Coral reefs are dynamic systems whose composition is highly influenced by unpredictable biotic and abiotic factors. Understanding the spatial scale at which long-term predictions of reef composition can be made will be crucial for guiding conservation efforts. Using a 22-year time series of benthic composition data from 20 reefs on the Kenyan and Tanzanian coast, we developed Bayesian vector autoregressive state-space models for reef dynamics, incorporating among-site variability, and quantified their long-term behaviour. We estimated that if there were no among-site variability, the total long-term variability would be approximately one-third of its current value. Thus, our results showed that among-site variability contributes more to long-term variability in reef composition than does temporal variability. Individual sites were more predictable than previously thought, and predictions based on current snapshots are informative about long-term properties. Our approach allowed us to identify a subset of possible climate refugia sites with high conservation value, where the long-term probability of coral cover ≤0.1 (as a proportion of benthic cover of hard substrate) was very low. Analytical results show that this probability is most strongly influenced by among-site variability and by interactions among benthic components within sites. These findings suggest that conservation initiatives might be successful at the site scale as well as the regional scale. For exaA good candidate for such a model is the vector autoregressive model of order 1 or VAR(1) . This isOrbicella annularis-dominated reefs in Belize suggested a similar coral cover threshold of 0.05–0.1  model for regional dynamics of East African coral reefs, including random site effects and measurement error, and use it to answer four key questions about spatial and temporal variability. How important is among-site variability in the dynamics of benthic composition, relative to within-site temporal variability? How much variability is there among sites in the probability of low (≤0.1) coral cover? Which model parameters have the largest effects on the probability of low coral cover in the region? How informative is a single snapshot in time about the long-term properties of a site?Surveys of 20 spatially distinct reefs in Kenya and Tanzania  were conMillepora), macroalgae and other . Millepora were included in the coral category because they are important calcareous framework builders and may have calcification rates similar to those of branching corals (Each of the 30 sites was visited at least twice (data from sites visited once were omitted), with a maximum of 20 visits. In total, there were 289 site visits. In each, a version of line-intercept sampling  was usedg corals . Sand anThe three cover values form a three-part composition, a set of three positive numbers whose sum is 1 . It is oi, transect j, time t were represented by the vector yi,j,t = T, in which the first coordinate yi,j,t1, was proportional to the natural log of the ratio of algae to coral, and the second coordinate yi,j,t2, was proportional to the natural log of the ratio of other to the geometric mean of algae and coral , an approach used in other recent models of coral reef dynamics  with location vector equal to the corresponding xi,t, and unknown scale matrix H and degrees of freedom ν, so that yi,j,t has mean vector xi,t if ν > 1, and covariance matrix νH∕(ν − 2) if ν > 2  against number of time points. It is worth emphasizing that all parameters other than αi and xi,t are common to all reefs, and thus estimated from all 289 site visits.We estimated all model parameters and checked model performance using Bayesian methods implemented in the Stan programming language , as desct → ∞), the true transformed composition x∗ of a randomly-chosen site will converge to a stationary distribution, provided that all the eigenvalues of B lie inside the unit circle in the complex plane  and the stationary among-site covariance Z∗ (which depends on B and Z).This stationary distribution is the multivariate normal vector i, the value of αi is fixed and the stationary distribution is given by B, a and αi, and whose stationary covariance matrix is Σ∗. Note that B, which describes intra- and inter-component interactions on an annual time scale, affects all the parameters of both stationary distributions, and therefore affects both within- and among-site variability in the long term. Also, the back-transformation of the stationary mean μ∗ of the transformed composition, rather than the arithmetic mean vector of the untransformed composition, is the appropriate measure of the centre of the stationary distribution  so as to maintain fairly stable reef composition, with an increase in each transformed component tending to be counteracted in the following year-to-year change. Each column of A represents the effect of a unit increase in a component of transformed composition on the year-to-year change in transformed composition ) suggested that some aspect of the process leading to variation in measured composition among transects at a given site was varying substantially over space or time, although we cannot determine the mechanism.For all three components of variability , variation in algal cover was larger than variation in coral or other. This can be seen in the shapes of the back-transformed unit ellipsoids of concentration . The amoq0.1) in the long term was 0.12 ). The corresponding site-specific probabilities qi0.1, varied from 8 × 10−5 to 0.52 but were low for most sites, with a strong negative relationship between probability of low coral cover and observed mean coral cover  than on fringing reefs  among nearby sites, while the possibility of negative spatial autocorrelation at scales of around 300 km may reflect the generally low values of qi0.1, for Tanzanian patch reefs, separated from sites in the north of the study area with generally higher qi0.1, by approximately 300km . The four parameters to which q0.1 was most sensitive were (in descending order: ζ21 (among-site covariance between transformed components 1 and 2), b22 (effect of component 2 on next year’s component 2), ζ22 (among-site variance of component 2), and b12 (effect of component 2 on next year’s component 1). Although there was substantial variability among Monte Carlo iterations in the values of these derivatives, the rank order of magnitudes was fairly consistent . The signs of the effects of each parameter on q0.1, sensitivities for coral cover thresholds 0.05 and 0.2, and elasticities, are discussed further in Both among-site variability and internal dynamics, particularly of other relative to algae and coral (component 2), were important in determining the probability nsistent . All fount ; component 2 posterior mean 0.82, 95% HPD interval ). This is consistent with the negative relationship between long-term probability of coral cover ≤0.1 and observed mean coral cover . Thus, wt → ∞), among-site variability dominated within-site temporal variability in East African coral reefs. In consequence, the long-term probability of coral cover ≤0.1 varied substantially among sites. This suggests that it is in principle possible to make reliable decisions about the conservation value of individual sites based on a survey of multiple sites at one point in time, and to design conservation strategies at the site level. This was not the only possible outcome: if within-site temporal variability dominated among-site variability, among-site differences would be neither important nor predictable in the long term.In the long term (as The dominance of among-site variability has important implications for conservation. There was clear evidence for the existence of a stationary distribution of long-term reef composition in East Africa. The overall shape of this distribution  was simi2, but not at larger spatial scales. They argued that it would therefore be appropriate to focus management actions at the km2 scale. Also, it may be easier to persuade local communities to accept management at such scales than at larger scales (In our study, the sites with the highest long-term conservation value are those with very low long-term probabilities of coral cover ≤0.1 , a thresr scales .Millepora into a single category. Comparisons between models with this level of aggregation and models in which corals are separated into functional groups show that aggregation can hide important biological differences (e.g. Orbicella showed less year-to-year variability than a site dominated by short-lived species (Acropora and Montipora, and relative increases in resistant genera such as Porites (In this study, we aggregated all scleractinians and ces e.g. . These d species . These d Porites . Priorit Porites , or in w Porites . Clearly Porites . Separat Porites  may be tOur analyses were based on the long-term consequences of current environmental conditions, and may therefore not be relevant if environmental conditions change. It is therefore better to view a stationary distribution under current conditions as a “speedometer” that tells us about the long-term outcome if these conditions were maintained, rather than as a prediction  p. 30. FIn conclusion, our analysis extends the broadly-applicable vector autoregressive approach to community dynamics (reviewed by 10.7717/peerj.3290/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.3290/supp-2Data S1Click here for additional data file.


PINK1 (PTEN-induced putative kinase 1) gene is the causal gene for recessive familial type 6 of Parkinson’s disease (PARK6), which is an early-onset autosomal recessive inherited neurodegenerative disease. PINK1 has been reported to exert both autophosphorylation and phosphorylation activity, affecting cell damage under stress and other physiological responses. However, there has been no report on the identification of PINK1 autophosphorylation sites and their physiological functions.(1) We adopted mass spectrometry assay to identify the autophosphorylation site of PINK1, and autoradiography assay was further conducted to confirm this result. (2) Kinase activity assay was used to compare the kinase activity of both Ser465 mutant PINK1 and disease-causing mutant PINK1. (3) We use Pulse-chase analysis to measure whether Ser465 may affect PINK1 degradation. (4) Immunocytochemistry staining was used to study the PINK1 subcellular localization and Parkin transition in subcellular level.In our study, we identified the 465th serine residue (Ser465) as one of the autophosphorylation sites in PINK1 protein. The inactivation of Ser465 can decrease the kinase activity of PINK1. Either dissipated or excessive Ser465 site phosphorylation of PINK1 can slow down its degradation. PINK1 autophosphorylation contributes to the transit of Parkin to mitochondria, and has no effect on its subcellular localization. PARK6 causal mutations, T313 M and R492X, display the same characteristics as Ser465A mutation PINK1 protein, such as decreasing PINK1 kinase activity and affecting its interaction with Parkin.Ser465 was identified as one of the autophosphorylation sites of PINK1, which affected PINK1 kinase activity. In addition, Ser465 is involved in the degradation of PINK1 and the transit of Parkin to mitochondria. T313 M and R492X, two novel PARK6 mutations on Thr313 and Arg492, were similar to Ser465 mutation, including decreasing PINK1 phosphorylation activity and Parkin subcellular localization. Parkinson’s disease (PD) is one of the most pervasive neurodegenerative diseases. Currently, the etiology and mechanism of PD are not fully clear. But, aging, environmental and genetic factors appear to play the key role in PD , 7, 21.PINK1 (PTEN-induced putative kinase 1) gene is the causative gene for PARK6, which was cloned in 2004 by Valente et al.  ATP were added, and incubated in 30 °C water bath for 30 min. Then 5XSDS was added, and boiled at 100 °C for 5 min. SDS-PAGE was carried out as described previously, and protein bands were stained with brilliant coomassie blue R250 for 5 min, and decolorized for another 5 min. The plastic wrapped gel and a film in the X-ray film cassette were placed in a dark room. The film was exposed at −80 °C for 8-16 h, followed by developing and fixing.The proteins were purified by the same method described before. 2.4 μL of 10X kinase reaction buffer, 19.6 μL of purified protein supernatant, 10 μCi of ).[10] have also confirmed that PINK1 can phosphorylate Parkin and jointly transfer to the mitochondria, which were gathered in clusters around the nucleus. The decreased kinase activity of these mutant PINK1 proteins may lead to the abnormal physiological processes.Our study also investigated the influence of PINK1 autophosphorylation on Parkin. Both wild-type PINK1 protein and S465D mutant protein can promote the transfer of Parkin to clustered mitochondria, while S465A, T313 M, R492X mutant PINK1 had a relatively weaker role in the interaction with Parkin. S465A mutant can disable the autophosphorylation of PINK1, repressing the transfer process; While mutant S465D possesses the phosphorylation ability, completing the same process as the wild-type protein. We can infer that PINK1 autophosphorylation contributes to the transit of Parkin to mitochondria. For the S465A, T313 M and R492X mutant protein, the inhibition of transfer process may be related to the reduced kinase activity. Some studiesPINK1 causative mutations in our study, T313 M and R492X, which can decrease the kinase activity and affect the interaction with Parkin in the same way as Ser465A, hinting that the change of autophosphorylation activity of PINK1 may be one of the mechanisms of the two disease-causing mutations.In summary, protein phosphorylation is a crucial aspect of intracellular signaling. Our study elucidated that PINK1 protein autophosphorylation can affect its kinase activity and protein stability, and influent PINK1 and Parkin protein interaction. We also included two


Immunoglobulin E and its interactions with receptors FcϵRI and CD23 play a central role in allergic disease. Omalizumab, a clinically approved therapeutic antibody, inhibits the interaction between IgE and FcϵRI, preventing mast cell and basophil activation, and blocks IgE binding to CD23 on B cells and antigen-presenting cells. We solved the crystal structure of the complex between an omalizumab-derived Fab and IgE-Fc, with one Fab bound to each Cϵ3 domain. Free IgE-Fc adopts an acutely bent structure, but in the complex it is only partially bent, with large-scale conformational changes in the Cϵ3 domains that inhibit the interaction with FcϵRI. CD23 binding is inhibited sterically due to overlapping binding sites on each Cϵ3 domain. Studies of omalizumab Fab binding in solution demonstrate the allosteric basis for FcϵRI inhibition and, together with the structure, reveal how omalizumab may accelerate dissociation of receptor-bound IgE from FcϵRI, exploiting the intrinsic flexibility and allosteric potential of IgE. DK ≈10−10m) that such cells are sensitized with pre-bound IgE, requiring only the presence of an allergen to cross-link IgE/FcϵRI complexes and elicit an immediate reaction. CD23 is a homotrimer, and thus the intrinsically lower affinity of each IgE-binding C-type lectin-like “head” domain (DK ≈10−7m) can be enhanced by an avidity effect when binding to aggregated IgE in immune complexes, nearly matching that of FcϵRI for IgE (Immunoglobulin E (IgE) antibodies play a crucial role in allergic disease, binding to allergens through their Fab arms and expressing their effector functions by binding to receptors for the Fc region . The two2 domain pair folded back onto the Cϵ3 and Cϵ4 domains (2 domain pair and that of Fcϵ3–4 (the region comprising only the Cϵ3 and Cϵ4 domains), was found to become even more acute in the crystal structure of IgE-Fc bound to sFcϵRIα, the soluble extracellular domains of the IgE-binding α-chain of the receptor 2 domain pair could “flip” over from one side of the Fcϵ3–4 region to the other  was found to bind to the Cϵ3 domain of receptor-bound IgE and accelerate its dissociation from FcϵRI  . It bind18–om FcϵRI . The cryom FcϵRI , 23. Omaom FcϵRI  and the om FcϵRI ; this coWe report here the crystal structure of the complex between IgE-Fc and a Fab derived from omalizumab. The structure of the complex reveals substantial conformational changes in IgE-Fc, revealing the mechanism of action of omalizumab, both for receptor inhibition and accelerated dissociation of IgE from FcϵRI. Solution studies demonstrate that these mechanisms exploit the intrinsic flexibility of IgE.L domain framework region (S81R and Q83R) and one in the Cκ domain (L158P) , with the purpose of disrupting favorable crystal contacts observed in the omalizumab Fab crystal structure. We term this omalizumab-derived Fab FabXol3.Despite extensive efforts, crystallization trials for IgE-Fc in complex with the omalizumab Fab resulted in selective crystallization of the Fab fragment only. Others have reported a similar failure to crystallize the complex with IgE-Fc . The recsupplemental Movie S1). Two FabXol3 molecules (Fab1 and Fab2) bind to an asymmetric, partially bent IgE-Fc molecule, and the Fab engages one edge of the exposed face of each Cϵ3 domain . The overall structure of IgE-Fc in complex with FabXol3 is compared with that of the constrained Fcϵ3–4 molecule in complex with the omalizumab Fab (2 domain pair (A), which is absent in the Fcϵ3–4 molecule (B), but also shows that the Cϵ3 domains adopt a markedly open conformation (C and E), one that cannot be adopted by the disulfide-bonded Cϵ3 domains in the Fcϵ3–4 complex (D and F). The bending of the (Cϵ2)2 domain pair and the opening of the Cϵ3 domains are described in detail below.We determined the crystal structure of the complex between IgE-Fc and FabXol3 to 3.7 Å resolution .Each FabXol3 molecule engages one edge of the exposed face of the Cϵ3 domain . The interface with IgE-Fc is similar to that reported for the constrained Fcϵ3–4 molecule . Both thA).The FabXol3 heavy chain contacts, which differ slightly between the two interfaces, may be summarized as follows: Gly-32 and Tyr-33 (CDRH1) form van der Waals interactions with Ala-377 and Ser-378 (Cϵ3), whereas Tyr-54 (CDRH2) contacts Gly-379–Pro-381 (Cϵ3). The CDRH3 residues contribute the largest contact area and undergo a significant conformational change upon complex formation, when compared with unbound Fab structures , 20, 24.Fig. 3A). Arg-419 is within hydrogen-bonding distance of the Tyr-31 (CDRL1) and Asp-32 (CDRL1) main chain carbonyl oxygen atoms, in addition to contacting the Asp-32, Asp-34, and Tyr-36 side chains (forming a hydrogen bond with the Tyr-36 hydroxyl group). Asp-32 also forms van der Waals interactions with Thr-373 and Thr-421 (Cϵ3). By contrast, only two CDRL2 residues contribute to the interface, Tyr-53 (CDRL2) contacts Gln-417 (Cϵ3), and both Tyr-53 and Tyr-57 form van der Waals interactions with Met-430 (Cϵ3); Tyr-57 also forms a hydrogen bond with the Met-430 backbone. As for the heavy chain interaction, there are slight differences in the light chain contacts for Fab1 and Fab2.Arg-419 also plays a key role in the interaction with the FabXol3 light chain A. Arg-41B) and are of similar size at ∼715 and ∼753 Å2, respectively. The Cϵ3 domain residues shared between the two interfaces include Ser-375–Gly-379, Gln-417, Arg-419, Arg-427, and Met-430, but although FabXol3 forms more intimate contacts with the receptor-binding Cϵ3 FG loop, the DARPin E2_79 interface extends in the opposite direction to include the Cϵ3–4 domain linker.The binding sites on the Cϵ3 domain for FabXol3 and the recently described DARPin E2_79  overlap Fig. 32, but aϵFab engages IgE-Fc at a site centered on Arg-393 in Cϵ3, and it also contacts residues in the Cϵ2 domain and the Cϵ2-Cϵ3 linker . No. No16).  Asn-394 .2 domain pair folded back onto the Cϵ3 and Cϵ4 domains (A and B), the Cϵ2 domain of one chain (chain B) contacting the Cϵ4 domain of the other (chain A)  , 8. IgE-chain A) , and theC and C–F, and Movies S1 and S2). The site to which Fab1 binds is exposed in free, acutely bent IgE-Fc, but further unbending of IgE-Fc, to just over 90°, is required to render the site occupied by Fab2 accessible. This unbending of IgE-Fc in the FabXol3 complex is associated with opening of both Cϵ3 domains to create an almost symmetrical Fcϵ3–4 region .In contrast to the aϵFab complex, in which IgE-Fc adopts a fully extended, linear conformation , IgE-Fc Figs. 1supplemental Fig. S2).In a recent molecular dynamics simulation exploring unbending of IgE-Fc to an extended structure, it was found that although the acutely bent conformation observed in the crystal structure of free IgE-Fc occupied the lowest energy basin, another distinct and well defined energy basin, corresponding to partially bent IgE-Fc conformations, was observed . The parEmax = 91%, IC50 = ∼1 μm) when two molecules of aϵFab are bound to IgE-Fc . These experiments support the hypothesis that binding of FabXol3 induces large-scale conformational changes in IgE-Fc, consistent with a partially bent conformation.To test whether we could observe these conformational changes in solution, we performed intramolecular FRET measurements with IgE-Fc labeled with donor and acceptor fluorophores in the Cϵ2 domains and at the C termini, respectively. We have previously shown that binding of aϵFab results in a nearly fully extended IgE-Fc structure ; data heo IgE-Fc . In contsupplemental data). In the FabXol3/IgE-Fc complex, the Cϵ3 domains are positioned further away from one another than in any other crystal structure containing IgE-Fc or Fcϵ3–4 and thus adopt the most open conformation observed thus far ; this conformation is significantly more open than the conformation for FcϵRI-bound IgE-Fc .In crystal structures of IgE-Fc and the Fcϵ3–4 sub-fragment, the Cϵ3 domains adopt a range of different orientations , 25, 29,11–A and B)  , 14, 31.C–E). Moreover, superposition of the FabXol3/IgE-Fc and sFcϵRIα/IgE-Fc . Asp-32 (CDRL1) contacts Thr-421; Gly-33 (CDRL1) contacts Pro-426, Arg-427, and Ala-428; and Asp-34 (CDRL1) contacts Arg-427 and Ala-428. These interactions alter the position of the Cϵ3 domain FG loop and would further compromise the binding of IgE to FcϵRI.Also in contrast to CD23, the binding sites for omalizumab and FcϵRIα do not actually overlap, although FabXol3 CDRL1 residues are positioned immediately adjacent to the FcϵRIα-binding Cϵ3 domain FG loop. This loop, in chain B, contributes to a hydrophobic “proline sandwich” interaction, in which Pro-426 in Cϵ3 packs between two tryptophan residues of FcϵRIα E. Asp-32Thus, omalizumab binding stabilizes a conformation of IgE-Fc, which is incompatible with FcϵRI binding.Recently, binding of omalizumab to FcϵRIα-bound Fcϵ3–4 has been reported , althougA–C). Not surprisingly, in competition binding experiments, all three molecules competed for the same binding sites and showed broadly similar binding affinities; the FabXol3 construct demonstrates slightly higher affinity compared with FabXol and intact omalizumab (A–C). Consistent with the crystal structure, two FabXol3 molecules bind to IgE-Fc; the binding is clearly biphasic with a high-affinity (∼1 nm) interaction observed at low ligand concentrations and a second (weaker) binding site (∼30 nm) observed at higher concentrations (D). It might be speculated that the higher affinity interaction corresponds to the binding of Fab1, which would have unimpeded access to a bent IgE-Fc molecule, whereas the lower affinity and slower on-rate corresponds to Fab2, but we cannot be definitive about this.We characterized the IgE-Fc/FabXol3 interaction in two different ways, either by directly immobilizing FabXol3, FabXol , or intact omalizumab on an SPRalizumab , A–C. Cotrations D. It migDK ∼1 nm, kon ∼1.2 × 106m−1 s−1, and koff ∼ 8 × 10−4 s−1). This 1:1 FabXol3/IgE-Fc complex, captured on the SPR biosensor surface, could then be used to measure the binding of the second FabXol3 molecule, the binding of which is significantly weaker  than the first (E). Again, the slower association rate constant measured for the second (weaker) interaction would be consistent with the Fab binding to the less accessible of the two binding sites, i.e. the Fab2 site in the crystal structure.A sandwich-style SPR experiment allowed the two FabXol3-binding sites to be observed and characterized separately. Using this approach, FabXol3 was covalently immobilized on a sensor surface, and IgE-Fc was flowed over this surface. At low concentrations, under these conditions, the high-affinity site dominates the interaction, and the binding curves can be described by monophasic interaction kinetics . Mechanistically, FabXol3 affects both the number of available binding sites (Bmax) and the apparent DK value of the IgE-Fc/FcϵRIα interaction. Reduction in Bmax values is indicative of an allosteric inhibitory process, and a decrease in the apparent affinity of the interaction is most commonly associated with direct competition for a shared binding site (i.e. orthosteric inhibition) but can also be seen for some allosteric inhibitors . The data indicate that although the binding of IgE-Fc to FcϵRIα did not significantly change the affinity of FabXol3 for IgE-Fc, it did markedly change the number of available binding sites for FabXol3 in the population of FcϵRIα-bound IgE-Fc molecules. We compared the DK and Bmax binding values for an IgE-Fc molecule captured by an anti-His tag antibody with one captured by sFcϵRIα, and we found that FcϵRIα-bound IgE-Fc had less than 10% of the FabXol3-binding sites compared with the His tag captured IgE-Fc, which, as expected, showed binding levels consistent with 2:1 stoichiometry (B). Therefore, it is not that omalizumab does not bind to mast cell-bound IgE because the FcϵRIα and omalizumab-binding sites overlap, or because of steric clashes between two ligands bound to adjacent sites  and, furthermore, that FabXol3 does this more efficiently than FabXol, and much more efficiently than intact omalizumab (D). One Fab engages the IgE-Fc/sFcϵRIα complex but does not accelerate the dissociation of IgE-Fc from sFcϵRIα. Strikingly, it appears that accelerated dissociation occurs only after occupancy of the second binding site (i.e. the low-affinity site). The (FabXol3)2/IgE-Fc/sFcϵRIα tetramolecular complex must alter the energy landscape of IgE-Fc in such a way as to markedly reduce the energy barrier for IgE-Fc/sFcϵRIα dissociation, resulting in a rapid dissociation of this otherwise very stable complex.Kim et al.  reported  and is consistent with our intramolecular FRET measurements , and it is therefore possible that IgE-Fc can undergo further substantial changes in conformation even when in complex with omalizumab.IgE-Fc is predominantly bent in solution 4–, 26–28, 26– domains , 8. Rece domains . A molecmulation   to open , 25, 29. to open , 12; in  to open , 13, 14, to open , 10. ComHowever, inhibition of FcϵRI binding is mechanistically different. In the FabXol3 complex, the Cϵ3 domains adopt a more open conformation than seen in any previous structure, so much so that the two sub-sites of interaction between IgE-Fc and FcϵRI, one involving each Cϵ3 domain, cannot engage simultaneously. In addition to large scale domain motions, local conformational changes induced by FabXol3 binding, such as those in the FcϵRI-binding FG loop, may also contribute to this inhibition. Although there is a possibility of steric clashes if FabXol3 and FcϵRIα bind simultaneously to IgE-Fc, the crystal structure of the FabXol3/IgE-Fc complex demonstrates that omalizumab's mechanism of inhibition is principally allosteric.Bmax) when in complex with FcϵRIα. The inhibition of IgE binding to FcϵRI by omalizumab has frequently been interpreted in terms of direct competition for overlapping sites, but there have been reports that indicate that omalizumab can bind to receptor-bound IgE  NaN3, and concentrated to 23 mg/ml.Omalizumab human IgGr et al. . IgE-Fc r et al. . Omalizu−1 in a running buffer of 20 mm HEPES, pH 7.4, 150 mm NaCl, and 0.005% (v/v) surfactant P-20 . Most experimental binding measurements were performed at 25 °C; some binding experiments were performed over a range of temperatures (5–35 °C) to control the degree of accelerated dissociation in the system; low temperatures minimize this phenomenon, and higher temperatures increase it. In all cases, standard double referencing data subtraction methods were used . Specific surfaces were prepared either by covalently coupling proteins using the amine coupling protocol , with coupling densities <300 resonance units, or capturing His-tagged proteins using an anti-His sensor surface. For capturing His-tagged ligands, an anti-His tag monoclonal antibody was employed and immobilized according to the manufacturer's instructions . In binding experiments, association times of 180–240 s were typically used, and dissociation components were monitored for at least 500 s. Injections were performed at a flow rate of 25 μl minatures 5– °C to com sodium bicarbonate, 50 mm NaCl, pH 9.3, with a 5-fold molar excess of terbium chelate isothiocyanate (Invitrogen). After a 3-h incubation at room temperature with agitation, excess unreacted fluorophore was removed by dialyzing into PBS . sFcϵRIα-IgG4-Fc fusion protein (α-γ) (IgE-Fc was labeled with donor fluorophore by reacting 4 mg/ml protein in 100 min (α-γ)  was labem terbium-labeled IgE-Fc and 0–20 nm Alexa Fluor 647-labeled sFcϵRIα-IgG4-Fc with a range of concentrations of FabXol3. Assays were conducted in 384-well hi-base white plates (Greiner BioOne) using Lanthascreen buffer (Invitrogen) as a diluent. The plate was left to incubate overnight at room temperature and read by an Artemis plate reader (Berthold Technologies). TR-FRET ratios were then calculated for each well as the emission of acceptor at 665 nm divided by the emission of donor at 620 nm multiplied by 10,000.TR-FRET inhibition assays were performed by competing 1 net al.  with an excitation wavelength of 330 nm and emission wavelengths of 485 and 520 nm. Each sample was measured in quadruplicate, and in at least two separate experiments.Measurements of intramolecular FRET were performed essentially as described in Drinkwater et al. , using aet al. . The antm sodium fluoride, and the drop contained 100 nl of protein solution and 300 nl of reservoir. Despite extensive efforts at optimization, the diffraction quality of the crystals could not be further improved beyond that used for this study. Crystals typically started to grow after a few days and often dissolved in their drops, but they could be stabilized in 4 m trimethylamine N-oxide, which was successfully used as a cryoprotectant.Crystals up to 400 μm in length were grown at 18 °C using the sitting drop vapor diffusion method. The reservoir contained 50 μl of 4% (w/v) PEG 8000 and 0.03 Data were collected at beamlines I02 and I03 at the Diamond Light Source . Integration was performed using XDS  as imple2WQR (supplemental Fig. S4. Interfaces were analyzed with PISA (The structure was solved by molecular replacement with PHASER  and MOLR2WQR  and a 1.2WQR , 24 as s2WQR  and late2WQR  and alte2WQR . The qua2WQR  and POLY2WQR . Data prith PISA , figuresith PISA , and movith PISA , PyMOL (ith PISA , and theith PISA  for PyMOT. C., A. J. H., J. M. M., and B. J. S. designed the experiments. A. M. D., E. G. A., A. H. K., J. D., M. O. Y. P., A. J. B., A. J. H., and J. M. M. performed the experiments. A. M. D., E. G. A., A. H. K., J. D., B. P. C. A. N. M., M. O. Y. P., T. C., A. J. B., G. C., M. W., A. J. H., J. M. M., and B. J. S analyzed and/or discussed the data. A. M. D., B. P. C., A. J. H., J. M. M., and B. J. S wrote the manuscript. All authors approved the manuscript.


We applied genetically encoded unnatural amino acids (Uaas) and monitored the functional changes in living cells with photo-cross-linkers specifically incorporated at the ifenprodil binding interface between GluN1 and GluN2B subunits. We report constraining the NTD domain movement, by a light induced crosslinking bond that introduces minimal perturbation to the ligand binding, specifically impedes the transduction of ifenprodil but not Zn2+ inhibition. Subtle distance changes reveal interfacial flexibility and NTD rearrangements in the presence of modulators. Our results present a much richer dynamic picture of allostery than conventional approaches targeting the same interface, and highlight key residues that determine functional and subtype specificity of NMDARs. The light-sensitive mutant neuronal receptors provide complementary tools to the photo-switchable ligands for opto-neuropharmacology.Allostery is essential to neuronal receptor function, but its transient nature poses a challenge for characterization. The N-terminal domains (NTDs) distinct from ligand binding domains are a major locus for allosteric regulation of NMDA receptors (NMDARs), where different modulatory binding sites have been observed. The inhibitor ifenprodil, and related phenylethanoamine compounds specifically targeting GluN1/GluN2B NMDARs have neuroprotective activity. However, whether they use differential structural pathways than the endogenous inhibitor Zn Ligand-gated ion channels (LGICs) in response to the binding of chemical messengers, such as neurotransmitters, mediate synaptic transmissions. LGICs are membrane proteins composed of multiple subunits. Allosteric modulators selectively binding at sites distinct from agonists binding domains can affect channel function. Understanding the mechanisms of allosteric regulation and their impacts in gating is an important goal in neurophysiology and neuropharmacology. Recently, the electron microscopic structures and high-resolution crystal structures of several full-length LGICs122+ are two kinds of subunit-selective allosteric inhibitors that bind at distinct sites of NTDs of NMDARs. Although much information has been gained on the functional roles of ifenprodil and Zn2+ in the NMDARs allosteric signaling, whether and how they use differential structural pathways for inhibition remains unclear. Synthetic compounds ifenprodil and related phenylethanolamine derivatives, which specifically inhibit GluN1/GluN2B receptors, have been intensely studied for their potential use in the treatment of various neurological disorders and diseases892+ binding site which is in the GluN2B-NTD cleft revealed by the isolated GluN2B NTD structure112+ acting as an endogenous allosteric modulator plays a key role in physiology by shaping NMDAR synaptic currents and in pathology by modulating pain processing7A functional hallmark of NMDARs is that allosteric regulations by binding of small molecules at the interface of the N-terminal domains (NTDs)462+ and ifenprodil induced allosteric inhibition, we set out to apply the photo-chemical approach established previously15p-azido-L-phenylalanine (AzF) is introduced at a specific position in the receptor. Functional changes can be detected if the light-stimulated crosslinking causes structural rearrangements. The distance-dependent crosslinking may provide information for interfacial contacts. Previously we have applied the strategy at the Zn2+ 2+ and ifenprodil binding. In this case, AzF as a light-sensitive crosslinking probe is decisively advantageous to reveal the specific functional role GluN1/GluN2B interface in the transduction of ifenprodil inhibition, which conventional approaches failed to reveal. These findings pave the way for guiding the design of subtype-specific compounds with therapeutic value for neurological disorders and diseases. We further discuss the potential applications of the identified light-sensitive NMDARs in the context of opto-neurophamarcology.To differentiate the mechanisms between Zno25-6981-bound (phenylethanoamine derivative) forms of the structures  were chosen  and lower-lobe (LL) domains. In both ifenprodil-bound and Rructures 89, the ie chosen . In ordeXenopus laevis oocytes four DNA plasmids containing genes encoding the AzF aminoacyl tRNA-synthetase (AzF-RS), the orthogonal suppressor tRNA (Yam), the wild-type GluN1, and the GluN2 subunit with an amber stop codon mutation at the desired position18We co-injected into uv) were compared to currents before UV illumination (Io) and yielded the relative current changes (Iuv/Io). Among eight maximally activated GluN1/GluN2B-AzF mutants, two mutants (GluN2B-I82AzF and GluN2B-F114AzF) showed robust UV induced potentiation  condition, based on the observation that similar UV potentiation effect can be produced in the resting state . In this2 LED light source, 100% UV illumination evoked a 1.84 ± 0.11 (n = 3) relative current after three minutes of illumination (on = 49.5 s).We then investigated the relationship between the light power and current potentiation by modulating UV intensities. Using a 42 mW/cmmination . DecreasIn order to determine the heterodimer formation between subunits of GluN1 and GluN2B-F114AzF through photo-cross-linking, we performed western-blotting experiments . To faciTo reveal the cross-linking partner on the opposite GluN1 subunit, we combined the classical mutagenesis with the UV functional assay. Upon UV excitation, AzF usually generates a nitrene radical and forms a covalent linkage with a nearby atom at a distance of 3–6 Å2021823o) using MK-801, a specific open-pore blocker of NMDARso), this MK-801 assay is classically used to index NMDAR channel activity. Surprisingly, before UV treatment, MK-801 inhibition kinetics of the mutant was significantly slowed down compared to wild-type receptors, indicating a marked decrease of Po , suggesting the moderate change in glutamate affinity has no influence on the Po of the mutant.To determine if the PUV light . WithoutUV light . After U2+ bind at spatially distinct locations at the GluN1/GluN2B NTDs , indicating that AzF and crosslinking had no significant influence on ifenprodil binding. In addition, it suggests that the significant decrease in the ifenprodil maximal inhibition for the mutant before and after UV is not due to the change of ifenprodil binding, but rather an intrinsic functional feature of the interface.Ifenprodil and ZnN2B NTDs . To explN2B NTDs . After U2+ inhibition, we have performed Zn2+ titrations before and after UV treatment on the GluN1/GluN2B-F114AzF receptors. IC50 was slightly increased to 1.72 ± 0.60 μM for the UV treated mutant receptors, compared to 0.72 ± 0.12 μM of the wild-type  indicating the interface has minimal effect on the Zn2+ binding. In addition, there was no change in the maximal inhibition. Maximal inhibition is associated with the transduction of the allosteric modulation to gating. Our comparisons between ifenprodil and Zn2+ affinities using the light-sensitive mutant suggest the transduction of ifenprodil inhibition, but not Zn2+, is reduced  and compared to the relative current observed without UV   . Surpris; n = 7) . One exp; n = 7) .+uv/I0 values  as measured in the agonists alone (τon = 49.5 s). Taken together, our results reveal two mechanisms: (1) Functional changes induced by light and allosteric modulations are independent and not mutually exclusive; (2) Since AzF mediated crosslinking is strictly distance dependence1420212+ subtly separating the interface while spermine enhancing it by favoring crosslinking geometry , which is slightly faster than our previously identified inhibiting AzF mutant (τon = 80 s)on = 3–5.6 s)on = 0.3 s)on = 0.1–8 ms)The UV potentiation effect is remarkable, because it drastically contrasts the inhibition effect of ifenprodil binding. It provides the first evidence for a possible mechanism to positively regulate the receptor function at the ifenprodil binding interface, which is a dynamic transition from low Pp-benzoyl-L-phenylalanine, Bpa), we have found that when inserting Bpa which is bulkier than AzF  was purchased from Chem-Impex International  and p-benzyol-L-phenylalanine from Bachem , respectively. HEPES, l-glutamate, glycine, DTPA, spermine were obtained from Sigma–Aldrich. D-APV and (+)-MK-801 were purchased from Ascent Scientific . Ifenprodil was purchased from Synthélabo (France).B. stearothermophilus Tyr-tRNACUA and has been described previouslyPlasmid pSVB.Yam carrying the gene encoding the amber suppressor tRNA was derived from Xenopus laevis oocytes after nuclear injection of 36 nl of a mixture cDNAs encoding various GluN1 and GluN2 subunits . For Uaa incorporation, oocytes were co-injected with a 36 nl mixture of cDNAs containing GluN1, GluN2, Yam, and aaRS as follows, unless otherwise indicated in the text: GluN1-wt (60 ng/μl), GluN2A-F115AzF (60 ng/μl), Yam (5 ng/μl), AzF-RS (2.5 ng/μl); or, GluN1-wt (80 ng/μl), GluN2B-F114AzF (80 ng/μl), Yam (10 ng/μl), AzF-RS (5 ng/μl). After injection, oocytes were incubated at 19 °C in a Barth solution 2, 0.41 mM CaCl2, 0.82 mM MgSO4, 2.4 mM NaHCO3, 10 mM HEPES, pH adjusted to 7.6 with NaOH) supplemented with gentamicin sulfate (50 ng/ml) and D-APV (50 μM). AzF was dissolved with sonication in Barth solution (stock solution at 10 mM), and diluted (1 mM) for oocytes incubation.Oocytes were prepared and injected as described previously2, and 5 mM HEPES, pH adjusted to 7.3 with KOH. Other than for the glutamate sensitivity assay (100 μM glycine in the presence of various concentrations of glutamate), NMDAR-mediated currents were induced by applying glutamate (100 μM) and glycine (100 μM), which activate the receptors at the maximum level. Currents were recorded and measured at a holding potential of −60 mV at room temperature.For all experiments, the standard external solutions contained: 100 mM NaCl, 300 mM BaCl2 stock (in 1% HCl). In all zero-Zn2+ control solutions, diethylenetriamine-pentaacetic acid  was added to chelate trace Zn2+ and other heavy metalson) were obtained by fitting currents with a single-exponential component to a time window corresponding to 10–90% of maximum inhibition. Each τon was then normalized to the mean τon of wt receptors measured the same day.Glutamate, ifenprodil, zinc dose response curve experiments were performed and analyzed as previously describedSample preparation, non-reducing SDS-PAGE, and immunoblotting were performed as describedXenopus oocytes, online UV light treatment with PE-2 light source (CoolLED) using a 365 nM filter channeled through optical fiber was directly applied to the dark hemisphere of the oocytes in the recording chamber. Total power measured at a distance of 200 mm from the source is 105 mW (42 mW/cm2), as reported by the manufacturer. For measurements in presence of agonists and allosteric modulators, the UV application duration was ~3 min. In the absence of agonist, the UV application duration was ~5 min.For all functional recordings in For western immunoblotting, oocytes expressing wt NMDA receptors or AzF mutant receptors were transferred to a 96-well plate containing Barth solution free of AzF and agonists  on ice. Cells were irradiated for 30 min with a hand-held VL-6LC UV lamp  placed on top of the plate. After UV treatment, cells were subject to western blotting analysis.How to cite this article: Tian, M. and Ye, S. Allosteric regulation in NMDA receptors revealed by the genetically encoded photo-cross-linkers. Sci. Rep.6, 34751; doi: 10.1038/srep34751 (2016).


Stimulation of TNFR1 by TNFα can promote three distinct alternative mechanisms of cell death: necroptosis, RIPK1-independent and -dependent apoptosis. How cells decide which way to die is unclear. Here, we report that TNFα-induced phosphorylation of RIPK1 in the intermediate domain by TAK1 plays a key role in regulating this critical decision. Using phospho-Ser321 as a marker, we show that the transient phosphorylation of RIPK1 intermediate domain induced by TNFα leads to RIPK1-independent apoptosis when NF-κB activation is inhibited by cycloheximide. On the other hand, blocking Ser321 phosphorylation promotes RIPK1 activation and its interaction with FADD to mediate RIPK1-dependent apoptosis (RDA). Finally, sustained phosphorylation of RIPK1 intermediate domain at multiple sites by TAK1 promotes its interaction with RIPK3 and necroptosis. Thus, absent, transient and sustained levels of TAK1-mediated RIPK1 phosphorylation may represent distinct states in TNF-RSC to dictate the activation of three alternative cell death mechanisms, RDA, RIPK1-independent apoptosis and necroptosis. TNFα can promote three distinct mechanisms of cell death: necroptosis, RIPK1-independent and dependent apoptosis. Here the authors show that TNFα-induced phosphorylation of RIPK1 in the intermediate domain by TAK1 plays a key role in regulating this decision. Within minutes after cells stimulated by TNFα, RIPK1 is recruited into the TNFR1 signaling complex  together with signaling molecules such as TRADD, TRAF2 and cIAP1/2 to decide if a cell and ultimately, an organism, may live or die through apoptosis or necroptosis. Apoptosis may be mediated by binding of RIPK1, independent of its kinase activity, with FADD, an adaptor protein for caspase-8, which in turn promotes the activation of caspase-8 and executes apoptosis by triggering mitochondrial damage and the cleavage of downstream caspases such as caspase-3. Under apoptotic deficient conditions, RIPK1 may be activated to promote necroptosis by interacting with RIPK3 which in turn promotes the phosphorylation of MLKL to mediate the execution of necroptosis.RIPK1, a member of the receptor-interacting protein (RIP) serine-threonine kinase family, has emerged as a key upstream regulator that controls multiple downstream signaling pathways of TNFR13. Activated TAK1 mediates the phosphorylation of IKKβ to promote the formation of the IKK complex consisting of IKKα/β/γ(NEMO)4. Although the best characterized function of TAK1 and the IKK complex including NEMO is to mediate the activation of NF-κB pathway, recent studies have unveiled that deficiencies in TAK1, NEMO, IKKα/β or the loss of cIAP1/2 can sensitize cells to RIPK1-dependent apoptosis (RDA) independently of their roles in NF-κB activation6. On the other hand, in cells deficient for A20, an important ubiquitin-editing enzyme for RIPK1, or TAB2, which regulates the activation of TAK1, RIPK1 may be activated to interact with RIPK3 to mediate necroptosis8. It is not clear, however, how activated RIPK1 might be directed to mediate two alternative modes of cell death, RDA or necroptosis, that both occur in a RIPK1 kinase-dependent manner.Ubiquitination of RIPK1 by cIAP1/2 in TNF-RSC is involved in mediating the activation of NF-κB by recruiting TAB1/2 to promote the activation of the TAK1 1. The kinase activity of RIPK1 may be activated upon stimulation of TNFR1 by TNFα under selective conditions, which leads to multiple deleterious consequences including cell death and inflammation. Inhibition of RIPK1 kinase activity using improved necrostatin-1 , a highly specific small molecule inhibitor of RIPK1, and the use of RIPK1 kinase-dead mutant mice, have shown efficacy in a wide range of animal models of human diseases11. Small molecule inhibitors of RIPK1 are under clinical and preclinical development targeting human diseases. However, the molecular mechanism that controls the activation of RIPK1 kinase activity remains unclear.RIPK1 contains an N-terminal kinase domain, an intermediate domain and a C-terminal death domainHere we show that the intermediate domain of RIPK1 is phosphorylated transiently by TAK1 upon TNFα stimulation in wild-type (WT) cells in vitro and in vivo. While Ser321 (S321) phosphorylation of RIPK1 by TAK1 has no effect on the NF-κB activation, the loss of S321 phosphorylation promotes the binding of RIPK1 to FADD and RDA. On the other hand, the sustained TAK1-mediated phosphorylation of RIPK1 in multiple sites of the intermediate domain including S321 promotes its interaction with RIPK3 to mediate necroptosis. Our results elucidate the molecular mechanism of interaction between TAK1 and RIPK1, two critical mediators in the TNFα signaling pathway, distinct from their roles in the activation of the NF-κB pathway, and the mechanism by which the levels of RIPK1 phosphorylation control the cellular choices for alternative cell death mechanisms.12. S321 site is evolutionarily conserved in RIPK1 proteins from species including mouse, human, rat and cattle . We probed western blots of cell lysates from mouse embryonic fibroblast (MEF) cells stimulated with TNFα for different periods of time. After stimulation of TNFα, the RIPK1 band showed a specific up-shift within 5 min TNFα stimulation and the shift reduced after 30–60 min treatment. This shifted band was recognized by anti-p-S321-RIPK1 Fig. . The phoOn the basis of the temporal profile of RIPK1 S321 phosphorylation upon stimulation by TNFα, we focused on characterizing the involvement of TAK1 and TBK1 as possible kinases responsible for phosphorylating RIPK1 S321 in response to TNFα stimulation. We treated L929 and BV-2 cells with TNFα in the presence of TAK1 inhibitor  or TBK1 inhibitor (BX795). The treatment with either TAK1 or TBK1 inhibitors blocked TNFα-stimulated S321 phosphorylation in L929 cells 14. To generate TAK1-deficient MEFs, we excised the floxed genomic fragment by infecting TAK1F/F MEFs with virus expressing Cre and selected single colonies lacking the expression of TAK1 (TAK1F/F Cre). In TAK1F/F Cre MEFs, RIPK1 S321 phosphorylation in response to TNFα was substantially abolished , a well-established biomarker for liver damage. C57BL/6 mice are known to be tolerogenic to rAAV gene delivery to the liver23. Only a basal level of ALT release was found in the plasma of mice received control (GFP-expressing) AAV. Interestingly, while low levels of ALT release were detected in the plasma of mice transduced with RIPK1 WT, significantly elevated levels of ALT were found in the plasma of mice that received RIPK1 S321A, but not S321E, AAV  MEFs  MEFs. We found that the activity of caspase-8 in S321A(A/A) MEFs, but not WT or S321E(E/E) MEFs, was stimulated by TNFα alone Fig. . Consist25. Since S321 is only one amino acid beyond the usual 4-amino acid motif required for caspase recognition, we tested if the phosphorylation of S321 or its single point mutant might affect the cleavage by caspase-8 in vitro. However, S321A mutation had no effect on the cleavage by caspase-8 in vitro while S321E only showed weak increase of cleavage in vitro  by caspase-8 during TNFα mediated apoptosis27. To determine if the activation of RIPK1 might be affected by the phosphorylation of S321, we compared the phosphorylation of RIPK1 S166 in WT and S321A(A/A) MEFs stimulated by TNFα, TNFα/CHX or TNFα/CHX/zVAD. Stimulation of TNFα/CHX/zVAD is known to activate RIPK1 and necroptosis26. Consistently, the phosphorylation of RIPK1 S166 in WT MEFs was only detected upon stimulation by TNFα/CHX/zVAD together, but not by TNFα/CHX or TNFα alone  MEFs also showed an increased sensitivity to necroptosis induced by TNFα/CHX/zVAD, which was inhibited by the addition of Nec-1s Fig. . On the Since the activation of caspase-8 is mediated by the interaction of RIPK1 with FADD, we next characterized the interaction of RIPK1 and FADD in WT and S321A(A/A) MEFs by co-immunoprecipitation. We found that compared to that of WT MEFs, the interaction of RIPK1 and FADD in S321A(A/A) MEFs after treatment with TNFα/CHX was significantly increased. Furthermore, the treatment with Nec-1s inhibited the interaction of FADD and RIPK1 in S321A(A/A) MEFs but not in WT MEFs induced by TNFα/CHX Fig. . Taken t8; however, the mechanism is unclear. Given the enhanced phosphorylation on RIPK1 S321 in TAB2 KO cells, we hypothesized that hyperphosphorylation of RIPK1 by TAK1 might promote necroptosis. To test this hypothesis, we first characterized complex IIa formation and RIPK1 cleavage in S321E(E/E) MEFs induced by TNFα/CHX. When treated with TNFα/CHX, caspase-8-dependent RIPK1 cleavage was significantly reduced in S321E(E/E) MEF cells compared to WT  quadruple mutant as a model for sustained RIPK1 phosphorylation in the intermediate domain. We found that the expression of RIPK1 EEEE mutant in RGC-5 RIPK1 KO cells was sufficient to induce spontaneous RIPK1 kinase activation as shown by the detection of RIPK1 S166 phosphorylation and its interaction with RIPK3 in the absence of TNFα stimulation Fig. . IncreasIn this manuscript, we demonstrate a novel mechanism by which phosphorylation of the intermediate domain of RIPK1 by TAK1 dictates alternative cell death mechanisms. We show that dysregulation of RIPK1 phosphorylation by TAK1, including both inhibition or hyperphosphorylation, promotes the activation of RIPK1. On the other hand, TAK1-mediated phosphorylation in the intermediate domain of RIPK1 dictates whether RIPK1 interacts with FADD to form the complex IIa to mediate apoptosis in RIPK1-dependent or -independent manner, or with RIPK3 to form the necrosome (complex IIb) to drive necroptosis. Specifically, the lack of RIPK1 S321 phosphorylation by TAK1 promotes its interaction with FADD to mediate RDA; whereas excessive phosphorylation of RIPK1 by TAK1 in the intermediate domain promotes its interaction with RIPK3 while suppressing its binding with FADD to promote necroptosis. In WT cells, TNFα-induced transient phosphorylation of RIPK1 by TAK1 in the presence of CHX is sufficient to block the activation of RIPK1 and drives RIPK1-independent apoptosis. On the other hand, the absence of S321 phosphorylation on RIPK1 in TNFα alone or TNFα/CHX-stimulated S321A(A/A) cells promotes the activation of RIPK1 and RIPK1-dependent caspase activation to mediate RDA. Finally, the sustained phosphorylation of RIPK1 by TAK1 modeled by RIPK1 EEEE mutant suppresses RIPK1-FADD interaction but promotes the interaction of RIPK1 and RIPK3 to promote the activation of necroptosis in the absence of TNFα stimulation. Thus, absent, transient and sustained levels of TAK1-mediated RIPK1 phosphorylation in TNF-RSC may represent three distinct states to dictate the execution of three alternative cell death mechanisms, RDA, RIPK1-independent cell death and necroptosis, by regulating the interaction of RIPK1 with FADD in kinase-dependent or -independent manner to mediate apoptosis, or with RIPK3 to mediate necroptosis Fig. .κB kinase (IKK) and activation of NF-κB pathway, which has a powerful pro-survival role by inducing the expression of target genes that can block apoptosis, promote cell proliferation and stimulate inflammatory responses29. Our study defines a novel molecular contribution of TAK1 that controls the activity of RIPK1 kinase in mediating apoptosis, which is consistent, but distinct, from the role of TAK1 in promoting cell survival and inflammation in mediating NF-κB pathway activation. Supporting this idea, S321A mutation has no effect on the activation of NF-κB when cells are stimulated by TNFα, as blocking NF-κB activation by the addition of CHX, Smac mimetic, or TAK1 inhibitor, 5Z-7, is still required to significantly induce apoptosis in S321A(A/A) MEFs.In TNFα-stimulated cells, TAK1 is recruited to TNFR1 in a RIPK1-dependent manner to promote the phosphorylation of ITNFAIP3, is an important ubiquitin-editing enzyme recruited to TNF-RSC to terminate multiple downstream events including NF-κB-mediated transcriptional response, RIPK1-mediated signaling and ultimately, the disassembly of the TNFR1 signaling complex30. K63 ubiquitination of RIPK1 is abnormally elevated in A20-deficient cells31. Since K63 ubiquitination of TNF-RSC is critical for mediating the activation of TAK14, the defective removal of K63 ubiquitination from TNF-RSC in A20-deficient cells are predicted to lead to sustained TAK1 activation. However, since increased activation of TAK1 in A20-deficient cells is expected to promote sustained activation of NF-κB, it has been puzzling as to why A20 deficiency also sensitizes cells to necroptosis7. Our study reveals a previously unexpected link between A20-regulated ubiquitination of TNF-RSC and TAK1-mediated RIPK1 phosphorylation, which provides mechanistic insights as how A20 deficiency might promote the activation of RIPK1/RIPK3 complex to mediate necroptosis independent of NF-κB activation. Recent genome-wide association studies have identified single-nucleotide polymorphisms at the TNFAIP3/A20 locus in humans that are linked to susceptibility/resistance to inflammatory and autoimmune diseases32. Our study also suggests the possible clinical application of phospho-S320 in human RIPK1  as a biomarker for RIPK1-dependent cell death and inflammation.Our results demonstrate that deficiencies in TAB2 or A20 promote the phosphorylation of RIPK1 S321. Elevated levels of RIPK1 phosphorylation in the intermediate domain in TAB2 KO MEFs drive the interaction of RIPK1 and RIPK3 to promote necroptosis. On the other hand, A20, encoded by the gene The following commercial antibodies and reagents were used in this study: TAK1, Cell Signaling Technology (5206); RIPK1, Cell Signaling Technology (3493) and BD Biosciences (610459); TBK1, Cell Signaling Technology (3504); p-IKKα/β, Cell Signaling Technology 2697); IKKβ, Cell Signaling Technology (8943); IκBα, Santa Cruz (sc-371); p-p38, Cell Signaling Technology (9211); p38, Cell Signaling Technology (9212); CYLD, Cell Signaling Technology (8462); FADD, Abcam (ab124812) and Santa Cruz (6036); α-Tubulin, Sigma-Aldrich (T9026); β-actin, Santa Cruz (81178). α-Tubulin and β-actin antibodies were used with 5000-fold dilution and other antibodies were used with 1000-fold dilution. Uncropped scans of the most important blots were provided as Supplementary Fig. ; IKKβ, CThe phospho-peptides, VLQRMFpSLQHDC (S321) and CVPLPPpSRpSNSEQPG (S332/334), were synthesized and coupled to KLH carrier protein via Cys at the C terminus. Polyclonal anti-p-S321 or p-S332/334 RIPK1 antibodies were produced in rabbits against p-peptide antigen by ProteinTech.24, are transfected individually into HEK293 cells with a packaging plasmid and adenovirus helper plasmid. The recombinant viruses were purified by standard CsCl gradient sedimentation method and desalted by dialysis33. The quality of vectors was tested by qPCR titration for DNase resistant vector genome concentration, silver-stained SDS-polyacrylamide gel analysis to establish the purity of each lot, electron microscopic analysis to independently test full/empty virus ratios33.rAAV vector plasmids carrying the vector genomes with WT, S321A or S321E RIPK1 gene expression cassettes under the control of human thyroxine binding globulin (TBG) promoter, which directs efficient and sustaining transgene expression in liver-specific pattern2 in air until transplantation. Thereafter, 20–25 injected embryos were transferred into oviducts of pseudopregnant ICR females at 0.5 d.p.c. The pups were identified by both enzyme digestion and DNA sequencing. The positive mice were mated with WT C57BL/6 mice to produce heterozygous S321A or E mutant progenies, which are then backcrossed with C57BL/6 mice.Super-ovulated female B6D2F1 mice (7–8 weeks old) were mated to B6D2F1 vasectomy males, and zygotes were collected from oviducts. Cas9 mRNA and sgRNA were prepared by in vitro transcription. Single-stranded oligonucleotides (ssODN) were synthesized by Sangon. 100 ng/μl Cas9 mRNA, 50 ng/μl sgRNA and 100 ng/μl ssODN were mixed in M2 medium (Sigma) and injected into the cytoplasm of zygote using a microinjector . The injected zygotes were cultured in KSOM with amino acids at 37 °C under 5% COTarget sequence:5′-TTTGACCTGCTCGGAGGTAA-3′S321A ssODN :GCATTGCAGCATGACTGTGTACCCTTGCCGccgagcaggtcaaattcaggtaactcacctattcgttcatttgcatactc-3′5′-cattacagaaagagtatccagatcaaagcccagtgctgcagagaatgtttS321E ssODN:GAATTGCAGCATGACTGTGTACCCTTGCCGccgagcaggtcaaattcaggtaactcacctattcgttcatttgcatactc-3′5′-cattacagaaagagtatccagatcaaagcccagtgctgcagagaatgtttThe founder mice carrying S321A or S321E mutant were backcrossed with C57BL/6 mice to produce heterozygous mice. WT and homozygous S321A(A/A) or WT and homozygous S321E(E/E) embryonic fibroblast cells were isolated from littermates of E13.5 embryos of heterozygous crosses. All experiments on mice were conducted according to the protocols approved by the Harvard Medical School Animal Care Committee and Institutional Animal Care and Use Committee of the Interdisciplinary Research Center on Biology and Chemistry of Chinese Academy of Sciences.F/F MEFs were immortalized spontaneously in culture and infected by virus for the expression of Cre recombinase to generate TAK1-deficient MEF. RIPK1 S321A and S321E MEFs were immortalized by viral infection and subsequent expression of SV-40 large T antigen.TAK1Cell lysates were collected and mixed with substrate for caspase-8  and the activity was determined according to the manufacturer’s instructions.35S]-methionine labeling using TNT T7 Quick coupled transcription/translation kit (Promega). In vitro cleavage assay was performed in cleavage buffer containing 20 mM HEPES (pH = 7.4), 100 mM NaCl, 20 mM dithiothreitol and 0.5% NP-40 at 37 °C for 1 h34.RIPK1 WT, S321A and S321E plasmids were transcribed and translated in vitro with ATP11. When indicated in the text, 1 µM 5Z-7 was added to the reaction mixture. The reactions were carried out at 30 °C for 60 min and stopped by boiling in SDS-PAGE loading buffer at 95 °C for 5 min. Similar protocol was used for RIPK1 S321A phosphorylation assay. To confirm anti-p-S321-RIPK1 antibody specificity, 293T cells transiently expressing FLAG-RIPK1 WT or S321A were treated with 5Z-7 (0.5 µM) and Nec-1s (10 µM) to minimize RIPK1 phosphorylation background. RIPK1 WT and S321A mutant were purified and incubate with recombinant TAK1 or IKKα in kinase buffer without [γ-32P]ATP at 30 °C for 30 min.Kinase-dead FLAG-RIPK1 variants with or without additional mutations or kinase-dead IKKβ were transiently expressed in 293T and immunoprecipitated with anti-FLAG M2-agarose beads. Protein bound FLAG beads were incubated with 100 ng recombinant TAK1 in kinase buffer containing 20 mM HEPES (pH = 7.3), 10 mM MgClt-test. Differences were considered statistically significant if P < 0.05 (*), P < 0.01 (**), P < 0.001 (***) or not significant (n.s.). At least three independent biological repeats were included in each data point. Each experiment was repeated at least three times.Data are expressed as the mean ± s.e.m. Error bar, s.e.m. Pairwise comparisons between two groups were performed using the Student’s The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information Files.Supplementary Information


Sik3-deficient mice (-/-Sik3), we observed that circadian rhythmicity of the metabolisms was phase-delayed. -/-Sik3 mice also exhibited other circadian abnormalities, including lengthening of the period, impaired entrainment to the light-dark cycle, phase variation in locomotor activities, and aberrant physiological rhythms. Ex vivo suprachiasmatic nucleus slices from -/-Sik3 mice exhibited destabilized and desynchronized molecular rhythms among individual neurons. In cultured cells, Sik3-knockdown resulted in abnormal bioluminescence rhythms. Expression levels of PER2, a clock protein, were elevated in Sik3-knockdown cells but down-regulated in Sik3-overexpressing cells, which could be attributed to a phosphorylation-dependent decrease in PER2 protein stability. This was further confirmed by PER2 accumulation in the -/-Sik3 fibroblasts and liver. Collectively, SIK3 plays key roles in circadian rhythms by facilitating phosphorylation-dependent PER2 destabilization, either directly or indirectly.Salt-inducible kinase 3 (SIK3) plays a crucial role in various aspects of metabolism. In the course of investigating metabolic defects in  Cry1 and Cry2 knockout (KO) mice, respectively, demonstrate shortened and lengthened periods of the locomotor activity rhythms in constant dark condition  exhibit shortened circadian periods, and the phase was advanced in behavioral and physiological rhythms . A growing body of evidence suggests that oscillation of the circadian clock is generated by the transcriptional-translational feedback loop composed of the circadian clock genes and their products, including the transcriptional activators CLOCK and brain/muscle ARNT-like protein 1 (BMAL1), and their negative regulators PERIODs (PER1/2/3) and CHRYPTOCHROMEs (CRY1/2). These fondition . Tau mut rhythms . These let al. reported that a point mutation in the clock gene Per2 results in familial advanced sleep phase syndrome (FASPS) in a human kindred (Sik3 knockout (KO) mice  mice . SIK1-3 KO) mice . PreviouKO) mice . Sik3-/-er birth . Furtherer birth . In the Sik3-/- mice, we observed that the average oxygen consumption rhythm, representing the circadian rhythmicity of metabolism, was significantly phase-delayed by approximately 6 hr compared with wild-type (WT) littermates  littermates. In 12 hr light-dark (LD) cycle conditions (LD 12:12), +/+Sik3 mice exhibited nocturnal activity profiles in which they were entrained to the LD cycle, with activity onset at light-off  and the activity offset at light-on (ZT0). In stark contrast, Sik3-/- mice failed to become completely entrained to the LD cycle , although they did still retain nocturnality (Sik3-/- mice have a defect in a light-input pathway. In constant dark (DD) conditions, the free-running period of the wheel-running activity rhythms of the Sik3-/- mice (24.17 ± 0.09 hr [n = 13]) was significantly longer than that of +/+Sik3 mice (23.80 ± 0.05 hr [n = 15]) . These data indicated that SIK3 plays a crucial role in controlling and sustaining robust circadian behavioral rhythmicity in addition to contributing to the photic entrainment. Notably, Sik3-/- mice were relatively less active at night than +/+Sik3 mice and more active during the day, which resulted in lower amplitude in the locomotor activity rhythms  . The abied in LD . Acropha rhythms . It shouSik3-/- mice were crossed with Per2::Luciferase knockin (KI) mice (LucPer2) expressing PER2-luciferase fusion protein in place of PER2, which monitors the circadian oscillation of PER2-luciferase fusion protein both in SCN explants and in cultured cells . Although the average periods of the individual SCN cells in the Sik3-/- and +/+Sik3 slices were comparable with one another, the variation (standard deviation) was significantly greater in the Sik3-/- cells than in the +/+Sik3 cells , and cellular rhythms were monitored via a Bmal1-luc reporter gene (Sik3-knockdown (Sik3-KD) cells were significantly shorter than those of WT controls  cells examined during 1 week of monitoring, with a characteristic of longer trough-to-peak period and shorter peak-to-trough period  in NIH3T3 cells resulted in a significant reduction in PER2 protein levels, but not PER1, compared with control cells , PER2 levels decreased rapidly in SIK3-OX cells compared with the control cells, indicating that SIK3 accelerates PER2 degradation  isolated either from +/+ mice . Expressoth high , left. Wous mice , right, SIK3-catalyzed phosphorylation of PER2 was examined via an in vitro kinase assay. The up-shifted band of PER2 was significantly increased after incubation of PER2 with SIK3, and the total amount of PER2 was reduced in a manner dependent on the amount of SIK3 added . PER2 prPer2 and Sik3 were incubated with the CSNK1 inhibitor IC261. IC261 did not affect PER2 degradation mediated by SIK3, suggesting that SIK3-dependent PER2 degradation is independent of CSNK1-mediated PER2 degradation  fluctuating circadian periods and phases both in vivo  im in vivo , (iii) r in vivo , and , as previously shown, and also because average blood glucose level in -/-Sik3 mice is significantly lower than that of controls, it may be a requirement for at least some of the KO mice to ‘keep seeking food’ even during a resting period, which may result in a change in rhythm strength. We believe that further studies, such as those investigating Sik3 conditional KO mice or transgenic rescue mice, would enable us to distinguish the dual effects on behavioral rhythms (i.e. effects from the circadian clock and metabolism).As shown in -/-Sik3 mice exhibited quite intriguing behaviors as mentioned above. It appears that, after transition from DD to LD, a part of the locomotor activities free-run even in the light phase (red dotted lines), but the rest of the activities were gradually entrained to LD cycles  is also a target of SIK3.It has previously been shown that constitutive expression of  in vivo . This ma mutants , may lea mammals , and Sikmalities . These pSik1-KD mice  was proposed as a target of SIK1, which contributes to light-induced phase reset  resulted in significant decrease in wake time caused by extended non-REM (NREM) sleep (Sleepy mutants in which a point mutation resulting in a lack of a protein kinase A (PKA) recognition site of the SIK3 protein, the authors also described that locomotor activity rhythms in the mutants were normal. Although the results were in contrast to our present data, the discrepancy could be explained by a difference in introduced mutations, that is, a partial deletion in the Sleepy mutants versus null mutation in our Sik3 KO mice.Strikingly, Funato M) sleep . In the Drosophila suggested effects of SIK3 homolog on coupling of different oscillator cells and with alternative period-lengthening and shortening in different cell types  was damped in Sik3-knockdown flies under constant darkness, while they did not propose a role for SIK3 in the phosphorylation and degradation of dPER. Instead, they proposed HDAC4 as a target of SIK3 since HDAC4 is one of well-established targets of SIK3 in fly. Thus, it is possible that SIK3 regulates the mammalian circadian clock by modulating activity of HDAC4 besides PER2 or that the effect of SIK3 on PER2 degradation is mediated by some kinases or degradation proteins, which are transcriptionally regulated by HDAC4.More recently, a study in ll types . Interesout mice . Fujii e-/-Sik3 mice (RRID: MGI:5317926) has been reported previously  were originally generated by Joseph Takahashi (2) of each group was monitored for two days using the Oxymax system , and the average was calculated every hour (n = 6). Food consumption and rectal temperature rhythms were measured manually and averaged every 6 hr (n = 12 each).All animals were cared for in accordance with Law No. 105 and Notification No. 6 of the Japanese Government, and experimental protocols involving the mice were approved by the relevant Ethics Committees at Kindai University and Nagoya University. Mice were maintained at 23 ± 1°C under LD 12:12 hr or constant darkness (DD conditions). The derivation of the eviously . Brieflyakahashi . All micakahashi . Analyseakahashi . BrieflySCR_014309).Mice were individually housed in translucent polypropylene cages under 12:12 hr LD (200 lux) and DD conditions, and locomotor activity was assessed by an area sensor . Activity was continuously monitored and analyzed using ClockLab software  by in vitro transcription using either T7 or SP6 polymerase . Frozen mouse brain sections (40 μm thick) were hybridized with riboprobe overnight, and exposed to Kodak film . The Sik3 cDNA (550 bp) was amplified via PCR and subcloned into a pGEM-T Easy Vector (Promega). The plasmids were linearized with NcoI to synthesize riboprobe.Riboprobe was labeled with CVCL_1926) transfected with Bmal1-luc in a 35 mm cell culture dish were used. NIH3T3 cell line was authorized as follows: identification of animal species was done by PCR and isozyme analysis and mouse strain was identified by SSLP analysis.For bioluminescence recording, NIH3T3 cells . Two hours after the DEX treatments, the culture medium was replaced with a recording medium, DMEM culture medium including 10 mM HEPES (pH 7.0), 10% fetal calf serum (FCS) and 0.1 mM luciferin, and circadian bioluminescence rhythm was monitored in Kronos . For single-cell time-lapse imaging, LV 200  was used for SCN slices. SCN slices from adult Sik3-/-Luc/Luc; Per2  and Sik3+/+Luc/Luc; Per2 mice were prepared using a Microslicer  and incubated on MilliCell membrane  in a 35-mm petri dish with culture medium . Exposure time for a single image was 55 min, and time-lapse images were captured for five consecutive days. Movies were produced using MetaMorph software .For SCN imaging, adult For each pixel of the bioluminescence movie, the time series was extracted. Each data-set was detrended and analyzed via the chi-squared periodogram . Only peCVCL_1926) and HEK293  were authorized by Riken Cell Bank and ATCC respectively as follows. NIH3T3 was authorized at Riken Cell Bank by PCN and isozyme analysis for identification of animal species, and by SSLP analysis for mouse strain. HEK293T/17 was authorized at ATCC by STR . These cell lines were cultured and passaged under 5% CO2 in DMEM  containing 1.8 mg/ml NaHCO3, 4.5 mg/ml glucose, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% FCS . Mycoplasma contamination was examined for both cell lines.NIH3T3  cDNA cloned into pcDNA 3.1. Mutations (Lys to Met at K37 or Thr to Glu at T163) were introduced into mSik3 using site-directed mutagenesis. Flag-Csnk1e (CKIε) and Flag-Csnk1d−ΚN (Kinase Negative) have been described previously (Sik3 gene and a control vector were purchased from QIAGEN (Germany). The following sequences were used: mSik3 sh#1 (5′- CTGCA GGCAC AAGTG GATGA A-3′), mSik3 sh#2 (5′- AGCAG CAACC CGAGA ACTGT T-3′), mSik3 sh#3 (5′- CCCAA CTTTG ACAGG TTAAT A-3′), mSik3 sh#4 (5’- TGCCA CCACG TTCAG TAGAA A-3′), and control sh (5′- GGAAT CTCAT TCGAT GCATA C-3′). The target sequences were inserted into a pGeneClip neomycin vector (Promega).NIH3T3 and HEK293 cells were transiently transfected using Lipofectamine Plus reagent  and Lipofectamine 2000 reagent (Invitrogen), respectively, in accordance with the manufacturer’s instructions. The pEGFP-C1 plasmid  was used as a transfection control. A vector encoding 6xMyc epitope-tagged PER2 (termed myc-PER2 in the figures) was kindly provided by Dr. Louis J. Ptacek . For Flag-SIK3, an oligonucleotide encoding the FLAG epitope sequence was fused to the 5′-end of full-length mouse eviously . Several2; pH 7.4]) for 1 hr at 37°C then incubated 1 hat 37°C or overnight at 4°C with a primary antibody in the blocking solution. Signals were visualized via an enhanced chemiluminescence detection system . The blot membrane was subjected to densitometric scanning, and the band intensities were quantified using Image Quant software . The primary antibodies used were anti-β-actin (Sigma), anti-Flag (Sigma), anti-myc , anti-GFP (Santa Cruz Biotechnology), and anti-PER2 (Millipore for Proteins separated by SDS-PAGE were transferred to polyvinylidene difluoride membrane (Millipore). The blots were blocked in a blocking solution . The lysate was incubated with the precipitating antibody for 2 hr at room temperature or overnight at 4°C, followed by incubation with 20 μl of Protein G-Sepharose beads  for 2 hr at 4°C. Beads were washed three times with IP buffer and subjected to immunoblotting.Transfected cells were lysed for 30 min in ice-chilled immunoprecipitation (IP) buffer  for the time periods specified in the figures NIH3T3 cells were transfected with myc-PER2 and Flag-SIK3 expression vectors or Sik3-KD vectors, and cultured for 48 hr. Immunoprecipitated myc-PER2 were incubated with 400 IU λPPase (Sigma) in Lambda Protein Phosphatase Buffer (Sigma) containing 2 mM MnCl2 for 30 min at 30°C in the presence of Protein G Sepharose. When samples were sequentially subjected to kinase assay, λPPase had been removed by washing PER2 bind to Protein G Sepharose in kinase assay buffer three times. Na3VO4 was used for a phosphatase inhibitor.NIH3T3 cells were transfected with myc-PER2 and Flag-SIK3 expression vectors or 2, 100 mM NaCl, 1 mM DTT, 10% glycerol, 1 mM ATP; pH 7.4) at 30°C for 30 min or 75 min. When samples were sequentially subjected to phosphatase assay, SIK3 had been removed by washing PER2 bind to Protein G Sepharose in phosphatase assay buffer three times.NIH3T3 cells were transfected with myc-PER2 vector and cultured for 48 hr. Immunoprecipitated myc-PER2 were incubated with GST-tagged recombinant human SIK3 (aa.1–307)  in kinase assay buffer  experiments, quantification of PER2 protein was independently done twice using triplicates, and two different cell lines (NIH3T3 and HEK 293) were used in the same series of experiments for confirmation. Phosphorylation of PER2 in SIK3-OX 3T3 cells were examined by two independent experiments, whereas PER2 degradation was done once (n = 3). Circadian rhythms in KD cells were analyzed twice, each of which shRNAs, #1, 3, and 4 were used.Number of animals used for experiments are all independent repeats. In KD experiments in  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.eLife. Your article has been favorably evaluated by a Senior Editor and three reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for submitting your article "Salt-inducible kinase 3 regulates the mammalian circadian clock by destabilizing PER2 protein" for consideration by The reviewers have discussed the reviews with one another and have raised some significant concerns that we feel must be addressed.Summary:The study by Hayasaka et al. reports a role for the salt-inducible kinase 3 (SIK3) in the circadian clock of mice. SIK3 KO mice display slightly longer periods in locomotor activity rhythms and less efficient entrainment to light dark cycles. The circadian periods of SCN neurons within a cultured slice, however, are not significantly different from wild-type, yet the phase coherence of individual neurons seems to be affected. In contrast to behavior, circadian periods of NIH3T3 fibroblasts with RNAi-mediated knockdown of SIK3 are shorter than in control cells. The authors then test the effect of SIK3 levels on the abundance and stability of PER2 and find that overexpressing SIK3 (but not a kinase-dead version) decreases ectopically expressed PER2 levels by destabilization, and knockdown of SIK3 increases ectopically expressed PER2 levels. In addition, the authors show that SIK3 overexpression leads to an increase in overexpressed PER2 phosphorylation, and the resulting destabilization is independent from CK1 phosphorylation and proteasomal degradation.Essential revisions:The study is potentially interesting, but all three of the reviewers were concerned about the central hypothesis that PER2 is the relevant substrate for the circadian effects of this kinase. The effects of RNAi knock-down or overexpression on PER2 levels in tissue culture are very strong, and yet the period lengthening in the SIK3 knock-out mouse are very small (and go in the opposite direction from those seen with RNAi in NIH 3T3 cells) and therefore inconsistent with the strong effects seen in tissue culture manipulations.1) Why did the authors investigate PER2 as a target for Sik3? Did they investigate any other circadian targets and fail to find effects on those? Could the direct target be something besides PER2 that produces a downstream effect on PER2?-/- cells rather than overexpression or RNAi studies. These studies could include immunoblot analysis of PER2 levels in the SIK3-/- knock-out mice or in primary fibroblasts from SIK3-/- mice to show that these levels are significantly higher than in wild type mice, with blunted oscillations and effects on the PER2 phosphorylation profile. The plots of overall per2-luciferase levels  could also be informative about differential effects on PER2. Primary fibroblasts derived from the SIK3-/- per2-luciferase mice (and SIK3+/+ per2 luciferase control fibroblast) could be assessed for short or long period oscillations, rather than the NIH 3T3 cells with RNAi kd, and an interaction of PER2 with SIK3 in normal circadian cells would also be indicative of PER2 relevance . Identification of a SIK3 phosphorylation site in PER2 and demonstration of a destabilizing effect of phosphorylation at these sites would be a seminal finding, although we do not insist that this finding be produced for this manuscript.2) There was a consensus that some additional findings are needed to strengthen the conclusion that PER2 is a relevant target, and that these findings should employ the SIK33) In any event, additional findings in circadian cells that do not involve overexpression or RNAi are needed, and if the additional findings do not show strong effects on PER2 the authors must discuss PER2 as a target of Sik3 in a speculative manner.eLife. Your revised article has been favorably evaluated by a Senior Editor and a Reviewing Editor.Thank you for resubmitting your work entitled "Salt-inducible kinase 3 regulates the mammalian circadian clock by destabilizing PER2 protein" for further consideration at The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:Drosophila clock has been published  114(32):E6669-E6677) since the initial submission of this manuscript. This manuscript is not cited or discussed in the revised version of this manuscript under review, but it needs to be, particularly since some of the findings  are also found in this revised manuscript.Hayasaka et al. have made a thorough effort to revise the manuscript according to the suggestions of the previous reviewers, and the paper is much improved. However, there are still a few points that need to be addressed. Most importantly, a paper describing the role of the SIK3 kinase in the Drosophila manuscript does not propose a role for SIK3 in the phosphorylation and degradation of PER. Instead, HDAC4 is proposed as a target. The Drosophila paper did not investigate thoroughly an effect on PER levels and phosphorylation, so it may have missed them (feel free to suggest this), or alternatively the Drosophila clock may work differently from the mammalian clock with regard to this kinase. Nevertheless, I think the authors of this revised submitted manuscript need to be cautious about claiming direct regulation of PER2 phosphorylation and stability by SIK3 kinase activity. Clearly, PER2 is less stable and more phosphorylated with catalytically active SIK3 overexpression and more stable and less phosphorylated with SIK3 knock-down or expression of the inactive kinase, but their attempts to identify a SIK3 target site in PER2 or a protein degradation mechanism that mediates this effect were not successful. The "in vitro" phosphorylation of immunoprecipitated PER2 by SIK3 kinase in Figure 5A by their own admission involves other factors besides PER2 and SIK3 , so it might also involve an intermediate kinase that responds to SIK3 addition to phosphorylate PER2. I think it is fair to say that the kinase activity of SIK3 leads to the phosphorylation and degradation of PER2, but not necessarily directly. It could be mediated by other kinases or other clock components, which do not appear to have been assessed extensively as targets for Sik3 (just PER1). If HDAC4 is a target for SIK3, its activity could modulate the expression of many other kinases and degradation proteins that contribute to the effect on PER2. I recommend modifying the last sentence of the Abstract to "Collectively, the results indicate that SIK3 plays key roles in circadian rhythms by facilitating phosphorylation-dependent destabilization of PER2, either directly or indirectly." Also, offer the alternative of an indirect regulation of PER2 phosphorylation and degradation status by SIK3 in the Results presentation of the Figure 5 data and in the Discussion, which now suggests only that PER2 is directly phosphorylated by SIK3.In contrast with this revised manuscript, the  Essential revisions:The study is potentially interesting, but all three of the reviewers were concerned about the central hypothesis that PER2 is the relevant substrate for the circadian effects of this kinase. The effects of RNAi knock-down or overexpression on PER2 levels in tissue culture are very strong, and yet the period lengthening in the SIK3 knock-out mouse are very small (and go in the opposite direction from those seen with RNAi in NIH 3T3 cells) and therefore inconsistent with the strong effects seen in tissue culture manipulations.1) Why did the authors investigate PER2 as a target for Sik3? Did they investigate any other circadian targets and fail to find effects on those? Could the direct target be something besides PER2 that produces a downstream effect on PER2?Thank you for these insightful comments and questions. We investigated other proteins, including some clock proteins, and initially found that PER2 protein levels were largely affected by SIK3. Therefore, we commenced the PER2 study and found that, in in vitro kinase assays, incubation of PER2 purified from cells and recombinant SIK3 enhanced PER2 phosphorylation, indicating that PER2 is a direct substrate of SIK3-catalyzed phosphorylation. However, we do not exclude the possibility that other clock/clock-related protein(s) is/are also a target of SIK3. In the revised manuscript, we add the following statement to the Discussion:“In addition, the present study showed that PER2 protein is a substrate of SIK3-mediated phosphorylation; however, this does not exclude the possibility that other clock/clock-related protein(s) is also a target of SIK3.”-/- cells rather than overexpression or RNAi studies. These studies could include immunoblot analysis of PER2 levels in the SIK3-/- knock-out mice or in primary fibroblasts from SIK3-/- mice to show that these levels are significantly higher than in wild type mice, with blunted oscillations and effects on the PER2 phosphorylation profile. The plots of overall per2-luciferase levels  could also be informative about differential effects on PER2. Primary fibroblasts derived from the SIK3-/- per2-luciferase mice (and SIK3+/+ per2 luciferase control fibroblast) could be assessed for short or long period oscillations, rather than the NIH 3T3 cells with RNAi kd, and an interaction of PER2 with SIK3 in normal circadian cells would also be indicative of PER2 relevance . Identification of a SIK3 phosphorylation site in PER2 and demonstration of a destabilizing effect of phosphorylation at these sites would be a seminal finding, although we do not insist that this finding be produced for this manuscript.2) There was a consensus that some additional findings are needed to strengthen the conclusion that PER2 is a relevant target, and that these findings should employ the SIK3-/-; PER2::LUC+/- and SIK3+/+; PER2::LUC+/- embryos. In western blot analysis, we found that endogenous PER2 protein levels were markedly elevated in SIK3-/- MEFs at all of the time-points studied. This result strengthened our conclusion that SIK3 promotes the degradation of PER2 protein. This finding is included in revised manuscript as the new Figure 4A.Thank you for these comments and constructive suggestions. As mentioned in the previous response, we established mouse embryonic fibroblast (MEF) cell lines from SIK3-/-; PER2::LUC+/- mouse was available for this experiment. Unfortunately, no SIK3+/+; PER2::LUC+/- littermates were produced; therefore, we used a Sik3+/-; PER2::LUC+/- littermate as a control in this experiment. Of note, we observed a dramatic increase in PER2 protein level in SIK3-KO mouse liver compared with control SIK3-heterozygous mice .In addition, we attempted to detect/determine PER2 protein levels in SIK3-KO mouse liver. As mentioned, many SIK3-KO mice died on the day of birth, and only one SIK3In response to the latter comment, we tried to identify PER2 phosphorylation site(s) responsible for control of its degradation rate. For this purpose, we prepared four single amino-acid (SA) mutants of the PER2 protein, in which each of four potential phosphorylation sites  by SIK3 was mutated to Alanine . These phosphorylation sites were identified by MS analysis in cell culture . Among all sites, S624 was identified by omics studies most frequently  and S659 is a well-known site regulating PER2 protein stability . We also considered the kinase consensus sequence of SIK3 (LXR[T/S]XpSXXX), especially regarding S971 and S981. Because the responsible kinases for all four sites are undetermined, we focused on these sites as candidates for SIK3-mediated phosphorylation.We compared the steady state protein levels of the mutants with wild-type (WT)-PER2 in the presence of co-expressed SIK3 in HEK293T17 cells. However, no significant stabilization of PER2 protein was detected in any of the single SA mutations of PER2 . Combina3) In any event, additional findings in circadian cells that do not involve overexpression or RNAi are needed, and if the additional findings do not show strong effects on PER2 the authors must discuss PER2 as a target of Sik3 in a speculative manner.-/- cells  and SIK3-/- liver , which strongly suggest that PER2 is the most promising SIK3 substrate candidate.Thank you, these are valid points. As mentioned above, we observed markedly elevated expression levels of PER2 in SIK3[…] I recommend modifying the last sentence of the Abstract to "Collectively, the results indicate that SIK3 plays key roles in circadian rhythms by facilitating phosphorylation-dependent destabilization of PER2, either directly or indirectly." Also, offer the alternative of an indirect regulation of PER2 phosphorylation and degradation status by SIK3 in the Results presentation of the Figure 5 data and in the Discussion, which now suggests only that PER2 is directly phosphorylated by SIK3.Thank you for these insightful comments. While we have tried to indicate the direct effect of SIK3 on PER2, we agree that our data is not strong enough to conclude that. According to your suggestion, we have modified our statements in the Abstract for the role of SIK3 as follow according to the reviewers’ suggestion:“Collectively, SIK3 plays key roles in circadian rhythms by facilitating phosphorylation-dependent destabilization of PER2, either directly or indirectly.”In addition, we cited the paper that reviewers had suggested  114(32): E6669-E6677) and added the following statement to the Discussion:Drosophila suggested effects of SIK3 homolog on coupling of different oscillator cells and with alternative period-lengthening and shortening in different cell types . […] Thus, it is possible that SIK3 regulates the mammalian circadian clock by modulating activity of HDAC4 besides PER2 or that the effect of SIK3 on PER2 degradation is mediated by some kinases or degradation proteins, which are transcriptionally regulated by HDAC4.”“More recently, a study in


Since Cdc25A degradation is tightly related to its phosphorylation status, we further examined whether CPX alters Cdc25A phosphorylation. The results showed that CPX treatment increased the phosphorylation of Cdc25A (S76 and S82), but only Cdc25A-S82A mutant was resistant to CPX-induced degradation. Furthermore, ectopic expression of Cdc25A-S82A partially conferred resistance to CPX inhibition of cell proliferation. Therefore, our findings indicate that CPX inhibits cell proliferation at least in part by promoting Cdc25A degradation.Ciclopirox olamine (CPX), an off-patent fungicide, has recently been identified as a novel anticancer agent. However, the molecular mechanism underlying its anticancer action remains to be elucidated. Here we show that CPX inhibits cell proliferation in part by downregulating the protein level of Cdc25A in tumor cells. Our studies revealed that CPX did not significantly reduce Cdc25A mRNA level or Cdc25A protein synthesis, but remarkably promoted Cdc25A protein degradation. This resulted in inhibition of G The immunocomplexes were subjected to SDS-PAGE, transferred to a PVDF membrane, and finally autoradiographed at −80°C. NIH Image J was used for semi-quantitative analysis of the intensities of the bands.eviously . BrieflyTo determine the effect of CPX on Cdc25A protein degradation, Rh30 or MDA-MB-231 cells, grown in the growth medium to 70% confluence, were treated with cycloheximide (CHX) , in the presence or absence of CPX (10 μM), for 0-24 h, followed by Western blotting with antibodies to Cdc25A and β-tubulin (loading control), respectively. NIH Image J was used for semi-quantitative analysis of the intensities of the bands.Western blotting was performed as described previously . NIH Imat-test for multiple comparisons. A level of P < 0.05 was considered to be statistically significant.Results were expressed as mean values ± standard error (mean ± SE). The data were analyzed by one-way analysis of variance (ANOVA) followed by post-hoc Dunnett's