![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
RESEARCH COMMUNICATION
1 Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA; 2 Department of Plant Pathology and Microbiology, Faculty of Agricultural Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel
 |
Abstract |
|---|
 Top Abstract Results and DiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
[Keywords: frequency; phosphorylation; phosphatase; PP1; PP2A]
Received September 15, 2003; revised version accepted December 15, 2003.
; Young and Kay 2001). Several kinases, including casein kinase I (CKI) and CKII, have been shown to phosphorylate and regulate the key clock components in eukaryotic systems (Kloss et al. 1998; Sugano et al. 1999; Lowrey et al. 2000; Gorl et al. 2001; Yang et al. 2002).
FREQUENCY (FRQ), WHITE COLLAR-1 (WC-1), and WC-2 proteins are three key components in the Neurospora frq-wc-based circadian oscillator (Loros and Dunlap 2001). In addition to being the circadian blue light photoreceptor (Froehlich et al. 2002; He et al. 2002), in the dark, WC-1 forms a heterodimeric complex with WC-2 through their PAS domains and activate frq transcription by binding its promoter (Crosthwaite et al. 1997; Talora et al. 1999; Cheng et al. 2001b, 2002, 2003; Froehlich et al. 2003). FRQ, the negative element of the feedback loop, inhibits its own transcription through interactions with the WC complex (Aronson et al. 1994; Cheng et al. 2001a; Denault et al. 2001; Froehlich et al. 2003). FRQ protein is immediately phosphorylated after its synthesis and becomes extensively phosphorylated prior to its degradation by the ubiquitin/proteasome pathway (Garceau et al. 1997; Liu et al. 2000; He et al. 2003). In constant darkness, FRQ is not only robustly rhythmic in its cellular concentration, but also in its phosphorylation states, so that the level and the phosphorylation status of FRQ define the time of the clock during a circadian cycle (Garceau et al. 1997).
Phoshorylation of FRQ is mediated by CKI, CKII, and a calcium/calmodulin-dependent kinase (Gorl et al. 2001; Yang et al. 2001, 2002, 2003). Molecular, genetic, and biochemical experiments have shown that phosphorylation of FRQ has several functions. Mutations of the FRQ phosphorylation sites lead to the stabilization of the FRQ protein and long period rhythms of the clock (Liu et al. 2000; Gorl et al. 2001; Yang et al. 2003). In strains with the CKII catalytic subunit or one of its regulatory subunits disrupted, the protein level of FRQ is high and more stable, and the clock function is either completely abolished or severely affected (Yang et al. 2002, 2003). These data indicate that phosphorylation of FRQ promotes its degradation. In addition, we have shown previously that the CKII-mediated FRQ phosphorylation regulates the FRQ-WC interaction and is important for the closing of the circadian negative feedback loop (Yang et al. 2002, 2003). In a CKII mutant, frq mRNA levels are high despite the high FRQ levels.
In contrast to the well-characterized clock functions of several kinases in various circadian systems, little is known about the potential roles of protein phosphatases. Unlike the large number of protein kinases in eukaryotes, there are only a few highly conserved catalytic subunits of protein phosphatases (Virshup 2000; Cohen 2002). Protein phosphatase 1 (PP1) and PP2A are two major eukaryotic serine/threonine protein phosphatases. The catalytic subunits of the Neurospora PP1 and PP2A are 87% and 85% identical to their homologs in human, respectively. Protein phosphatases carry out their diverse cellular functions with the help of a large number of regulatory proteins, which form heteromultimeric complexes with the catalytic subunits and regulate their activity, specificity, and cellular localization. In this study, we show that PP1 and PP2A are important regulators of the Neurospora circadian clock. Surprisingly, down-regulation of these two phosphatases leads to distinct phenotypes of the circadian clock.
|
Results and Discussion |
|---|
|
TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
; Zeke et al. 2003), we used repeat-induced point mutation (RIP; Cambareri et al. 1989) to obtain partially functional mutants of these two genes. RIP introduces random, but exclusively G-C to A-T point mutations during sexual cycle. Whereas we are unable to obtain any viable mutant of pph-1, one mutant of the ppp-1 gene (ppp-1RIP) was isolated. Sequencing of endogenous ppp-1 gene revealed four missense mutations (all G to A mutations) in the coding region of the gene, that is, Asp 71-Asn, Glu 84-Lys, Met 183-Ile, and Val 223-Ile. Among them, Asp 71 is conserved in all known PP1 catalytic subunit genes, whereas Glu 84, Met 183, and Val 223 are variable in some PP1 homologs. To examine whether these mutations led to the decrease of the phosphatase activity of PP1, phosphatase assay (see supplemental materials) was performed using extracts prepared from cultures grown in LL, a condition that the clock is not running. The 32P-labeled phosphorylase a is used as the substrate of the assay in the presence or absence of inhibitor-2, a specific protein inhibitor for PP1 (Krebs and Fischer 1962; Yatzkan et al. 1998; Cohen 2002). As shown in Figure 1A, the inhibitor-2-sensitive phosphatase acitivity was significantly reduced in the ppp-1RIP strain, indicating that the mutations resulted in a partial functional PPP-1 protein. The ppp-1RIP strain exhibits near normal appearance in slants and its growth rate is only 
20% slower than the wild-type strain (Fig. 1B), suggesting that the 70% decrease in PP1 activity did not severely affect its essential cellular functions and that the amount of PPP-1 is probably in excess in a wild-type strain, or, alternatively, other PPs can partially compensate for the reduction in PP1 activity.
|
1 h shorter than that of the wild-type strain (P value = 2.5E-06; Fig. 1B). More prominently, the phase of the first conidiation band in the mutant was 3 h advanced compared with that of the wild-type strain (P value = 6.5E-11). To further confirm the advanced phase of the ppp-1RIP strain, race tube assays were performed under light/dark cycles (12 h dark/12 h light). As seen in Figure 1C, although the period of the mutant was entrained to 24 h by the light/dark cycles, the phases of the conidiation peaks in the mutants were 4 h earlier than those of the wild-type (P value = 2.3E-08). To monitor the molecular rhythms in DD, Western blot and Northern analyses were performed to examine the expression of FRQ protein and frq mRNA in the ppp-1RIP strain. The rhythms of protein level and phosphorylation states of FRQ were robust in the ppp-1RIP strain, but a significantly advanced phase was observed as compared with the wild-type strain (Fig. 2A; Supplemental Fig. 1). At DD12 (12 h in constant darkness) and DD32, when FRQ proteins in the wild-type strain were extensively phosphorylated, the newly synthesized hypophosphorylated FRQ forms were seen in the mutant, indicating an advanced phase. Taken together, these data suggest that PP1 is a regulator of the Neurospora circadian clock.
|
), between the wild-type and the ppp-1RIP trains. As shown in Figure 2B, the disappearance of FRQ in the ppp-1RIP strain was faster than in the wild-type strain. Similar results were obtained in seven independent CHX experiments (Fig. 2B) and in experiments that measured the FRQ degradation rate after a light to dark transition (data not shown). Interestingly, the faster FRQ degradation in the mutant appears to be mostly due to the lack of a lag between the addition of CHX and FRQ degradation in the first 3 h of the CHX treatment. Together, these data indicate that FRQ is less stable in the ppp-1RIP strain, explaining its advanced phase and the shorter period of the clock.
The eukaryotic PP2A holoenzyme consists of a tightly associated core complex containing the PP2A catalytic subunit (PPH-1 in Neurospora; C subunit) and a scaffolding subunit (A subunit). This dimeric core can form trimeric complexes with a third variable regulatory subunit (B subunit), which regulates the activity, specificity, and the localization of the holoenzyme (Virshup 2000). To study the role of one of the PP2A holoenzymes in the Neurospora circadian clock, we studied the circadian clock phenotype in a mutant (rgb-1RIP), in which one of the Neurospora PP2A B subunits (RGB-1) had been disrupted by RIP (Yatzkan and Yarden 1999). RGB-1 is a highly conserved B subunit of PP2A found in fungi, plants, insects, and mammals. The disruption of the rgb-1 gene led to slow growth, abnormal morphology, and defects in several developmental processes. Sequencing of the RIP-inactivated allele of rgb-1 revealed that in addition to many missense mutations, there are two premature stop codons at amino acids 156 and 219. Thus, this mutant is not expected to make any functional RGB-1 protein. Phosphatase assays showed that the total phosphatase activity was 20% lower in the rgb-1RIP strain than in the wild-type (Fig. 3A). This data suggests that the loss of RGB-1 protein lead to the decrease of the PP2A activity.
|
10% of the wild type) and poor production of aerial hyphae and conidia, the circadian conidiation rhythm in the rgb-1RIP strain was not easily observed by race tube assay. However, hyphae-banding rhythms could be seen in some race tubes in constant darkness (Fig. 3B). These banding rhythms did not appear to exhibit regular period length; at times, the period was 24 h, but sometimes periods were significantly longer than 1 d. This irregular banding pattern suggests that the circadian clock probably does not function properly in the rgb-1RIP strain. To examine whether the FRQ degradation rate was increased in the rgb-1RIP strain, we measured the degradation rates of FRQ after the addition of CHX (Fig. 3C) or following a light to dark transition (data not known). Our results indicated that the degradation rate of FRQ was not increased in the rgb-1RIP strain.
Rhythmic Western blot analyses were then performed to examine whether the clock was functional at the molecular level in the mutant strain. As shown in Figure 4A, a low-amplitude FRQ protein oscillation was seen in the mutant in constant darkness, but the period of the oscillation was 4-6 h longer than that of the wild type. In addition, the overall levels of FRQ protein in DD in the rgb-1RIP strain were significantly lower than those in the wild-type strain. Similar results were obtained in multiple independent experiments.
|
How do PP1 and PP2A regulate the Neurospora clock? The increase of FRQ degradation rate in the ppp-1RIP strain suggests that PP1 may directly dephosphorylate FRQ to inhibit its degradation. On the other hand, the low levels of frq RNA and FRQ protein in the rgb-1RIP strain suggest that the normal circadian feedback loop is impaired in this mutant. Thus, it is likely that PP2A may also dephosphorylate FRQ directly. To test these possibilities, Myc-tagged PPP-1 (Myc-PPP-1) or PPH-1 (Myc-PPH-1) was expressed in a wild-type Neurospora strain (wild type, Myc-PPP-1 or wild type, Myc-PPH-1). To examine whether these Myc-tagged phosphatases expressed in Neurospora can dephosphorylate the endogenously expressed phosphorylated FRQ, total extracts of either strain were mixed with extracts of a frq null strain expressing the Myc-tagged FRQ protein (frq10, Myc-FRQ; Cheng et al. 2001a). As a negative control, the extracts of the frq10, Myc-FRQ strain were mixed with a wild-type strain (containing no Myc-tagged protein). After immunoprecipitation using Myc antibody, they were incubated in phosphatase assay buffer. As shown in Figure 5A, the inclusion of the Myc-PPP-1 extracts led to the reduction of the extensively phosphorylated Myc-FRQ species. The inclusion of the Myc-PPH-1 also led to the gel mobility-shift changes of Myc-FRQ (Fig. 5B). However, unlike that observed in the extracts containing Myc-PPP-1, the presence of Myc-PPH-1 resulted in the appearance of hypophosphorylated FRQ species that were not normally seen in the control sample. Such hypophosphorylated FRQ forms were similar to those previously observed in the CKII mutants or when FRQ was treated by 
phosphatase (Garceau et al. 1997; Yang et al. 2002). Similar results were obtained in three independent experiments. These data suggest that both PP1 and PP2A expressed in Neurospora can directly dephosphorylate endogenous FRQ in vitro. The difference in Myc-FRQ phosphorylation patterns after PP1 or PP2A treatment suggests that these phosphatases might dephosphorylate FRQ at distinct sites.
|
The evidence presented here indicates that PP1 and PP2A have different roles in the regulation of the Neurospora circadian clock. PP1 influences the clock by regulating the stability of FRQ, a role that is predicted from the function of FRQ phosphorylation in promoting FRQ degradation. Unlike PP1, the PP2A holoenzyme containing RGB-1 does not appear to play a role in regulating FRQ stability. In the rgb-1RIP strain, the levels of FRQ protein and frq mRNA are low, and the clock oscillates with a low amplitude and long period. The low levels of FRQ and frq are in contrast to what was observed previously in a CKII mutant (Yang et al. 2002), suggesting that the function of PP2A opposes that of CKII, probably by preventing the closing of the negative feedback loop. The direct dephosphorylation of the endogenous FRQ by the Neurospora expressed PP1 and PP2A in vitro and changes in FRQ phosphorylation profile in the rgb-1RIP strain further suggest that both PP1 and PP2A may regulate the clock by desphosphorylating FRQ. Therefore, PP1 and PP2A may function in coordination with the FRQ kinases to determine the phosphorylation status of FRQ, which in turn defines the time in a circadian cycle. However, it is also possible that these phosphatases regulate the clock indirectly by dephosphorylating other proteins, such as by affecting the activity of the FRQ kinases. In the rgb-1RIP strain, its slow growth and developmental phenotype may also contribute to its clock phenotypes. But, we have previously shown that there is no direct relationship between severe growth and developmental phenotypes and the function of the clock (Yang et al. 2002, 2003).
The functions of the eukaryotic PP1 and PP2A are regulated by numerous regulatory subunits (Virshup 2000; Cohen 2002). Furthermore, it has been shown that PP2A can form complexes with CKII or a calcium-calmodulin-dependent kinase (Heriche et al. 1997; Westphal et al. 1998), suggesting that phosphorylation and dephosphorylation of cellular proteins are tightly coupled. Interestingly, both CKII and a calcium-calmodulin-dependent kinase were shown to be FRQ kinases in Neurospora (Yang et al. 2001, 2002), raising the possibility that the functions of CKII and CAMK-1 in FRQ phosphorylation are regulated by PP2A.
The roles of CKI and CKII appear to be conserved in various eukaryotic circadian systems (Kloss et al. 1998; Price et al. 1998; Sugano et al. 1999; Lowrey et al. 2000; Gorl et al. 2001; Lin et al. 2002; Yang et al. 2002, 2003; Akten et al. 2003). Recent evidence in Drosophila and Neurospora further demonstrated that the proteasome-mediated degradation of the phosphorylated clock proteins are both mediated by a conserved F-box/WD40-repeat-containing protein (Grima et al. 2002; Ko et al. 2002; He et al. 2003). These studies suggest that the posttranscriptional regulators mediating phosphorylation and degradation of clock proteins may be the evolutionary links among different eukaryotic circadian systems. Therefore, it is tempting to speculate that the highly conserved PP1 and PP2A enzymes will play important roles in other eukaryotic circadian systems.
|
Materials and methods |
|---|
|
TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
A bd,a strain and a wild-type Neurospora strain (FGSC 987, 74-OR23-1A) were used as control strains in this study. Because both strains contained wild-type clocks, for simplicity, they were both called wild-type strains. All other strains described in this study contain the bd mutation, except for the rgb-1RIP strain, which was described previously (Yatzkan and Yarden 1999). The FGSC 987 strain (without the bd mutation) was used as the control strain in experiments described in Figures 4 and 5C, while the bd,a strain was used as the control strain in the rest of the study. Liquid culture conditions were the same as described previously using medium containing 1 x Vogel's and 2% glucose (Aronson et al. 1994). Conidiation rhythms were examined on race tubes containing glucose/arginine medium (1 x Vogel's, 0.1% glucose, 0.17% arginine, 50 ng/mL biotin, and 1.5% agar). Densitometric analysis of race tubes and calculations of period length and phase were performed as previously described (Roenneberg and Taylor 2000).
Mutation of ppp-1 in Neurospora by repeat-induced point mutation
A PCR fragment containing the PP1 ORF was cloned into pDE3dBH and introduced into the his-3 locus of 301-6 by transformation. A positive transformant was crossed with a wild-type strain. DNA sequencing was performed to identify strains in which the endogenous ppp-1 gene was mutated. The bd, ppp-1RIP strain grows on histidine-free medium and contains only one copy of the ppp-1 gene.
Expression of Myc-tagged PPP-1 and PPH-1 in
Neurospora A PCR fragment containing the entire ORF and the 3' UTR of PPP-1 or PPH-1 was cloned into pqa.5Myc (He et al. 2003) to create pqa-Myc-PPP-1 or pqa-Myc-PPH-1. The resulting plasmids were transformed into a wild-type strain (301-6) at the his-3 locus. The expression of Myc-PPP-1 or Myc-PPH-1 in these transformants was confirmed by Western blot analysis using a monoclonal c-Myc antibody (9E10, Santa Cruz Biotechnology).
Immunoprecipitation, followed by dephosphorylation reaction
For immunoprecipitation, 50 µg of Neurospora total extracts of frq10, Myc-FRQ was mixed with 1 mg extracts of wild type, Myc.PPP-1, wild type, Myc.PPH-1, or wild type strain. The extracts were then incubated at 4°C for 2 h with the monoclonal c-Myc antibody (2 µg; Santa Cruz Biotechnology). Subsequently, protein G agarose beads (10 µL) were added, and the mixture was incubated at 4°C for 1-2 h. After centrifugation, the agarose beads were washed four times with ice-cold extraction buffer and once with phosphatase buffer before they were resuspended in 30 µL of phosphatase buffer and incubated at 30°C for 1 h. Dephosphorylation reactions were stopped by the addition of SDS-polyacrylamide sample buffer and subjected to Western blot analysis.
|
Acknowledgments |
|---|
|
TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
|
Footnotes |
|---|
Supplemental material is available at http://www.genesdev.org.
3 Corresponding author.
E-MAIL Yi.Liu{at}UTSouthwestern.edu; FAX (214) 648-7891. 
|
References |
|---|
|
TopAbstractResults and DiscussionMaterials and methodsAcknowledgmentsReferences |
|---|
Aronson, B., Johnson, K., Loros, J.J., and Dunlap, J.C. 1994. Negative feedback defining a circadian clock: Autoregulation in the clock gene frequency. Science 263: 1578-1584.
Cambareri, E.B., Jensen, B.C., Schabtach, E., and Selker, E.U. 1989. Repeat-induced G-C to A-T mutations in Neurospora. Science 244: 1571-1575.
Cheng, P., Yang, Y., Heintzen, C., and Liu, Y. 2001a. Coiled-coil domain mediated FRQ-FRQ interaction is essential for its circadian clock function in Neurospora. EMBO J. 20: 101-108.[CrossRef][Medline]
Cheng, P., Yang, Y., and Liu, Y. 2001b. Interlocked feedback loops contribute to the robustness of the Neurospora circadian clock. Proc. Natl. Acad. Sci. 98: 7408-7413.
Cheng, P., Yang, Y., Gardner, K.H., and Liu, Y. 2002. PAS domain-mediated WC-1/WC-2 interaction is essential for maintaining the steady state level of WC-1 and the function of both proteins in circadian clock and light responses of Neurospora. Mol. Cell. Biol. 22: 517-524.
Cheng, P., Yang, Y., Wang, L., He, Q., and Liu, Y. 2003. WHITE COLLAR-1, a multifunctional Neurospora protein involved in the circadian feedback loops, light sensing, and transcription repression of wc-2. J. Biol. Chem. 278: 3801-3808.
Cohen, P.T. 2002. Protein phosphatase 1—Targeted in many directions. J. Cell. Sci. 115: 241-256.
Crosthwaite, S.K., Dunlap, J.C., and Loros, J.J. 1997. Neurospora wc-1 and wc-2: Transcription, photoresponses, and the origins of circadian rhythmicity. Science 276: 763-769.
Denault, D.L., Loros, J.J., and Dunlap, J.C. 2001. WC-2 mediates WC-1-FRQ interaction within the PAS protein-linked circadian feedback loop of Neurospora. EMBO J. 20: 109-117.[CrossRef][Medline]
Dunlap, J.C. 1999. Molecular bases for circadian clocks. Cell 96: 271-290.[CrossRef][Medline]
Froehlich, A.C., Liu, Y., Loros, J.J., and Dunlap, J.C. 2002. White Collar-1, a circadian blue light photoreceptor, binding to the frequency promoter. Science 297: 815-819.
Froehlich, A.C., Loros, J.J., and Dunlap, J.C. 2003. Rhythmic binding of a WHITE COLLAR-containing complex to the frequency promoter is inhibited by FREQUENCY. Proc. Natl. Acad. Sci. 100: 5914-5919.
Garceau, N., Liu, Y., Loros, J.J., and Dunlap, J.C. 1997. Alternative initiation of translation and time-specific phosphorylation yield multiple forms of the essential clock protein FREQUENCY. Cell 89: 469-476.[CrossRef][Medline]
Gorl, M., Merrow, M., Huttner, B., Johnson, J., Roenneberg, T., and Brunner, M. 2001. A PEST-like element in FREQUENCY determines the length of the circadian period in Neurospora crassa. EMBO J. 20: 7074-7084.[CrossRef][Medline]
Grima, B., Lamouroux, A., Chelot, E., Papin, C., Limbourg-Bouchon, B., and Rouyer, F. 2002. The F-box protein slimb controls the levels of clock proteins period and timeless. Nature 420: 178-182.[CrossRef][Medline]
He, Q., Cheng, P., Yang, Y., Wang, L., Gardner, K.H., and Liu, Y. 2002. White collar-1, a DNA binding transcription factor and a light sensor. Science 297: 840-843.
He, Q., Cheng, P., Yang, Y., He, Q., Yu, H., and Liu, Y. 2003. FWD1-mediated degradation of FREQUENCY in Neurospora establishes a conserved mechanism for circadian clock regulation. EMBO J. 22: 4421-4430.[CrossRef][Medline]
Heriche, J.K., Lebrin, F., Rabilloud, T., Leroy, D., Chambaz, E.M., and Goldberg, Y. 1997. Regulation of protein phosphatase 2A by direct interaction with casein kinase 2
. Science 276: 952-955.
Kloss, B., Price, J.L., Saez, L., Blau, J., Rothenfluh, A., and Young, M.W. 1998. The Drosophila clock gene double-time encodes a protein closely related to human casein kinase Ie. Cell 94: 97-107.[CrossRef][Medline]
Ko, H.W., Jiang, J., and Edery, I. 2002. Role for Slimb in the degradation of Drosophila Period protein phosphorylated by Doubletime. Nature 420: 673-678.[CrossRef][Medline]
Krebs, E.G. and Fischer, E.H. 1962. Phosphorylase b kinase from rabbit skeletal muscle. Methods Enzymol. 5: 373-376.[CrossRef]
Lin, J.M., Kilman, V.L., Keegan, K., Paddock, B., Emery-Le, M., Rosbash, M., and Allada, R. 2002. A role for casein kinase 2 in the Drosophila circadian clock. Nature 420: 816-820.[CrossRef][Medline]
Liu, Y., Garceau, N., Loros, J.J., and Dunlap, J.C. 1997. Thermally regulated translational control mediates an aspect of temperature compensation in the Neurospora circadian clock. Cell 89: 477-486.[CrossRef][Medline]
Liu, Y., Loros, J., and Dunlap, J.C. 2000. Phosphorylation of the Neurospora clock protein FREQUENCY determines its degradation rate and strongly influences the period length of the circadian clock. Proc. Natl. Acad. Sci. 97: 234-239.
Loros, J.J. and Dunlap, J.C. 2001. Genetic and molecular analysis of circadian rhythms in NEUROSPORA. Annu. Rev. Physiol. 63: 757-794.[CrossRef][Medline]
Lowrey, P.L., Shimomura, K., Antoch, M.P., Yamazaki, S., Zemenides, P.D., Ralph, M.R., Menaker, M., and Takahashi, J.S. 2000. Positional syntenic cloning and functional characterization of the mammalian circadian mutation tau. Science 288: 483-492.
Price, J.L., Blau, J., Rothenfluh, A., Adodeely, M., Kloss, B., and Young, M.W. 1998. double-time is a new Drosophila clock gene that regulates PERIOD protein accumulation. Cell 94: 83-95.[CrossRef][Medline]
Roenneberg, T. and Taylor, W. 2000. Automated recordings of bioluminescence with special reference to the analysis of circadian rhythms. Methods Enzymol. 305: 104-119.[Medline]
Sugano, S., Andronis, C., Ong, M.S., Green, R.M., and Tobin, E.M. 1999. The protein kinase CK2 is involved in regulation of circadian rhythms in Arabidopsis. Proc. Natl. Acad .Sci. 96: 12362-12366.
Talora, C., Franchi, L., Linden, H., Ballario, P., and Macino, G. 1999. Role of a white collar-1-white collar-2 complex in blue-light signal transduction. EMBO J. 18: 4961-4968.[CrossRef][Medline]
Virshup, D.M. 2000. Protein phosphatase 2A: A panoply of enzymes. Curr. Opin. Cell Biol. 12: 180-185.[CrossRef][Medline]
Westphal, R.S., Anderson, K.A., Means, A.R., and Wadzinski, B.E. 1998. A signaling complex of Ca2+-calmodulin-dependent protein kinase IV and protein phosphatase 2A. Science 280: 1258-1261.
Yang, Y., Cheng, P., Zhi, G., and Liu, Y. 2001. Identification of a calcium/calmodulin-dependent protein kinase that phosphorylates the Neurospora circadian clock protein FREQUENCY. J. Biol. Chem. 276: 41064-41072.
Yang, Y., Cheng, P., and Liu, Y. 2002. Regulation of the Neurospora circadian clock by casein kinase II. Genes & Dev. 16: 994-1006.
Yang, Y., Cheng, P., He, Q., Wang, L., and Liu, Y. 2003. Phosphorylation of FREQUENCY protein by casein kinase II is necessary for the function of the Neurospora circadian clock. Mol. Cell. Biol. 23: 6221-6228.
Yatzkan, E. and Yarden, O. 1995. Inactivation of a single-2A phospho-protein phosphatase is lethal in Neurospora crassa. Curr. Genet. 28: 458-466.[CrossRef][Medline]
____. 1999. The B regulatory subunit of protein phosphatase 2A is required for completion of macroconidiation and other developmental processes in Neurospora crassa. Mol. Microbiol. 31: 197-209.[CrossRef][Medline]
Yatzkan, E., Szoor, B., Feher, Z., Dombradi, V., and Yarden, O. 1998. Protein phosphatase 2A is involved in hyphal growth of Neurospora crassa. Mol. Gen. Genet. 259: 523-531.[CrossRef][Medline]
Young, M.W. and Kay, S.A. 2001. Time zones: A comparative genetics of circadian clocks. Nat. Rev. Genet. 2: 702-715.[CrossRef][Medline]
Zeke, T., Kokai, E., Szoor, B., Yatzkan, E., Yarden, O., Szirak, K., Feher, Z., Bagossi, P., Gergely, P., and Dombradi, V. 2003. Expression of protein phosphatase 1 during the asexual development of Neurospora crassa. Comp. Biochem. Physiol. B. 143: 161-170.
CiteULike 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  J. C. Chiu, J. T. Vanselow, A. Kramer, and I. Edery The phospho-occupancy of an atypical SLIMB-binding site on PERIOD that is phosphorylated by DOUBLETIME controls the pace of the clock Genes & Dev., July 1, 2008; 22(13): 1758 - 1772. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Huang, S. Chen, S. Li, J. Cha, C. Long, L. Li, Q. He, and Y. Liu Protein kinase A and casein kinases mediate sequential phosphorylation events in the circadian negative feedback loop Genes & Dev., December 15, 2007; 21(24): 3283 - 3295. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Bhasin, S. R. Cunha, M. Mudannayake, M. S. Gigena, T. B. Rogers, and P. J. Mohler Molecular basis for PP2A regulatory subunit B56{alpha} targeting in cardiomyocytes Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H109 - H119. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Fang, S. Sathyanarayanan, and A. Sehgal Post-translational regulation of the Drosophila circadian clock requires protein phosphatase 1 (PP1) Genes & Dev., June 15, 2007; 21(12): 1506 - 1518. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. W. Jolma, G. Falkeid, M. Bamerni, and P. Ruoff Lithium Leads to an Increased FRQ Protein Stability and to a Partial Loss of Temperature Compensation in the Neurospora Circadian Clock. J Biol Rhythms, October 1, 2006; 21(5): 327 - 334. [Abstract] [PDF]
|
|
|
|
|
|
M. Merrow, G. Mazzotta, Z. Chen, and T. Roenneberg The right place at the right time: regulation of daily timing by phosphorylation Genes & Dev., October 1, 2006; 20(19): 2629 - 2633. [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Dunlap Proteins in the Neurospora Circadian Clockworks J. Biol. Chem., September 29, 2006; 281(39): 28489 - 28493. [Full Text] [PDF]
|
|
|
|
|
|
Q. He, J. Cha, Q. He, H.-C. Lee, Y. Yang, and Y. Liu CKI and CKII mediate the FREQUENCY-dependent phosphorylation of the WHITE COLLAR complex to close the Neurospora circadian negative feedback loop Genes & Dev., September 15, 2006; 20(18): 2552 - 2565. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Liu and D. Bell-Pedersen Circadian Rhythms in Neurospora crassa and Other Filamentous Fungi. Eukaryot. Cell, August 1, 2006; 5(8): 1184 - 1193. [Full Text] [PDF]
|
|
|
|
 |
|
 C. L. Partch, K. F. Shields, C. L. Thompson, C. P. Selby, and A. Sancar Posttranslational regulation of the mammalian circadian clock by cryptochrome and protein phosphatase 5 PNAS, July 5, 2006; 103(27): 10467 - 10472. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Brunner and T. Schafmeier Transcriptional and post-transcriptional regulation of the circadian clock of cyanobacteria and Neurospora. Genes & Dev., May 1, 2006; 20(9): 1061 - 1074. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Schafmeier, K. Kaldi, A. Diernfellner, C. Mohr, and M. Brunner Phosphorylation-dependent maturation of Neurospora circadian clock protein from a nuclear repressor toward a cytoplasmic activator Genes & Dev., February 1, 2006; 20(3): 297 - 306. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Ruoff, J. J. Loros, and J. C. Dunlap The relationship between FRQ-protein stability and temperature compensation in the Neurospora circadian clock PNAS, December 6, 2005; 102(49): 17681 - 17686. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. He and Y. Liu Molecular mechanism of light responses in Neurospora: from light-induced transcription to photoadaptation Genes & Dev., December 1, 2005; 19(23): 2888 - 2899. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. He, P. Cheng, Q. He, and Y. Liu The COP9 signalosome regulates the Neurospora circadian clock by controlling the stability of the SCFFWD-1 complex Genes & Dev., July 1, 2005; 19(13): 1518 - 1531. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. He, H. Shu, P. Cheng, S. Chen, L. Wang, and Y. Liu Light-independent Phosphorylation of WHITE COLLAR-1 Regulates Its Function in the Neurospora Circadian Negative Feedback Loop J. Biol. Chem., April 29, 2005; 280(17): 17526 - 17532. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Mittag, S. Kiaulehn, and C. H. Johnson The Circadian Clock in Chlamydomonas reinhardtii. What Is It For? What Is It Similar To? Plant Physiology, February 1, 2005; 137(2): 399 - 409. [Full Text] [PDF]
|
|
|
|
|
|
E. Harms, S. Kivimae, M. W. Young, and L. Saez Posttranscriptional and Posttranslational Regulation of Clock Genes J Biol Rhythms, October 1, 2004; 19(5): 361 - 373. [Abstract] [PDF]
|
|
|
|
|
|
J. C. Dunlap and J. J. Loros The Neurospora Circadian System J Biol Rhythms, October 1, 2004; 19(5): 414 - 424. [Abstract] [PDF]
|
|
|
|
|
|
T. Nishiwaki, Y. Satomi, M. Nakajima, C. Lee, R. Kiyohara, H. Kageyama, Y. Kitayama, M. Temamoto, A. Yamaguchi, A. Hijikata, et al. From the Cover: Role of KaiC phosphorylation in the circadian clock system of Synechococcus elongatus PCC 7942 PNAS, September 21, 2004; 101(38): 13927 - 13932. [Abstract] |
) strains. (A) Western blot analysis showing the circadian oscillation of FRQ in DD. Densitometric analysis of the Western blot results is shown at bottom. Two independent experiments were performed and similar results were obtained (see Supplemental Fig. 1). (B) Western blot analysis showing that FRQ is less stable in the ppp-1RIP strains after the addition of CHX (10 µg/mL). Cultures were grown in LL. Densitometric analysis of the Western blot results from seven independent experiments is shown at bottom. Error bars, standard deviations. (*) P values for 3, 6, 9, and 12 h are 0.02, 0.003, 0.001, and 0.0009, respectively.
