Regulation of CRY protein stability by SCF-ubiquitin ligase
The data presented here illustrate the importance of protein complex purification for identifying new proteins involved in the generation of circadian oscillations. Previous studies on circadian complex purification using PER1 as a bait identified two new proteins; Nono and WDR5, involved in the regulation of circadian transcription (Brown et al., 2005).
Our finding of the S-phase kinase-associated protein 1 (SKP1), a component of the SCF (SKP-CULLIN-F-BOX protein) E3-ubiquitin ligase complex, being associated with CRYs is in line with recent studies that identified another SCF component, the F-box protein FBXL3 as CRY interacting protein (Busino et al., 2007; Godinho et al., 2007;
Siepka et al., 2007). Conjugation of ubiquitin, a highly conserved 76 residue peptide, proceeds in three steps. First, the ubiquitin activating enzyme E1 activates ubiquitin in a reaction that forms E1-S-ubiquitin thiol-ester. Ubiquitin conjugating enzymes E2 then transferes the activated ubiquitin moiety from E1, via an E2-S-ubiquitin intermediate, to the lysine residue of the substrate specifically bound to a member of ubiquitin ligase family E3 members. Among several classes of E3 enzymes, the SCF complex is the major type. F-box proteins are thought to provide the specificity of the SCF complex, frequently by recognizing phosphorylated substrates (Glickman and Ciechanover, 2002).
During the past few years, we have experienced a vast expansion of SCF complexes involved in circadian clock functions in various organisms. For example, the Drosophila F-box proteins Slimb and Jetlag ubiquitinate PER and TIM proteins, respectively, and target them for proteasomal degradation (Grima et al., 2002; Ko et al., 2002; Koh et al.,
2006). In mammals, the homolog of Slimb, beta transducin repeat containing protein (βTRCP) ubiquitinates both PER1 and PER2 (Eide et al., 2005; Reischl et al., 2007;
Shirogane et al., 2005), which leads to proteasome-dependent degradation. Inhibition of either FBXL3 or βTRCP activity lengthens the circadian period in a similar manner as I have described here for the the depletion of the adapter protein SKP1. Phosphorylation of PERs by casein kinase 1 epsilon (CK1δ/ε) is a prerequisite for the recognition and ubiquitination by the SCF-βTRCP complex. Further studies are required to address the identity of the signal that targets CRYs for ubiquitination-dependent proteasomal degradation by SCF-FBXL3. Possible candidates are the known CRY kinases, mitogen activated protein kinase (MAPK), CK1ε/δ, and glycogen synthase kinase 3β (GSK3β) (Eide et al., 2002; Harada et al., 2005; Sanada et al., 2004). In my opinion, GSK3β constitutes the best candidate, since it has been shown that mCRY2 phosphorylation by GSK3β leads to efficient degradation of CRY2 by the proteasomal pathway. Mutational analysis of phospho-acceptor sites within the CRY1 and CRY2 sequences should further clarify which of the phosphorylation sites are necessary for the interaction with SCF-FBXL3. It is now becoming evident that regulated temporal degradation of the proteins in the negative limb of the oscillator is an important feature in the maintenance of 24 hour circadian cycle length.
In the positive limb, CLOCK protein levels in mouse liver are almost constant throughout the day, but BMAL1 protein levels do exhibit an about 2 to 3-fold circadian accumulation (Lee et al., 2001). Since the half-lives of the core clock proteins are
relatively short when compared to bulk cellular proteins, it is likely that their degradation is tightly regulated. It would thus be interesting to examine whether BMAL1 and
CLOCK proteins are also subject to temporally regulated ubiquitination and degradation, and if so, to identify the kinases and ubiquitin ligases involved in these processes.
Regulation of CRY protein stability by OTUD4
In recent years, we have witnessed the identification of many novel enzymes, collectively called deubiquitinating enzymes (DUBs), which counteract ubiquitination by cleaving covalently attached ubiquitin moieties from target proteins (Ventii and Wilkinson, 2008).
There are five known groups of DUBs, ubiquitin specific proteases (USP), ubiquitin
C-terminal hydrolases, JAMM metalloproteinases, Machado-Joseph domain-containing proteins, and Ovarian tumor OTU-domain containing proteins. We have shown here that an OTU-domain containing protein, OTUD4, interacts with CRYs and negatively
regulates their stability. In a recent report, Sowa and co-workers purified 75 different DUB complexes, OTUD4 complex among them, and analyzed the associated proteins by mass spectrometry (Sowa et al., 2009). While they identified a number of OTUD4 associated proteins, neither CRY1 nor CRY2 proteins were among them. The
discrepancy between the studies performed by Sowa et al. and my own results might be due to the utilization of different cell lines used in these studies. While we used mouse fibroblast NIH-3T3 cells, a cell line known to exhibit robust circadian oscillations, Sowa and co-workers performed their purification experiments with extracts from human embryonic kidney (HEK) 293 cells, which do not have functional circadian clocks (Charna Dibner personal communication). OTUD4 has not yet been characterized in details, but we do have some information on the putative functions of a few related proteins. A20 and cezanne are OTU-domain DUBs involved in tumor necrosis factor alpha (TNFα) signaling, and recently Stanisic et al. reported OTUB1 as an estrogen receptor (ER) deubiquitinase and a negative regulator of ER target genes (Enesa et al., 2008; Stanisic et al., 2009; Wertz et al., 2004). The expression of OTUD4 seems to be ubiquitous, since transcripts were detected in all examined tissues; brain, heart, intestine, kidney, and liver. Since circadian clocks exist in almost all tissues, the ubiquitous
expression of OTUD4 is in keeping with a function in the rhythm generating clockwork circuitry. The OTUD4 protein spans 1107 amino acids, and the only predicted domain, the OTU-domain, maps to the N-terminal part encompassing amino acids 42 to 149.
Many DUBs bind ubiquitin with low affinity, and it has been proposed that DUBs acquire their substrate specificity by associating with other proteins in macromolecular complexes (Ventii and Wilkinson, 2008). The long C-terminal part may mediate such interactions. Expression of truncated OTUD4 forms combined with immunoprecipitation might thus help us to understand the role of the long C-terminal part in complex
formation with CRYs. By chance, we isolated two different splice variants of Otud4. The shorter protein variant differs from the longer one by missing glutamate 290 and residues from 431 to 473. It is conceivable that the protein products of the alternatively spliced
variants differ in their properties, such as subcellular localization, target specificity or target affinity. Our short hairpin RNAs did not discriminate between the different
transcripts. As a consequence, I could decide which OTUD4 form was responsible for the phenotype I observed. Based on the limited sequence information we gained from the peptide sequencing afforded by mass spectrometry, I speculate that the longer splice variant of OTUD4 was associated with CRY1. In fact, a peptide diagnostic for this form was recovered in the CRY complexes. RNAi mediated depletion experiments with transcript-specific shRNAs should clarify which of the two OTUD4 splice variants is involved in modulating CRY stability.
Intuitively one would expect that DUBs positively regulate the stability of their targets, since they should counteract ubiquitination. However, published reports provide evidence for opposite scenarios. A20 has an N-terminal OTU-domain with specificity for K-63 linked poly-ubiquitin chains and a C-terminal zinc finger ubiquitin ligase domain (Wertz et al., 2004). It is generally believed that K-48 linked poly-ubiquitin chains function as a signal for proteasomal degradations, whereas K-63 linked poly-ubiquitin chains on target proteins indicate regulation of other cellular function, such as
endocytosis of cell surface receptors, post-replicative DNA repair, stress response, and regulation of TNFα signalling (Glickman and Ciechanover, 2002). Accordingly, A20 first cleaves K-63 linked poly-ubiquitin chains from its target, receptor interacting protein (RIP), and subsequently adds K-48 linked poly-ubiquitin chains, which targets RIP for proteasomal degradation. Trabid is another example of an OTU-domain DUB with specificity for K-63 linked poly-ubiquitin chains (Tran et al., 2008). Trabid
deubiquitinates adenomatosis polyposis coli (APC) and positively regulates Wnt induced transcription.
Our observations on CRY stability could be explained in similar manner, if indeed OTUD4 removed K-63 linked poly-ubiquitin from CRYs and if this was followed by K-48 ubiquitination of CRYs by SCF-FBXL3 or by (an) yet unknown ubiquitin ligase(s). Ubiquitination assays with mutant ubiquitin forms that lack all other lysine residues except either lysine 48 or 63 should be performed to distinguish the mode of CRY ubiquitination. Alternatively, it might be possible to raise antibodies against the
different ubiquitin chains, since they differ in their topological structures (Varadan et al., 2004).
We have shown that OTUD4 depletion by shRNA has dramatic consequences on circadian transcription in our reporter system. The expression of BMAL1/CLOCK target genes; such as Rev-erbα−luc, Per2-luc, and Dbp-luc were severely blunted, and their circadian oscillation was dramatically attenuated. Since, the stability of CRYs was increased upon knocking-down OTUD4, we believe that the loss of circadian expression of CLOCK/BMAL1 target genes is due to elevated CRY protein levels, which would be expected to results in prolonged and increased repression. This would be in line with previous reports, which showed that overexpression of CRY1 impairs circadian rhythms in Rat1 cells (Ueda et al., 2005). Although, the experiments performed with clock gene promoter driven luciferase reporters clearly show that OTUD4 plays a role in the regulation of circadian transcription, future studies should address the regulation on the level of endogenous clock genes. Our initial efforts to study the effect of OTUD4 depletion on endogenous genes were hampered by the low viability of OTUD4 knock-down cells. Since circadian clocks are not required for survival of either cells or animals, the impaired viability of OTUD4-depleted cells indicated that OTUD4 may also
functions in processes unrelated to circadian rhythms but essential for cellular viability or proliferation. If so, conventional knock-out techniques would not necessarily prove unsuccessful in assessing OTUD4 functions. Generating mice with OTUD4 alleles flanked by loxP sites and crossing them to already existing strains with tissue specific, a tamoxifen-inducible CRE-recombinase would perhaps allow us to study the effect of OTUD4 loss-of-function in the generation of 24 hour oscillations (Imai et al., 2001). Our knowledge from previous studies in mutant mice and in tissue culture systems predicts that the tissue-specific ablation of OTUD4 would lead to an accumulation of CRY proteins, which in turn would lead to period lengthening or even a complete arrhythmicity in the targeted tissues and ex-vivo explants of them.
Regulation of circadian transcription by KRIP1/KAP1/TIF1β
I also found KRIP1, a corepressor protein, associated with CRY1. KRIP1 would be an attractive candidate for CRY-mediated repression since it had previously been shown to
function as a corepressor for Krüppel associated box zinc finger proteins (Friedman et al., 1996; Kim et al., 1996; Moosmann et al., 1996). KRIP1 also interacts with several factors that mediate gene silencing such as, histone deacetylase complex NuRD and N-coR1, Histone methyltransferase SETDB1, and heterochromatin protein 1 (HP1) (Lechner et al., 2000; Schultz et al., 2002; Schultz et al., 2001; Underhill et al., 2000). On the other hand, KRIP1 has also been reported to function as a coactivator for CCAAT/Enhancer Binding Protein (C/EBP), glucocorticoid receptor (GR), and nerve growth factor IB (NGFI-B) (Chang et al., 1998; Rambaud et al., 2009). Such Ying-Yang functions have been
observed for many different transcriptional regulatory proteins. Examples are the nuclear receptors GR(Newton and Holden, 2007) and thyroid receptor (Eckey et al., 2003), and heat shock transcription factor 1 (Cahill et al., 1996). Although the physical interaction between KRIP1 and CRY1 remains to be established more rigorously, the RNA
interference experiments I performed indicate that KRIP1 may well participate in the regulation of circadian transcription. Thus, KRIP1 depletion resulted in increased Bmal1-luc expression, without affecting its period length. The circadian transcription of Bmal1 is regulated by the transcriptional repressor REV-ERBα and the activators RORα/β/γ, which compete for the binding to two ROR response elements (ROREs) located within the Bmal1 promoter (Guillaumond et al., 2005; Preitner et al., 2002). The increased Bmal1 expression could therefore be explained if RORs accumulation was upregulated upon KRIP1 depletion. In this scenario, CRY1 would selectively recruit KRIP1 to the promoters of RORs through binding to the BMAL1/CLOCK complex. Of note, HP1 binds the BMAL1/CLOCK target gene Dbp promoter in a strictly circadian manner (Ripperger and Schibler, 2006). Ideally, studies with KRIP1 knock-out mice should be performed to address the question of whether and by what mechanism KRIP1 regulates circadian transcription. Unfortunately, KRIP1 knock-out mice die at an early embryonic developmental stage at embryonic day 5.5 indicating that this corepressor executes essential functions during embryogenesis (Cammas et al., 2000). Generation of mice harboring floxed Krip1 alleles -and crossing these animals to a mouse strain with tissue (e.g. liver) -specific tamoxifen inducible CRE-alleles should allow one to study circadian transcription in the absence of KRIP1 (Imai et al., 2001).
PSF
We also identified the PTB-associated splicing factor (PSF) in the complex with CRY1.
As indicated by its name PSF was initially identified as a splicing factor in a complex with poly-pyrimidine tract binding protein (PTB) (Patton et al., 1993). Following the cloning of PSF, several different functions have been associated to this protein, including DNA unwinding and repression of several nuclear receptors, such as thyroid hormone receptor, retinoid X receptor, androgen receptor, and progesterone receptor (Dong et al., 2005; Dong et al., 2007; Mathur et al., 2001; Straub et al., 1998). The repression of nuclear receptors by PSF is believed to function through the direct interaction with mSin3a and the recruitment of histone deacetylase complexes (HDAC). Interestingly, NONO, a closely related homologue and binding partner of PSF, was found associated with mouse PER1 in NIH-3T3 cells (Brown et al., 2005). Brown and coworkers also showed that depletion of NONO by RNA interference attenuates circadian transcription.
Furthermore, Drosophila flies with a strongly hypomorphic mutation in NONA, the fruit fly ortholog of NONO, are nearly behaviorally arrhythmic and display greatly attenuated timeless mRNA accumulation rhythm. While these results implicate a possible role of NONO in the control of circadian rhythms, the mechanism of this regulation remains obscure. Mice with mutated NONO alleles have also been established, but they exhibit normal circadian locomotor activity (Steve Brown, personal communication).
Advantages and disadvantages of identifying new clock proteins by biochemical complex purification
Proteins often function as component of large macromolecular complexes to perform specific tasks. The interaction of CRYs and PERs provides the basis of the negative regulation of BMAL1/CLOCK target genes. Biochemical studies have also shown that CRYs are present in high molecular weight complexes even in the absence of PERs (Brown et al., 2005). Thus characterizing CRY complexes may offer important insight into CRY protein function. Here I have shown that with the exception of PER1, many additional protein partners of CRY1 exist (Brown et al., 2005). Typical
immunoprecipitation experiments generate significant background of protein contaminants, due to the affinity of contaminant proteins with the resin used for the
tethering of the antibodies. The two step purification protocol we used significantly improved the signal to noise ratio. Despite this, the mass spectrometric analysis revealed hits for proteins which we believe to be contaminants, such as tubulin alpha and beta chains. In addition, there was a significant spillover of immunoglobulin in the second purification step, despite the mild peptide elution protocol we employed. Moreover, high levels of antibody-specific peptides can easily mask the presence of lower abundance peptides, perhaps explaining why FBXL3 peptides were not identified in our CRY complexes. To further improve the method and gain confidence in the specificity of the identified proteins, more quantitative proteomics technique could be used. Several methods for quantitative proteomics exist that rely on stable isotopes with known mass differences that can easily be discriminated by mass spectrometry (Goshe and Smith, 2003; Sechi and Oda, 2003). Stable isotope labeling with amino acids in cell culture (SILAC) is becoming one of the most popular methods used in protein complex purifications analysis (Ong et al., 2002).
This method uses 13C arginine, which is added to the culture medium of sample cells, whereas normal 12C arginine is supplied to control cells. After purification eluted samples are mixed and analyzed by mass spectrometry. Pairs of identical peptides of different stable-isotope composition can thus be differentiated, owing to their mass difference, and the relative protein abundances can thereby be calculated from the ratio of peak intensities in the mass spectrum. In these experiments, a one to one ratio indicates a contaminant, whereas specifically enriched proteins yield asymmetric peak heights. If I had to start this project again, I would definitely combine the SILAC procedure with the two step purification protocol, as it would considerably lower the number of false postitives and thus the number of proteins whose functional significance has to be scrutinized by loss-and gain-of function experiments.
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