Setting the Pace of the Neurospora Circadian Clock by Multiple Independent FRQ Phosphorylation Events

Setting the pace of the Neurospora circadian clock by multiple independent FRQ phosphorylation events

Chi-Tai Tanga,1, Shaojie Lia,b,1, Chengzu Longc, Joonseok Chaa, Guocun Huanga, Lily Lia,2, She Chend,3 and Yi Liua,4

aDepartment of Physiology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390; bKey Laboratory of Systematic Mycology and Lichenology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China; cNational Institute of Biological Sciences, 7 Life Science Park Road, Changping District, Beijing 102206, China; and dCollege of Life Science, Beijing Normal University, Beijing 100875, China

Communicated by Melanie H. Cobb, University of Texas Southwestern Medical Center, Dallas, TX, May 5, 2009 (received for review April 15, 2009) Protein phosphorylation plays essential roles in eukaryotic circa- that FRQ is phosphorylated at multiple sites. Although some dian clocks. Like PERIOD in animals, the Neurospora core circadian putative FRQ phosphorylation sites and domains have been protein FRQ is progressively phosphorylated and becomes exten- identified by deletion and mutation analyses, no FRQ phosphor- sively phosphorylated before its degradation. In this study, by ylation sites have been confirmed in vivo (22, 24, 25, 27). How using purified FRQ protein from Neurospora, we identified 43 in FRQ phosphorylation is temporally regulated by different ki- vivo FRQ phosphorylation sites by mass spectrometry analysis. In nases is not known. addition, we show that CK-1a and CKII are responsible for most To understand the function and regulation of FRQ phosphor- FRQ phosphorylation events and identify an additional 33 phos- ylation, we identified 43 in vivo phosphorylation sites by MS phorylation sites by in vitro kinase assays. Whole-cell metabolic analyses using purified FRQ from Neurospora. In vitro kinase isotope labeling and quantitative MS analyses suggest that circa- assays showed that CK-1a and CKII are responsible for most dian oscillation of the FRQ phosphorylation profile is primarily due FRQ phosphorylation, and they identified 33 additional putative to progressive phosphorylation at the majority of these newly FRQ phosphorylation sites. By performing whole-cell metabolic discovered phosphorylation sites. Furthermore, systematic muta- isotope labeling and quantitative MS analyses, we demonstrate tions of the identified FRQ phosphorylation sites led to either long that a majority of the phosphorylation sites are preferentially

or short period phenotypes. These changes in circadian period are phosphorylated when FRQ becomes hyperphosphorylated. Our GENETICS attributed to increases or decreases in FRQ stability, respectively. systematic mutagenesis of these phosphorylation sites reveals Together, this comprehensive study of FRQ phosphorylation re- that phosphorylation at different regions along FRQ regulates veals that regulation of FRQ stability by multiple independent the period of the clock by either promoting or inhibiting FRQ phosphorylation events is a major factor that determines the degradation. period length of the clock. A model is proposed to explain how FRQ stability is regulated by multiple phosphorylation events. Results Mapping In Vivo FRQ Phosphorylation Sites. To purify FRQ from mass spectrometry ͉ casein kinase ͉ frequency Neurospora for mapping phosphorylation sites, we created an expression construct in which a 5ϫ c-Myc tag and a 9ϫ His tag ukaryotic circadian oscillators from fungi to mammals are were inserted into the C-terminal end of the FRQ ORF. This Econtrolled by autoregulatory negative feedback loops (1–4). construct was transformed into a frq-null strain (frq10) (28). In the filamentous fungus Neurospora crassa, 2 protein com- Transformants exhibited robust circadian rhythms (Fig. S1A), plexes function in the core circadian negative feedback loop (5, indicating that epitope-tagged FRQ functions as the endogenous 6). WHITE COLLAR complex (WCC), formed by WC-1 and FRQ protein. The Myc-His-FRQ construct was also transformed WC-2, activates transcription of the frequency (frq) gene by into an fwd-1RIP strain (20). In the fwd-1 mutant, FRQ levels are binding to its promoter (7–13). On the other hand, FFC (con- elevated and FRQ exists as hyperphosphorylated forms. Myc- sisting of FRQ and the FRQ-interacting RNA helicase, FRH) His-FRQ was purified to near homogeneity from either a inhibits WCC activity by promoting the phosphorylation, and frq10,Myc-His-FRQ strain or a fwd-1RIP,Myc-His-FRQ strain by consequently repression, of frq transcription (12, 14–18). tandem affinity purification (Fig. S1B) (21). Protein bands Like the animal PERIOD (PER) proteins, FRQ is progres- corresponding to FRQ were excised and subjected to tryptic sively phosphorylated after its synthesis and becomes extensively digestion followed by MS analyses. phosphorylated before its disappearance, resulting in a robust MS analyses using purified Myc-His-FRQ from either the oscillation of its phosphorylation profile (19). One role of FRQ frq10,Myc-His-FRQ strain or the fwd-1RIP,Myc-His-FRQ strain phosphorylation is to promote FRQ degradation through the identified 43 in vivo FRQ phosphorylation sites at Ser and Thr ubiquitin-proteasome pathway mediated by ubiquitin E3 ligase residues. As shown in Fig. 1C, phosphorylation sites (amino acids SCFFWD-1. FWD-1 acts as the substrate-recruiting subunit that labeled in red) are located throughout FRQ. In many instances, recognizes and binds phosphorylated FRQ (20–22). Under normal conditions, FRQ is phosphorylated by CK-1a, CKII, and PKA (12, 16, 19, 23–25). In the ck-1a (casein kinase 1a), cka Author contributions: C.-T.T., S.L., C.L., S.C., and Y.L. designed research; C.-T.T., S.L., C.L., J.C., G.H., L.L., and S.C. performed research; C.-T.T., S.L., C.L., J.C., G.H., L.L., S.C., and Y.L. (catalytic subunit of CKII), and ckb-1 (regulatory subunit of analyzed data; and C.-T.T. and Y.L. wrote the paper. CKII) mutants, FRQ is hypophosphorylated and more stable The authors declare no conﬂict of interest. relative to the wild type, resulting in arrhythmia or long-period 1 phenotypes (12, 23, 25). These results suggest that CK-1a and C.-T.T. and S.L. contributed equally to this study. 2 CKII phosphorylate and promote FRQ degradation. In contrast, Present address: Department of Dermatology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390. PKA counters the role of casein kinases by stabilizing FRQ (12, 3To whom correspondence may be addressed. E-mail: [email protected]. 16). FRQ is also dephosphorylated and stabilized by protein 4To whom correspondence may be sent at: Department of Physiology, ND13.214A, Uni- phosphatases PP1 and PP4 (17, 26). versity of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX FRQ exists as many isoforms, with mobilities ranging from 120 75390-9040. E-mail: [email protected]. Ϸ kDa to 200 kDa when analyzed by SDS/PAGE (19). These This article contains supporting information online at www.pnas.org/cgi/content/full/ variations are due to differences in phosphorylation, suggesting 0904898106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904898106 PNAS Early Edition ͉ 1of6 Downloaded by guest on October 5, 2021 Fig. 1. Map of FRQ. FRQ ORF with the identiﬁed phosphorylation sites and known FRQ domains are indicated. Amino acids in red are identiﬁed in vivo FRQ phosphorylation sites. The asterisks above the residues indicate in vitro FRQ phosphorylation sites by CK-1a, CKA, or CK-1a and CKA combined. Short and long blue lines indicate sites and phosphopeptides preferentially phosphorylated in hyperphosphorylated FRQ samples detected by quantitative MS experiments, respectively. Underlined sequences (M1–19) indicate mutated phosphorylation sites.

2 or more of these sites are clustered, suggesting sequential also perform distinct roles in FRQ phosphorylation. On the phosphorylation events. Despite the large number of sites un- other hand, 10 phosphorylation sites were detected only when covered, it is certainly an underestimate. First, tryptic peptides both CK-1a and CKII were present in the kinase reaction, either too small or too large are difficult to detect by MS. Thus, indicating that they also cooperate with each other to phosphor- peptide coverage of FRQ by trypsin was limited (44–63%) ylate FRQ. When compared to the 43 identified in vivo sites, 30 depending on the experiments. For example, the regions around were phosphorylated by CK-1a, CKII, or when combined, indi- the FRQ-CK-1a interaction domain, the FRQ-FRH interaction cating that these 2 kinases play a major role in phosphorylating domain, the PEST-1 and PEST-2 region, and the C-terminal tail FRQ. Additional kinases, such as PKA and CHK2 (16, 29), may had very poor or no MS coverage. Second, although their phosphorylate at sites independent of casein kinases. Together, unphosphorylated forms could be detected, peptides with mul- our MS analyses have led to the identification of 76 confirmed tiple phosphorylation events are known to ionize poorly in MS and potential FRQ phosphorylation sites. experiments and are often difficult to detect. Finally, limited The in vitro phosphorylation by CKI and CKII revealed 33 amounts of purified Myc-His-FRQ (Ϸ1–2 ␮g) and dephosphor- additional potential FRQ phosphorylation sites. These addi- ylation in the cell during purification procedures also make tional sites include 12 sites spanning amino acids 211–289, S513, comprehensive identification difficult. Nonetheless, the 43 in and 519 (2 previously identified sites) (22), and a phosphoryla- vivo sites identified on FRQ represent the highest number of tion site in the PEST-1 domain. These results suggest that these phosphorylation events mapped for a clock protein. In contrast additional in vitro sites may also play important roles in FRQ to FRQ, only 1 phosphorylation site (Ser-21) was identified on phosphorylation in vivo. FRH despite its 60% peptide coverage by MS. Quantitative MS Identified Preferentially Phosphorylated Sites and FRQ Phosphorylation by CK-1a and CKII. We have shown that CK-1a Peptides in Hyperphosphorylated FRQ. We set out to understand and CKII are 2 major kinases that phosphorylate FRQ in vivo how these diverse phosphorylation events are regulated during (12, 23, 25). Although both kinases preferentially phosphorylate a circadian cycle. Because the FRQ phosphorylation profile sites around acidic residues, they target distinct substrate con- oscillates daily, we investigated whether some or all of the sensus sequences: (D/E/Sp/Tp)XX(S/T) for CKI and (S/T)XX(D/ identified FRQ phosphorylation sites are preferentially phos- E/Sp/Tp) for CKII (p stands for phosphorylated residue). Thus, phorylated when FRQ is hyperphosphorylated. We previously they may phosphorylate distinct sites on FRQ. Alternatively, one developed a quantitative MS method to study the regulation of kinase may serve as a priming kinase for the other to achieve protein phosphorylation by whole cell 15N metabolic labeling in proper FRQ phosphorylation. To understand the contribution Neurospora (16). By using this method, we can monitor the of each kinase for FRQ phosphorylation and to further identify regulation of multiple phosphorylation events by quantitatively phosphorylation sites, we phosphorylated recombinant full- comparing protein phosphorylation profiles between different length FRQ in vitro with recombinant Neurospora CK-1a, CKA time points or strains. FRQ becomes hyperphosphorylated (the catalytic subunit of CKII), or both combined. The phos- before its degradation in wild-type strains and accumulates as phorylated FRQ protein was subjected to MS analyses. hyperphosphorylated forms in the fwd-1RIP strain. Thus, we Identified in vitro FRQ phosphorylation sites for these kinases compared FRQ phosphorylation profiles in the frq10,Myc-His- are indicated by asterisks above the amino acid sequence in Fig. FRQ strains between 2 different time points [6 h in constant 1, and in Table S1. The MS peptide coverage for the in vitro darkness (DD) (15N-labeled) and DD18]. At DD6, FRQ is phosphorylated FRQ (Ϸ71%) was significantly higher than the mostly hyperphosphorylated as FRQ levels are decreasing, Myc-His-FRQ purified from Neurospora due to significantly whereas FRQ is less phosphorylated at DD18. We also com- more recombinant FRQ (Ͼ5 ␮g) used in the kinase assays. As pared the FRQ phosphorylation profiles in constant light (LL) a result, 63 phosphorylation sites were identified. These sites between the frq10,Myc-His-FRQ strain and the fwd-1RIP,Myc- were either phosphorylated by CK-1a, CKA, or in combination. His-FRQ (15N-labeled) strain. After the initial protein extrac- Among them, 43 could be phosphorylated by CK-1a alone, 28 by tion, equal amounts of total protein from the 15N-labeled sample CKII alone, and 18 were shared. These results indicate that and the control sample (14N) were mixed and Myc-His-FRQ was although activities of CK-1a and CKII overlap at some sites, they purified and analyzed by quantitative MS. Relative levels of Ͼ20

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904898106 Tang et al. Downloaded by guest on October 5, 2021 peptide was accompanied by a decrease of its unphosphorylated form. Importantly, all of these peptides contain identified in vivo and in vitro phosphorylation sites (Fig. 1). Among these peptides, 3 are clustered downstream of the FRQ nuclear localization signal (NLS) and 1 corresponds to the PEST-1 region. Although we could not detect the phosphorylated forms for some of these peptides, most likely due to their poor ionization, our results strongly suggest that these regions become extensively phosphorylated when FRQ is hyperphosphorylated. Taken together, these quantitative MS results demonstrate that progressive phosphorylation of the majority of the identified FRQ phosphorylation sites determines the circadian rhythm of FRQ phosphorylation profiles.

Multiple Independent FRQ Phosphorylation Events Determine the Circadian Period Length. To understand the functions of these FRQ phosphorylation events, we carried out systematic mutagenesis of the phosphorylation sites. Due to the large number of sites identified, we focused on sites that are preferentially phosphorylated when FRQ becomes extensively phosphorylated. Locations of these mutations are indicated in Fig. 1 (M1–M19). Corresponding Ser/Thr residues within the underlined regions were mutated to Ala. For M7, the region underlined was deleted because it contains 17 Ser/Thr residues, 7 of which were identified phosphorylation sites, and 2 were extensively phosphorylated tryptic peptides identified in the 15N

Fig. 2. Mapping phosphorylation sites and peptides preferentially phos- experiments. All constructs carrying FRQ mutations were trans- GENETICS phorylated in hyperphosphorylated FRQ by 15N metabolic labeling and quan- formed into the frq10 strain and homokaryon strains were titative MS. (A) MS spectrum of one FRQ phosphopeptide (with both 14N and obtained. A frq10 strain that carries the wild-type frq construct 15N forms) showing the increase of 15N-labeled peptide peaks in the purified (KAJ120) was used as the control. FRQ sample (DD6 vs. DD18). The m/z of the 15N/14N phosphopeptide pair and ⌬ Due to the partial coverage of MS analyses, we also mutated their mass difference ( mass) are indicated. Hyperphosphorylated FRQ was additional Ser/Thr residues in regions that were not covered by 15N labeled. (B) A table of identified phosphopeptides with significant enrich- ment of 15N peaks compared with unphosphorylated peptide controls. (C)MS our MS experiments but are highly conserved in fungal FRQ spectrum of one representative unphosphorylated peptide showing the dis- homologs. These residues reside downstream of the FRQ-CK-1a appearance of its 15N form in the purified FRQ. (D) Table showing the iden- binding domain (M9), in the PEST-1 region (M10), and the tified outliers of unphosphorylated peptides that have low 15N/14N ratios. C-terminal tail (M18, M19). As shown in Fig. 3A, comparison of FRQ phosphorylation profiles in LL cultures by Western blot analysis showed that hyperphosphorylated FRQ species detected 15N/14N unphosphorylated FRQ peptide pairs were used as in the KAJ120 strain disappeared in the M9, M10, and M18ϩ19 controls to determine relative amounts of FRQ protein contrib- (combination of mutations in M18 and M19) mutants in LL uted by each sample. As shown in Table S2, for a representative samples, suggesting that these regions contain in vivo phosphor- experiment (DD6 vs. DD18) the standard deviation for the ylation sites. relative levels of unphosphorylated FRQ peptides was Ͻ10%, The circadian conidiation rhythms of these mutants were indicating accuracy of the method. examined by race tube assays in comparison with the control Twenty-six FRQ phosphorylation sites were identified in these strain (KAJ120, period Ϸ22.3 h) (Fig. 3B). Importantly, no labeling experiments. The fewer number of phosphorylation mutants were arrhythmic and most exhibited robust conidiation sites identified was most likely due to the reduced amounts rhythms, indicating that there is no single phosphorylation event (Ϸ0.5–1 ␮g) of purified Myc-His-FRQ (limited by the cost of 15N that is essential for clock function. The M7 mutant exhibited the labeling). Although levels of some FRQ phosphopeptides were most severe circadian phenotype: a very long period (Ϸ32 h) for comparable between the control (14N) and hyperphosphorylated 3–4 days before becoming arrhythmic. (15N) samples, the phosphorylation levels of 19 sites were Period differences between the mutants and the control strain significantly increased in the 15N samples (Figs. 1 and 2 A and B), are summarized in Fig. 3C. Although a few mutants (M1, M4, indicating that a majority of FRQ sites are preferentially phos- M15, and M16) exhibited similar periods as the control, all other phorylated when FRQ becomes hyperphosphorylated. Several strains showed significantly different periods. Mutants M5–13, pairs of singly and doubly phosphorylated peptides were also which bear mutations in the central part of FRQ, showed identified (Fig. 2B), and in every case the 15N/14N ratios for the significantly longer periods (2–9 h) than the control. Mutations singly phosphorylated peptides were lower than doubly phos- causing the largest differences are clustered in 2 regions. One is phorylated peptides, demonstrating sequential phosphorylation downstream of the FRQ NLS (amino acids 211–283, M5, M6, events. M7), which is highly represented by phosphorylation sites. The Whereas the vast majority of unphosphorylated FRQ peptides other region is downstream of the FRQ-CK-1a interaction showed similar amounts in 15N and 14N samples, we noticed that domain (M9, M10, M11, M12, and M13), containing the PEST-1 the levels of some unphosphorylated peptides were dramatically domain. The PEST-1 region (mutated in M10) was also shown enriched in the 14N samples (Fig. 1, indicated by blue lines, and to promote FRQ degradation and is phosphorylated by CK-1a Fig. 2 C and D). Because phosphorylation of a peptide results in in vitro (24). Importantly, all of these sites except for M9 (not the decrease of its unphosphorylated form, disappearance of covered by MS) are preferentially phosphorylated when FRQ is unphosphorylated peptides in the 15N sample suggests that they extensively phosphorylated, suggesting that the phosphorylation are extensively phosphorylated in hyperphosphorylated FRQ. of these sites shortens the period of the clock. In addition, these As shown in Fig. S2, preferential phosphorylation of one FRQ sites are phosphorylated by CK-1a, CKA, or both combined,

Tang et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on October 5, 2021 Fig. 3. Mutational analyses of FRQ phosphorylation sites. (A) Western blot analysis showing the FRQ phosphorylation proﬁles of the indicated strains’ Fig. 4. Regulation of FRQ stability by multiple FRQ phosphorylation events. cultures in LL. Arrows indicate the extensively phosphorylated FRQ forms in (A) Western blot analysis showing the circadian expression of FRQ in the the KAJ120 strain. (B) Race tube assays showing the circadian conidiation wild-type and indicated mutant strains in DD. (B) Western blot and densito- rhythms of the phosphorylation mutants in DD. (C) Summary diagram show- metric analyses showing the pairwise comparisons of FRQ degradation kinet- ing the period differences between the KAJ120 strain and the phosphoryla- ics between the KAJ120 strain and indicated mutants. Each comparison was tion mutants. Error bars indicate standard deviation of the mean. independently performed at least 3 times in LL. Error bars indicate standard deviations. indicating that these 2 kinases play major roles in regulating circadian period. Regulation of FRQ Stability by Multiple Phosphorylation Events. We In contrast to these period-lengthening mutations, mutations examined the circadian rhythms of FRQ expression in mutants of sites in the C-terminal end of FRQ mostly resulted in with the most severe phenotypes (M7 and M9). As shown short-period circadian conidiation rhythms (M14, M17, M18, previously, the wild-type strain exhibited robust circadian and M19). Therefore, phosphorylation of FRQ at different rhythms of FRQ amount and phosphorylation profile (Fig. 4A). regions has opposing effects on period length. Interestingly, the In contrast, the oscillations of FRQ levels and phosphorylation sites mutated in M14, like those with period-lengthening effects profiles were severely impaired in the M7 and M9 mutants. Both when mutated, are also preferentially phosphorylated when mutants showed a long period and low amplitude oscillation of FRQ becomes extensively phosphorylated, suggesting that the FRQ levels, consistent with their long period phenotypes on race phosphorylation of these sites counters the effects of other sites. tubes. Moreover, after the light to dark transition, when FRQ The C-terminal tail of FRQ contains several Ser/Thr residues protein is rapidly degrading, the levels of FRQ in both M7 and that are highly conserved among the fungal FRQ homologs. M9 strains decreased very slowly, suggesting that FRQ degra- Both M18 and M19 in this region resulted in period shortening, dation was impaired. suggesting that the phosphorylation of the C-terminal tail is We then compared the FRQ stability in the M7, M9, and other important for promoting period-lengthening of the clock. Taken mutants with significant period changes with those in the together, the circadian conidiation rhythms of these mutants KAJ120 strain by cycloheximide (CHX) treatment (Fig. 4B). As strongly suggest that multiple independent phosphorylation expected, stability of FRQ in the M7 and M9 strains was events at different regions of FRQ play a major role in setting significantly increased compared with the KAJ120 strain. Other the circadian period. long-period mutants (M5, M8, and M13) also displayed in- S885 and S887 (mutation in M17) in the PEST-2 domain are creased FRQ stability. Thus, multiple independent phosphory- 2 previously identified, in vitro CK-1a sites. These residues were lation events at different regions of FRQ can promote FRQ thought to be essential for clock function due to their role in the degradation, which explains the long-period phenotypes of these positive feedback loop (27). In contrast, we found that that M17 mutants. exhibited a robust circadian conidiation rhythm with a 21-h In contrast, the M18 strain possesses the shortest period, and period, indicating that the phosphorylation of these 2 sites does the degradation rate of FRQ in this mutant was faster than in the not play a major role in clock function. control strain. This result suggests that FRQ phosphorylation at

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904898106 Tang et al. Downloaded by guest on October 5, 2021 ylation analyses led to the identification of 33 additional potential phosphorylation sites. In sharp contrast to the large number of phosphorylation sites identified on FRQ, only 1 phosphorylation site was identified on FRH (a protein that is 117 aa longer than FRQ and has 101 Ser/Thr residues). This result suggests that FRQ may possess a unique 3D structure that makes it extremely accessible to kinases. The fact that FRQ phosphorylation sites are located throughout its ORF suggests that FRQ exists as a nonglobular shape with most of the FRQ regions exposed at the protein surface. Consistent with this notion, previous gel-filtration chro- matography experiments showed that FRQ was eluted in frac- tions with a size of Ϸ1 million daltons, whereas its sedimentation size was much smaller in sucrose-gradient analysis (19). Our quantitative MS analyses showed that a majority of FRQ phosphorylation sites were preferentially phosphorylated in the extensively phosphorylated FRQ. Thus, the circadian rhythm of FRQ phosphorylation profile is due to progressive phosphorylation of multiple independent sites throughout the FRQ ORF. The progressive nature of phosphorylation is likely due to the substrate preference of the 2 casein kinases: The phosphorylation of one site will prime the phosphorylation of nearby sites. Our systematic mutagenesis of the identified phosphorylation Fig. 5. Both period-lengthening and period-shortening mutations contrib- sites showed that FRQ phosphorylation is the major determinant ute to period and FRQ stability. (A) Race tube analyses showing the circadian in circadian period length by controlling FRQ stability. Most of conidiation rhythms of the indicated strains. (B) Western blot analysis showing the mutants are long-period due to increased FRQ stability, the comparison of FRQ degradation kinetics between the M9 and M9ϩ18 suggesting that multiple phosphorylation events promote FRQ GENETICS mutants. degradation. Our study identified 2 major regions that mediate phosphorylation-dependent FRQ degradation. The first region is located downstream of the FRQ-CK-1a interaction domain the C terminus results in the stabilization of FRQ. Thus, from amino acid 500–690, which includes the previously iden- different phosphorylation events can have opposing effects on tified PEST-1 domain and the S513 (22, 24). The other region is FRQ stability. To examine the interplay between the opposing effects of located downstream of the NLS (amino acids 200–300), a region FRQ phosphorylation on period determination and FRQ sta- of FRQ that is enriched with phosphorylation sites. ϩ Phosphorylated FRQ is degraded through the ubiquitin- bility, we created a mutant (M9 18) that contains the mutations FWD-1 of M9 and M18. As shown in Fig. 5A, the M9ϩ18 strain exhibited proteasome pathway mediated by the SCF complex (20, conidiation rhythms with an intermediate period length between 21). FWD-1 binds FRQ through its WD-40 domain, a phos- the M9 and M18 mutants. In addition, the M9ϩ18 strain showed phopeptide-binding domain. The mammalian FWD-1 homolog ␾ ␾ a significantly increased rate of FRQ degradation relative to the beta-TRCPs recognize a DpSG XpS ( is any hydrophobic M9 mutant (Fig. 5B). These results suggest that the overall amino acid) consensus motif on their substrates (30). Sequence determination of circadian period length in Neurospora is con- comparison between FWD-1 and beta-TRCP showed that amino ␾ trolled by the stability of FRQ, which itself depends on the acid residues that interact with the DpSG XpS motif in beta- interaction between different phosphorylated events. TRCP are conserved in FWD-1, suggesting that FWD-1 also recognizes a similar motif as a high-affinity binding site. Al- Discussion though a DSGDKS sequence is found at the N-terminal end of In this study, we identified 43 in vivo FRQ phosphorylation sites FRQ, its phosphopeptide form was never detected and it is not by MS analysis. In addition, we mapped the in vitro FRQ present in the sFRQ form (19). The lack of such a motif in other phosphorylation sites by CK-1a and CKII. These analyses regions and the involvement of multiple phosphorylation events showed that CK-1a and CKII phosphorylate most of the iden- in FRQ degradation suggest that FWD-1 does not recognize tified in vivo FRQ phosphorylation sites, indicating that CK-1a FRQ by a single high affinity site. Based on our results, we and CKII are the 2 major FRQ kinases in vivo (12, 23, 25). Our propose that there are multiple suboptimal FWD-1 binding sites results also indicate that CK-1a and CKII are not redundant in on FRQ and phosphorylation of FRQ at multiple sites can FRQ phosphorylation despite their shared role in regulating progressively promote its binding with FWD-1 (Fig. 6). There- FRQ stability. They also work in tandem to ensure proper fore, the mutation of one site impairs, but does not completely phosphorylation of FRQ. Furthermore, our in vitro phosphor- abolish, the FRQ-FWD-1 interaction. The different period-

Fig. 6. A model explaining how multiple FRQ phosphorylation events control FRQ stability by regulating the interaction between FRQ and SCFFWD-1. Lollipops on FRQ symbolize phosphorylation events. Multiple phosphorylation events in the middle of FRQ promote its degradation via SCFFWD-1-dependent ubiquitination and degradation whereas the phosphorylation events near the C terminus stabilize FRQ.

Tang et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on October 5, 2021 lengthening effects of these sites also suggest that some sites play mines FRQ stability and sets period length of the clock. How- more important roles than others. ever, it is entirely possible that FRQ phosphorylation has PER proteins in animals also exhibit progressive and extensive additional functions other than regulating FRQ stability. phosphorylation before their degradation by a conserved The opposing effects of FRQ phosphorylation on its stability ubiquitin-proteasome pathway (20, 31, 32). In mammals, 2 are similar to those found for the mammalian PER2. The independent beta-TRCP-interacting domains were identified on inhibition of phosphorylation by CK1 inhibitors stabilizes PER2 PER1 and PER2, and neither domain contained the high- and lengthens the period (32, 35). However, the phosphorylation affinity DSG␾XS consensus motif (32, 33), suggesting a similar of PER2 by CK1 at the FASPS site, or by CK2 at the N-terminal degradation mechanism to that we propose for Neurospora.In sites, results in the destabilization of PER and shortening of the Drosophila, however, an atypical SLIMB-binding site on the period (35, 36). Together, these results suggest that the con- PERIOD motif in the N-terminal end of the PER protein was trasting effect of phosphorylation is a common theme in the recently shown to be a major DBT-dependent, Slimb-interacting eukaryotic circadian clocks. domain (34). Although other phosphorylation events in the N terminus also appear to contribute to the PER-Slimb interaction, Materials and Methods surprisingly, most of hyperphosphorylation events of Drosophila Strains and Growth Conditions. The bd;frq10;his-3 and fwd-1RIP,bd,his-3 strains were used as host strains for constructs with his-3 targeting vectors. The PER do not have major effects on its stability. 10 Although most FRQ phosphorylation events promote its control strain for most experiments was a frq strain transformed with KAJ120, a vector containing the wild-type frq gene. All constructs bearing FRQ degradation, mutations of sites at the C-terminal part result in phosphorylation mutations were transformed into the frq10 strain. Liquid period shortening by decreasing FRQ stability. A combination of cultures were grown in minimal medium (1ϫ Vogel’s, 2% glucose). Race tube a period-lengthening mutation with a period-shortening muta- assays were performed in media composed of 1ϫ Vogel’s salts, 0.1% glucose, tion led to an intermediate period length. Thus, FRQ phosphor- 0.17% arginine, and 50 ng/mL biotin. ylation plays opposing roles in regulating its stability. This idea Additional materials and methods are in the SI Text. is consistent with our previous observation that PKA can phosphorylate and stabilize FRQ (16). Therefore, the effects of ACKNOWLEDGMENTS. We thank Haiyan Yuan for technical assistance, other members of the laboratory for critical comments, and Joost W. Gouw from FRQ phosphorylation events that promote degradation are Utrecht University for assistance in setting up MSQuant software. This re- countered by both dephosphorylation and phosphorylation search was supported by grants from the National Institutes of Health and the events that stabilize FRQ. The balance of these 2 forces deter- Welch Foundation (to Y.L.).

1. Sehgal A (2004) Molecular biology of circadian rhythms (John Wiley & Sons, Hoboken, 20. He Q, et al. (2003) FWD1-mediated degradation of FREQUENCY in Neurospora estab- NJ). lishes a conserved mechanism for circadian clock regulation. EMBO J 22:4421–4430. 2. Young MW, Kay SA (2001) Time zones: A comparative genetics of circadian clocks. Nat 21. He Q, Cheng P, He Q, Liu Y (2005) The COP9 signalosome regulates the Neurospora Rev Genet 2:702–715. circadian clock by controlling the stability of the SCFFWD-1 complex. Genes Dev 3. Heintzen C, Liu Y (2007) The Neurospora crassa circadian clock. Adv Genet 58:25–66. 19:1518–1531. 4. Yu W, Hardin PE (2006) Circadian oscillators of Drosophila and mammals. J Cell Sci 22. Liu Y, Loros J, Dunlap JC (2000) Phosphorylation of the Neurospora clock protein 119:4793–4795. FREQUENCY determines its degradation rate and strongly influences the period length 5. Liu Y, Bell-Pedersen D (2006) Circadian rhythms in Neurospora crassa and other of the circadian clock. Proc Natl Acad Sci USA 97:234–239. filamentous fungi. Eukaryot Cell 5:1184–1193. 23. Yang Y, Cheng P, Liu Y (2002) Regulation of the Neurospora circadian clock by casein 6. Dunlap JC (2006) Proteins in the Neurospora circadian clockworks. J Biol Chem kinase II. Genes Dev 16:994–1006. 281:28489–28493. 24. Gorl M, et al. (2001) A PEST-like element in FREQUENCY determines the length of the 7. Crosthwaite SK, Dunlap JC, Loros JJ (1997) Neurospora wc-1 and wc-2: Transcription, circadian period in Neurospora crassa. EMBO J 20:7074–7084. photoresponses, and the origins of circadian rhythmicity. Science 276:763–769. 25. Yang Y, Cheng P, He Q, Wang L, Liu Y (2003) Phosphorylation of FREQUENCY protein 8. Cheng P, Yang Y, Liu Y (2001) Interlocked feedback loops contribute to the robustness by casein kinase II is necessary for the function of the Neurospora circadian clock. Mol of the Neurospora circadian clock. Proc Natl Acad Sci USA 98:7408–7413. 9. Froehlich AC, Loros JJ, Dunlap JC (2003) Rhythmic binding of a WHITE COLLAR- Cell Biol 23:6221–6228. containing complex to the frequency promoter is inhibited by FREQUENCY. Proc Natl 26. Yang Y, et al. (2004) Distinct roles for PP1 and PP2A in the Neurospora circadian clock. Acad Sci USA 100:5914–5919. Genes Dev 18:255–260. 10. Cheng P, Yang Y, Gardner KH, Liu Y (2002) PAS domain-mediated WC-1/WC-2 inter- 27. Schafmeier T, Kaldi K, Diernfellner A, Mohr C, Brunner M (2006) Phosphorylation- action is essential for maintaining the steady state level of WC-1 and the function of dependent maturation of Neurospora circadian clock protein from a nuclear repressor both proteins in circadian clock and light responses of Neurospora. Mol Cell Biol toward a cytoplasmic activator. Genes Dev 20:297–306. 22:517–524. 28. Aronson BD, Johnson KA, Dunlap JC (1994) The circadian clock locus frequency: A single 11. He Q, Liu Y (2005) Molecular mechanism of light responses in Neurospora: From ORF defines period length and temperature compensation. Proc Natl Acad Sci USA light-induced transcription to photoadaptation. Genes Dev 19:2888–2899. 91:7683–7687. 12. He Q, et al. (2006) CKI and CKII mediate the FREQUENCY-dependent phosphorylation 29. Pregueiro AM, Liu Q, Baker CL, Dunlap JC, Loros JJ (2006) The Neurospora checkpoint of the WHITE COLLAR complex to close the Neurospora circadian negative feedback kinase 2: A regulatory link between the circadian and cell cycles. Science 313:644–649. loop. Genes Dev 20:2552–2565. 30. Wu G, et al. (2003) Structure of a beta-TrCP1-Skp1-beta-catenin complex: Destruction 13. Belden WJ, Loros JJ, Dunlap JC (2007) Execution of the circadian negative feedback motif binding and lysine specificity of the SCF(beta-TrCP1) ubiquitin ligase. Mol Cell loop in Neurospora requires the ATP-dependent chromatin-remodeling enzyme 11:1445–1456. CLOCKSWITCH. Mol Cell 25:587–600. 31. Grima B, et al. (2002) The F-box protein slimb controls the levels of clock proteins period 14. Aronson B, Johnson K, Loros JJ, Dunlap JC (1994) Negative feedback defining a and timeless. Nature 420:178–182. circadian clock: Autoregulation in the clock gene frequency. Science 263:1578–1584. 32. Eide EJ, et al. (2005) Control of mammalian circadian rhythm by CKIepsilon-regulated 15. Schafmeier T, et al. (2005) Transcriptional feedback of Neurospora circadian clock gene proteasome-mediated PER2 degradation. Mol Cell Biol 25:2795–2807. by phosphorylation-dependent inactivation of its transcription factor. Cell 122:235– 33. Shirogane T, Jin J, Ang XL, Harper JW (2005) SCFbeta-TRCP controls clock-dependent 246. transcription via casein kinase 1-dependent degradation of the mammalian period-1 16. Huang G, et al. (2007) Protein kinase A and casein kinases mediate sequential phos- (Per1) protein. J Biol Chem 280:26863–26872. phorylation events in the circadian negative feedback loop. Genes Dev 21:3283–3295. 17. Cha J, Chang SS, Huang G, Cheng P, Liu Y (2008) Control of WHITE COLLAR localization 34. Chiu JC, Vanselow JT, Kramer A, Edery I (2008) The phospho-occupancy of an atypical by phosphorylation is a critical step in the circadian negative feedback process. EMBO SLIMB-binding site on PERIOD that is phosphorylated by DOUBLETIME controls the J 27:3246–3255. pace of the clock. Genes Dev 22:1758–1772. 18. Cheng P, He Q, He Q, Wang L, Liu Y (2005) Regulation of the Neurospora circadian clock 35. Vanselow K, et al. (2006) Differential effects of PER2 phosphorylation: Molecular basis by an RNA helicase. Genes Dev 19:234–241. for the human familial advanced sleep phase syndrome (FASPS). Genes Dev 20:2660– 19. Garceau N, Liu Y, Loros JJ, Dunlap JC (1997) Alternative initiation of translation and 2672. time-specific phosphorylation yield multiple forms of the essential clock protein 36. Maier B, et al. (2009) A large-scale functional RNAi screen reveals a role for CK2 in the FREQUENCY. Cell 89:469–476. mammalian circadian clock. Genes Dev 23:708–718.

6of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904898106 Tang et al. Downloaded by guest on October 5, 2021