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. 2004 Feb 1;18(3):255–260. doi: 10.1101/gad.1152604

Distinct roles for PP1 and PP2A in the Neurospora circadian clock

Yuhong Yang 1, Qun He 1, Ping Cheng 1, Philip Wrage 1, Oded Yarden 2, Yi Liu 1,3
PMCID: PMC338279  PMID: 14871927

Abstract

Phosphorylation of the Neurospora circadian clock protein FREQUENCY by several kinases promotes its degradation and is important for the function of the circadian feedback loop. Here, we show that FRQ is less stable in a ppp-1 (catalytic subunit of PP1) mutant, resulting in its advanced phase and short period. In contrast, FRQ stability is not altered in a rgb-1 (a regulatory subunit of PP2A) mutant, but levels of frq protein and mRNA are low, resulting in a low-amplitude and long-period oscillation of the clock. Furthermore, PP1 and PP2A expressed in Neurospora can dephosphorylate the endogenous FRQ in vitro, suggesting that these two phosphatases may differentially regulate FRQ and, consequently, the behavior of the circadian clock.

Keywords: frequency, phosphorylation, phosphatase, PP1, PP2A

Reversible phosphorylation is an important regulatory mechanism for many biological processes in eukaryotic organisms. The phosphorylation state of a protein is controlled dynamically by protein kinases and phosphatases. Previous studies have demonstrated that phosphorylation of circadian clock proteins is an essential posttranscriptional mechanism in the regulation of circadian clocks (Dunlap 1999; 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

To investigate the possible roles of PP1 and PP2A in the Neurospora circadian clock, we screened for mutants in which the catalytic subunit or a regulatory subunit of PP1 or PP2A is mutated. Because the catalytic subunits of PP1 and PP2A (encoded for by the ppp-1 and pph-1 genes, respectively) are essential for cell survival in Neurospora and other eukaryotes (Yatzkan and Yarden 1995; 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.

Figure 1.

Figure 1.

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Circadian conidiation rhythms of the ppp-1RIP strain show advanced phase and short period phenotypes. (A) Phosphatase activity assay showing the reduction of PP1 activity in the ppp-1RIP strain. Cultures were cultured and harvested in LL. I-2: assay containing 100 U of inhibitor-2. Error bars, standard deviations. (B) Race tube assay showing the circadian conidiation rhythms of the wild-type and the ppp-1RIP strains in DD. The black lines mark the growth fronts at 24-h intervals. The calculated period length and phase are shown below. (SD) Standard deviation. (C) Race tube assay in LD cycles. To show the difference of the position of the conidiation peaks between the two strains, the image of the ppp-1RIP mutant was enlarged so that it showed similar daily growth distance as the wild-type strain.

Examination of the circadian conidiation rhythm in constant condition (constant darkness, DD) by race tube assay revealed that the period length of the circadian clock in the ppp-1RIP strain was ∼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.

Figure 2.

Figure 2.

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Circadian rhythm of FRQ expression and its stability in the wild-type (•) and ppp-1RIP(○) 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.

Because one of the roles of protein phosphorylation in the Neurospora circadian clock is to promote FRQ degradation, we compared the degradation rates of FRQ after the addition of the protein synthesis inhibitor, cycloheximide (CHX; Liu et al. 1997), 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.

Figure 3.

Figure 3.

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Reduction of PP2A activity and the altered circadian conidiation rhythm in the rgb-1RIP strain. (A) Phosphatase activity assay showing the reduction of phosphatase activity in the rgb-1RIP strain. (B) Race tube assay showing the circadian conidiation rhythms of the wild-type and rgb-1RIP strains in DD. Representative results were shown. The black lines mark the growth fronts at 24-h intervals. (C) Western blot analysis comparing the stability of FRQ after CHX treatment in the wild-type (•) and rgb-1RIP (○) strains.

Because of the slow growth rate (∼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.

Figure 4.

Figure 4.

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Altered circadian rhythms of FRQ, frq, and ccg-1 in the rgb-1RIP strain. (A) Western blot analysis showing the oscillation of FRQ protein in DD. Densitometric analysis of the Western blot results from three independent experiments is shown at bottom. (•) Wild type; (○) rgb-1RIP. (B) Northern blot analysis showing the expression of frq and ccg-1 in DD in the wild-type and rgb-1RIP strains. Two independent experiments were performed and similar results were obtained (Supplemental Fig. 2). (C) Densitometric analysis of the results shown in B. (•) Wild type; (○) rgb-1RIP.

Northern blot analysis was performed to examine whether the low levels of FRQ protein was due to the low levels of frq mRNA. The level of frq mRNA was significantly lower in the rgb-1RIP strain in constant darkness, but a robust rhythm of frq levels was not evident in the 2-d experiment (frq may have a low amplitude oscillation; Fig. 4B,C; Supplemental Fig. 2). For the clock-controlled gene, ccg-1, a low-amplitude long period rhythm was seen in the mutant strain. Taken together, these data suggest that the circadian clock function was severely compromised in the rgb-1RIP strain due to a partial loss-of-function of PP2A. Thus, PP2A is also important for the regulation of the Neurospora circadian clock.

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 λ 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.

Figure 5.

Figure 5.

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Dephosphorylation of Myc-FRQ by the Neurospora expressed Myc-PPP-1 and Myc-PPH-1. (A,B) The extracts of frq10, Myc-FRQ strain was mixed with the extracts of the wild type, Myc-PPP-1, wild type, Myc-PPh-1, or wild-type strain. Cells were grown in LL. Immunoprecipitation (IP) was then performed using a c-Myc monoclonal antibody. Afterward, the IP products were incubated in phosphatase buffer and Western blot analysis was performed. The arrow in A marks the extensively phosphorylated Myc-FRQ species, while it indicates the hypophosphorylated FRQ forms in B. (C) Comparison of the FRQ phosphorylation profiles in the wild-type and rgb-1RIP strains. Cultures were grown in LL. The arrow indicates the hyperphoshorylated FRQ species seen in the rgb-1RIP strains.

To obtain in vivo evidence that FRQ is dephosphorylated by PP1 or PP2A, we compared the FRQ phosphorylation profiles of the phosphatase mutants and the wild-type strain in LL, a condition in which FRQ is evenly phosphorylated. No significant differences in FRQ phosphorylation profiles were observed between the wild-type and ppp-1RIP strains, probably due to the residual PP1 activity in the mutant. The near-normal growth and developmental phenotypes of the ppp-1RIP strain suggest that the function of PP1 was not severely impacted in the mutant. On the other hand, a hyperphosphorylated FRQ species that was not normally present in the wild-type strain was seen in the rgb-1RIP strain (Fig. 5C), suggesting that FRQ is a PP2A substrate in vivo. No significant changes in WC-1 and WC-2 phosphorylation patterns were detected in the mutants (Supplemental Fig. 3), but it is possible that the changes are subtle and could not be detected by Western analysis.

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

Stains, culture conditions, and race tube assay

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 × Vogel's and 2% glucose (Aronson et al. 1994). Conidiation rhythms were examined on race tubes containing glucose/arginine medium (1 × 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

We thank Lixin Wang for excellent technical assistance and Drs. Mark Mumby, Hongtao Yu, and Michael Young for advice. Supported by grants from National Institutes of Health and Welch Foundation to Y.L. and the Israel Science Foundation to O.Y. Y.L is the Louise W. Kahn endowed scholar in Biomedical Research at University of Texas Southwestern Medical Center.

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.

Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.1152604.

Supplemental material is available at http://www.genesdev.org.

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