinner O. Kelly G. Tapon N. Gstaiger M. Combined functional genomic and proteomic approaches identify a PP2A complex as a negative regulator of Hippo signaling.Mol. Cell. 2010; 39: 521-534Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, Kean et al., 2011Kean M.J. Ceccarelli D.F. Goudreault M. Sanches M. Tate S. Larsen B. Gibson L.C. Derry W.B. Scott I.C. Pelletier L. et al.Structure-function analysis of core STRIPAK Proteins: a signaling complex implicated in Golgi polarization.J. Biol. Chem. 2011; 286: 25065-25075Crossref PubMed Scopus (108) Google Scholar, Hwang and Pallas, 2014Hwang J. Pallas D.C. STRIPAK complexes: structure, biological function, and involvement in human diseases.Int. J. Biochem. Cell Biol. 2014; 47: 118-148Crossref PubMed Scopus (155) Google Scholar, Ashton-Beaucage et al., 2014Ashton-Beaucage D. Udell C.M. Gendron P. Sahmi M. Lefrançois M. Baril C. Guenier A.S. Duchaine J. Lamarre D. Lemieux S. Therrien M. A functional screen reveals an extensive layer of transcriptional and splicing control underlying RAS/MAPK signaling in Drosophila.PLoS Biol. 2014; 12: e1001809Crossref PubMed Scopus (47) Google Scholar). In Drosophila, STRIPAK complexes act as negative regulators of the HIPPO pathway (Ribeiro et al., 2010Ribeiro P.S. Josué F. Wepf A. Wehr M.C. Rinner O. Kelly G. Tapon N. Gstaiger M. Combined functional genomic and proteomic approaches identify a PP2A complex as a negative regulator of Hippo signaling.Mol. Cell. 2010; 39: 521-534Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) and as positive regulators of RAS/MAPK signaling (Ashton-Beaucage et al., 2014Ashton-Beaucage D. Udell C.M. Gendron P. Sahmi M. Lefrançois M. Baril C. Guenier A.S. Duchaine J. Lamarre D. Lemieux S. Therrien M. A functional screen reveals an extensive layer of transcriptional and splicing control underlying RAS/MAPK signaling in Drosophila.PLoS Biol. 2014; 12: e1001809Crossref PubMed Scopus (47) Google Scholar). CKA is also required for the dorsal closure during embryonic development where it complexes with KAY and JUN-RELATED ANTIGEN (JRA) transcription factors (Chen et al., 2002Chen H.W. Marinissen M.J. Oh S.W. Chen X. Melnick M. Perrimon N. Gutkind J.S. Hou S.X. CKA, a novel multidomain protein, regulates the JUN N-terminal kinase signal transduction pathway in Drosophila.Mol. Cell. Biol. 2002; 22: 1792-1803Crossref PubMed Scopus (63) Google Scholar), but it is unknown whether STRIPAK complexes are involved. A STRIPAK-independent STRIP role in neuronal endosome organization has recently been described (Sakuma et al., 2014Sakuma C. Kawauchi T. Haraguchi S. Shikanai M. Yamaguchi Y. Gelfand V.I. Luo L. Miura M. Chihara T. Drosophila Strip serves as a platform for early endosome organization during axon elongation.Nat. Commun. 2014; 5: 5180Crossref PubMed Scopus (22) Google Scholar). Catalytic MTS has been shown to promote high levels of nuclear PER (Sathyanarayanan et al., 2004Sathyanarayanan S. Zheng X. Xiao R. Sehgal A. Posttranslational regulation of Drosophila PERIOD protein by protein phosphatase 2A.Cell. 2004; 116: 603-615Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), and downregulation of either TWS or WDB regulatory subunit induces PER degradation in Drosophila S2 cells, indicating that they associate with MTS to control PER oscillations (Sathyanarayanan et al., 2004Sathyanarayanan S. Zheng X. Xiao R. Sehgal A. Posttranslational regulation of Drosophila PERIOD protein by protein phosphatase 2A.Cell. 2004; 116: 603-615Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). wdb or tws downregulation in S2 cells also induces CLK degradation (Kim and Edery, 2006Kim E.Y. Edery I. Balance between DBT/CKIepsilon kinase and protein phosphatase activities regulate phosphorylation and stability of Drosophila CLOCK protein.Proc. Natl. Acad. Sci. USA. 2006; 103: 6178-6183Crossref PubMed Scopus (108) Google Scholar), suggesting a CLK stabilization function for PP2A in vivo. Whether CKA-containing PP2A complexes (STRIPAK) contribute to clock mechanisms is not known. In this study, we identify both CKA and STRIP proteins as new components of the Drosophila molecular clock. We show that STRIPAK complexes promote the dephosphorylation of the CLK protein during daytime, whereas WDB-containing PP2A complexes control CLK protein levels. The NIG-Fly collection of RNAi lines was used to individually downregulate about 6,000 genes using the GAL4/UAS expression system (S.B. and F.R., unpublished data). We used the gal1118 GAL4 driver (Blanchardon et al., 2001Blanchardon E. Grima B. Klarsfeld A. Chélot E. Hardin P.E. Préat T. Rouyer F. Defining the role of Drosophila lateral neurons in the control of circadian activity and eclosion rhythms by targeted genetic ablation and PERIOD protein overexpression.Eur. J. Neurosci. 2001; 13: 871-888Crossref PubMed Google Scholar), which mostly targets the clock neurons expressing the pigment-dispersing factor (PDF) neuropeptide. The locomotor activity of gal1118>RNAi flies was monitored in constant darkness (DD), and flies with altered behavioral rhythms were selected. We identified Strip, which encodes the Drosophila ortholog of the mammalian striatin-interacting protein (STRIP) (Glatter et al., 2009Glatter T. Wepf A. Aebersold R. Gstaiger M. An integrated workflow for charting the human interaction proteome: insights into the PP2A system.Mol. Syst. Biol. 2009; 5: 237Crossref PubMed Scopus (215) Google Scholar, Goudreault et al., 2009Goudreault M. D’Ambrosio L.M. Kean M.J. Mullin M.J. Larsen B.G. Sanchez A. Chaudhry S. Chen G.I. Sicheri F. Nesvizhskii A.I. et al.A PP2A phosphatase high density interaction network identifies a novel striatin-interacting phosphatase and kinase complex linked to the cerebral cavernous malformation 3 (CCM3) protein.Mol. Cell. Proteomics. 2009; 8: 157-171Crossref PubMed Scopus (273) Google Scholar, Ribeiro et al., 2010Ribeiro P.S. Josué F. Wepf A. Wehr M.C. Rinner O. Kelly G. Tapon N. Gstaiger M. Combined functional genomic and proteomic approaches identify a PP2A complex as a negative regulator of Hippo signaling.Mol. Cell. 2010; 39: 521-534Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, Sakuma et al., 2014Sakuma C. Kawauchi T. Haraguchi S. Shikanai M. Yamaguchi Y. Gelfand V.I. Luo L. Miura M. Chihara T. Drosophila Strip serves as a platform for early endosome organization during axon elongation.Nat. Commun. 2014; 5: 5180Crossref PubMed Scopus (22) Google Scholar). gal1118>Strip-RNAi1 flies showed a 2-hr lengthening of the behavioral period and a ≥2.5-hr period lengthening was observed when Strip RNAi and dicer were co-expressed under the control of either gal1118 or the tim-gal4 driver that is expressed in all clock cells (Figure 1A; Table 1). The target specificity of the Strip RNAi was addressed by testing two additional non-overlapping RNAi sequences (Figure S1), which also induced long-period rhythms when expressed in the clock neurons (Table S1). Head extracts of tim>Strip-RNAi1 flies were tested for Strip mRNA levels and showed a 70% decrease, indicating that the RNAi indeed targeted the Strip transcripts (Figure S1). This experiment also showed that Strip mRNA levels do not cycle in head extracts. PDF-expressing small lateral neurons (s-LNvs) were not morphologically altered in the RNAi flies (Figure S1), and PER immunofluorescence analysis revealed a delayed oscillation (Figure 1B), in agreement with the long-period behavioral rhythms.Table 1Locomotor Activity Rhythms in DDGenotypenaNumber of tested flies.,bSee Experimental Procedures.% RbSee Experimental Procedures.,cPercentage of rhythmic flies.Period (hr) ±SEMbSee Experimental Procedures.Power ±SEMbSee Experimental Procedures.w; ; UAS-strip-RNAi1/+1610023.8 ± 0.1187 ± 19w; UAS-Cka-RNAi1/+329423.4 ± 0.0195 ± 10w; ; UAS-wdb-RNAi/+2910023.6 ± 0.1193 ± 11w; ; gal1118/+1610024.1 ± 0.1219 ± 11w; UAS-dcr2/+; gal1118/+1610024.0 ± 0.1216 ± 16w; tim-gal4/+1610024.1 ± 0.1205 ± 16w; tim-gal4/+; UAS-dcr2/+1610023.9 ± 0.1178 ± 15w; ; #221301110023.8 ± 0.1134 ± 23w; ; #22130/Df1610024.4 ± 0.1224 ± 15w; ; gal1118/UAS-Strip-RNAi11610026.0 ± 0.1129 ± 11w; UAS-dcr2/+; gal1118/UAS-Strip-RNAi1169426.8 ± 0.1100 ± 11w; tim-gal4/+; UAS-dcr2/UAS-Strip-RNAi1168826.6 ± 0.1126 ± 19w; Cka2, HS-Cka/CyO188324.6 ± 0.2101 ± 20w; Cka2, HS-Cka143626.9 ± 2.350 ± 9w; UAS-dcr2/UAS-Cka-RNAi1; gal1118/+1610025.5 ± 0.1264 ± 11w; tim-gal4/UAS-Cka-RNAi1; UAS-dcr2/+1610025.9 ± 0.1199 ± 17w; UAS-dcr2/+; gal1118/UAS-wdb-RNAi1610025.0 ± 0.1302 ± 8w; tim-gal4/+; UAS-dcr2/UAS-wdb-RNAi1610025.1 ± 0.1218 ± 13w; tim-gal4/UAS-dcr2;UAS-dbtK/R,dbtar/+16636.0 ± 0.0dOnly one fly.75 ± 0dOnly one fly.w; tim-gal4/UAS-Cka-RNAi1;UAS-dbtK/R,dbtar/UAS-dcr2162540.3 ± 0.441 ± 6w; tim-gal4/UAS-dcr2;UAS-dbtK/R,dbtar/UAS-Strip-RNAi1162528.0 ± 3.027 ± 2All males except for the Cka2,HS-Cka and Cka2, HS-Cka/CyO genotypes.a Number of tested flies.b See Experimental Procedures.c Percentage of rhythmic flies.d Only one fly. Open table in a new tab All males except for the Cka2,HS-Cka and Cka2, HS-Cka/CyO genotypes. A P-element insertion that is localized in the non-coding 5′ end of Strip (Figure S1) was mobilized to generate deletions of the gene by imprecise excision (see Supplemental Experimental Procedures). A 4-kb deletion encompassing most of the Strip gene was obtained, but Δ22130 homozygous mutants died at the pupal stage. The PDF-expressing neurons of Δ22130 L3 larvae did not show obvious morphological defects (Figure S1) and were used for analyzing PER protein oscillations. PER levels cycled with a slow pace in the null homozygous mutants, with a 6-hr delay compared to heterozygous larvae at DD4 (Figure S1). This indicates that a functional Strip gene is required to set the pace of the clock to 24 hr. To ask whether the circadian function of STRIP involves the STRIPAK complex, we tested the downregulation of the STRIP-associated PP2A regulatory subunit CKA. Expression of two non-overlapping Cka RNAi sequences in clock cells induced long-period rhythms, indicating that CKA was indeed involved in the behavioral clock (Figure 2A; Figure S2; Table 1; Table S1). Accordingly, tim>Cka-RNAi1 flies showed a 40% decrease of Cka mRNA levels, whereas no cycling of the Cka transcripts nor of the CKA protein was observed in wild-type head extracts (Figure S2). Cka2-null mutants are embryonic lethal, but viable adult flies could be obtained by providing daily heat-shock-controlled Cka expression until the emergence of the adults (Chen et al., 2002Chen H.W. Marinissen M.J. Oh S.W. Chen X. Melnick M. Perrimon N. Gutkind J.S. Hou S.X. CKA, a novel multidomain protein, regulates the JUN N-terminal kinase signal transduction pathway in Drosophila.Mol. Cell. Biol. 2002; 22: 1792-1803Crossref PubMed Scopus (63) Google Scholar). Emerged flies were then transferred to low temperature and did not show detectable CKA immunostaining in head extracts (Figure S2). Developmentally rescued Cka2 mutants were behaviorally tested at low temperature and became arrhythmic after a few days in DD (Table 1; Figure 2A). PDF-positive s-LNvs appeared normal in the mutants as well as in the RNAi flies (Figure S2). PER cycling was analyzed in the mutants. PER oscillations were marginally delayed during the first two DD days but were abolished with low immunostaining levels at DD6 (Figure 2B). Our data indicate th" @default.
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• W1540772015 title "Daytime CLOCK Dephosphorylation Is Controlled by STRIPAK Complexes in Drosophila" @default.
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