ATP/ADP binding to Kir6.2 shuts KATP. Mg-nucleotide binding to SUR1 stimulates KATP. In the absence of Mg2+, SUR1 increases the apparent affinity for nucleotide inhibition at Kir6.2 by an unknown mechanism. We simultaneously measured channel currents and nucleotide binding to Kir6.2. Fits to combined data sets suggest that KATP closes with only one nucleotide molecule bound. A Kir6.2 mutation (C166S) that increases channel activity did not affect nucleotide binding, but greatly perturbed the ability of bound nucleotide to inhibit KATP. Mutations at position K205 in SUR1 affected both nucleotide affinity and the ability of bound nucleotide to inhibit KATP. This suggests a dual role for SUR1 in KATP inhibition, both in directly contributing to nucleotide binding and in stabilising the nucleotide-bound closed state.Pancreatic ATP-sensitive K Bindinactivity , whereasity , we replaced a tryptophan at position 311 (W311) of Kir6.2 that is 26 Å from the location of the inhibitory nucleotide-binding site with ANAP  and a plasmid encoding an ANAP-specific tRNA/tRNA synthetase pair (pANAP). We also included a dominant negative eukaryotic ribosomal release factor (eRF-E55D) in our transfections, which has been shown to increase the yield of full-length, ANAP-labelled protein , reducing the The apparent affinity of Kir6.2*-GFP + SUR1 for TNP-ATP in unroofed membranes was 25.6 µM . This valeotides .ATP, we turned to patch-clamp fluorometry -type model  which hahibition . In our D and KA . The brotermined .In addition to the full MWC-type model we considered alternate models . These iATP and decreases sensitivity of the channel to inhibition by nucleotides  which increases leotides . C166 isleotides . Other mleotides .In unroofed membranes, Kir6.2*,C166S-GFP + SUR1 bound TNP-ATP with an d to PCF . Rundowncomplete . How cancomplete  suggest cting KA , Table 3ATP and influenced our results  to the ABC core structure . This lohibition , and a rhibition . Other mhibition  and PHHIhibition .ATP inhibition, both in contributing to nucleotide binding and in stabilising the nucleotide-bound closed state.We introduced a charge neutralization  and a charge reversal  mutation at this position and measured simultaneous nucleotide binding and current inhibition with PCF . The binmutation . Howeverutations . This suATP and concomitant measurements of channel current. Performing these measurements simultaneously allowed us to examine nucleotide regulation of KATP function in great detail. We used a Bayesian approach to fit models to our combined fluorescence/current data sets to extract meaningful functional parameters with a minimum of prior assumptions. Such insights would not be possible from experiments in which macroscopic currents or binding were measured in isolation.We have developed a novel approach that allows for site-specific measurement of nucleotide binding to KATP. FRET also provided the spatial sensitivity necessary to discriminate between nucleotide binding directly to Kir6.2 and to the nucleotide-binding sites of SUR1. We assume that any TNP-ATP bound non-specifically to our membranes would be too far from Kir6.2 to cause appreciable FRET. This assumption was confirmed by the lack of FRET between TNP-ATP and a Kir6.2*-GFP mutant (G334D), in which nucleotide binding was severely disrupted . Despite obtaining similar parameters, past experiments in which only ionic currents were measured, did not allow us to distinguish between competing gating models. Measuring currents and fluorescence simultaneously allowed for better model selection and aided in our ability to identify constrained parameters.We performed all our experiments on mutated, tagged channels using a fluorescent derivative of ATP. This allowed us to fit mechanistic models and readily compare between mutated constructs that affect nucleotide inhibition of K2 + SUR1 . The valATP based on experiments in which currents and binding were measured in separate preparations . At saturating concentrations, TNP-ATP stabilied the closed state of KATP by −7.9 kcal mol-1 (31.8 kJ mol-1). Thus, assuming excitatory and inhibitory processes are independent, inhibition would be expected to dominate under conditions at which all the nucleotide binding sites are occupied. This is consistent with published measurements of wild-type KATP in the presence of Mg2+.We compared the parameters derived for inhibitory nucleotide binding to those estimated for nucleotide activation of Karations . In those of Mg2+. In our e of ATP . This phtrations . Future ATP to nucleotide inhibition, and reduction in nucleotide sensitivity is broadly correlated with disease severity  and purinergic P2X receptors.Human Kir6.2 and SUR1 were subcloned into pcDNA4/TO and pCGFP_EU vectors for expression of wild-type and GFP-tagged constructs, respectively. pcDNA4/TO and pANAP were obtained from Addgene. peRF1-E55D and pCGFP_EU were kind gifts from the Chin Laboratory  and the Gouaux Laboratory  respectively. Amber stop codons and point mutations were introduced using the QuikChange XL system . All constructs were confirmed by DNA sequencing .TAG), SUR1, pANAP and eRF1-E55D in the ratio 0.5:1.5:1:1. Transfected cells were cultured in Dulbecco’s Modified Eagle Medium  + 10% foetal bovine serum, 100 U ml-1 penicillin and 100 µg ml-1 streptomycin  supplemented with 20 μM ANAP . Cells were incubated at 33 °C and in the presence of 300 µM tolbutamide to enhance protein expression and channel trafficking to the plasma membrane . Our working stock tested negative for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit . Cells were plated onto either poly-L-lysine coated borosilicate glass coverslips  or poly-D-lysine coated glass-bottomed FluoroDishes . ANAP-tagged Kir6.2 constructs were labelled using amber stop codon suppression as described by membrane . eRF1-E5membrane . Experimane  was inserted in the extracellular region following helix M1 of Kir6.2 between L100 and A101. Transfected HEK-293T cells grown in 6-well plates were harvested in cold PBS , pelleted at 0.2 x g for 2.5 min and resuspended in lysis buffer containing 0.5% Triton X-100, 100 mM potassium acetate, and a cOmplete protease inhibitor tablet , buffered to pH 7.4. After a 30 min benzonase (Sigma) treatment at room temperature, samples were mixed with a DTT containing reducing agent and loading buffer  and run on a precast Bis-Tris 4–12% poly-acrylamide gel at 200 V for 40 min. Proteins were wet transferred overnight onto polyvinylidene difluoride (PVDF) membranes  in 25 mM Tris, 192 mM glycine, 20% methanol, and 0.1% SDS at 10 V on ice. Membranes were blocked with 5% milk in TBS-Tw  before staining for 30 min with a 1:1000 dilution of rat anti-HA monoclonal antibody in TBS-Tw . After washing with TBS-Tw, membranes were incubated for 30 min with a 1:20,000 dilution of HRP-conjugated goat anti-rat polyclonal antibodies in TBS-Tw . Detection was performed using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher) and a C-DiGit Blot Scanner . Analysis was performed using custom code written in Python.To confirm our ability to express full-length Kir6.2*-GFP, we performed western blots for HA-tagged Kir6.2 constructs in detergent-solubilized HEK-293T cells . The HA TAG-HA or Kir6.2-W311TAG-HA-GFP were co-transfected with SUR1, pANAP, and eRF1-E55D, and cells were cultured in the presence of ANAP, the western blots were similar to wild-type Kir6.2-HA or Kir6.2-HA-GFP. Over 90% full-length Kir6.2*-HA-GFP was produced under these conditions . We consistently observed a lower molecular weight band as well. This band must correspond to an N-terminally truncated Kir6.2 product, as the apparent molecular weight shifted with addition of the C-terminal GFP tag. Based on the molecular weight, we predict that the truncated protein product initiated from a start codon in the first transmembrane domain. Therefore, we believe it is unlikely that this protein would form functional channels or traffic to the plasma membrane. When Kir6.2-W311nditions . We werenditions . In the nditions .Confocal imaging was performed using a spinning-disk system  mounted on an IX81 microscope  with a Plan Apo 60x oil immersion objective (NA = 1.4), provided by the Micron Advanced Bioimaging Unit, Oxford. Transfected HEK-293T cells were incubated for 15 min with 1 nM CellMask Deep Red (Thermo Fisher) to stain plasma membranes before washing with PBS and imaging. ANAP was excited with a solid-state laser at 405 nm. GFP and CellMask were excited with an argon laser at 488 nm and 633 nm respectively. Images were captured on an EMCCD camera  binned at 2 × 2 pixels and analysed using Python. A median filter with a box size of 32 × 32 pixels was applied to improve the signal-to-noise ratio by reducing background fluorescence.TAG-GFP was co-transfected with SUR1 along with pANAP and eRF1-E55D in the presence of ANAP, the ANAP and GFP fluorescence were co-localized at the plasma membrane. When wild-type Kir6.2-GFP was transfected under the same conditions, only GFP fluorescence was observed at the plasma membrane. ANAP fluorescence was diffuse and confined to the cytoplasm or intracellular structures. Thus, the plasma-membrane ANAP signal was specific for Kir6.2*-GFP.We examined the surface expression of our ANAP-labelled constructs using confocal microscopy . When KiWe measured surface expression of HA-tagged Kir6.2 subunits using an approach outlined by TAG-HA or Kir6.2-W311TAG-HA-GFP, suggesting that prematurely truncated constructs did not traffic to the plasma membrane. In the absence of SUR1, surface expression was weak for both wild-type and tagged constructs, despite the reported ability of Kir6.2-GFP to traffic to the plasma membrane in the absence of SUR1 .Epifluorescence imaging and spectroscopy were performed using a Nikon Eclipse TE2000-U microscope with a 60x water immersion objective  or a 100x oil immersion objective . Imaging of ANAP was performed using a 385 nm LED source  with a 390/18 nm band-pass excitation filter, an MD416 dichroic and a 479/40 nm band-pass emission filter . GFP was imaged using a 490 nm LED source (ThorLabs) with a 480/40 nm band-pass excitation filter, a DM505 dichroic, and a 510 nm long-pass emission filter . Fluorescence spectra were collected by exciting ANAP as above but using a 400 nm long-pass emission filter (ThorLabs), then passing emitted light through an IsoPlane 160 Spectrometer  with a 300 grooves mm2+-free conditions. Currents were leak corrected using the current remaining in bath solution containing 5 mM barium acetate at 60 mV, assuming a linear leak with a reversal potential of 0 mV. Inhibition was calculated and corrected for rundown by alternating test concentrations of nucleotide solution with nucleotide-free solution, then expressing the test currents as a fraction of the average of the control currents before and after the test solution as described previously  to a resistance of 1.5 MΩ to 2.5 MΩ when filled with pipette solution. Currents were recorded at −60 mV from excised inside-out patches using an Axopatch 200B amplifier equipped with a Digidata 1322A digitizer and using pClamp 10 software . Currents were low-pass filtered at 5 kHz and digitized at 20 kHz. The bath solution (intracellular) contained 140 mM KCl, 10 mM HEPES, 1 mM EDTA and 1 mM EGTA (pH 7.3 with KOH). The pipette solution (extracellular) contained 140 mM KCl, 10 mM HEPES and 1 mM EDTA (pH 7.4 with KOH). All experiments were carried out in Mgeviously .ATP, we do not know exactly how much relative movement would occur between a bound TNP-ATP and Kir6.2-W311. However, based on cryo-EM structures of apo and nucleotide-bound Kir6.2 we do not expect to see a change in the distance between these two positions  before analysis with R . Where aHill fits to fluorescence quenching were nonlinear least-squares fits to the following equation:The MWC-type models considered  were forWhen no ligand is present  were prepared in 100 mM KOH and stored at −20° C. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:ATP channels important for insulin secretion from the pancreas and neonatal diabetes. KATP channels are heteromers of KIR6.2 pore-forming subunits and SUR1 modulatory subunits, and they contain 3 different classes of ATP binding sites, two excitatory sites on SUR1 and one inhibitory site on KIR6.2. This study uses a set of powerful methods including noncanonical amino acid incorporation, fluorescence resonance energy transfer (FRET), and patch-clamp fluorometry (PCF) to simultaneously measure the binding of a fluorescent derivative of ATP (TNP-ATP) specifically to the inhibitory site in KIR6.2, and the inhibition of the ionic current. Somewhat surprisingly, the authors found that the inhibition of the current occurs with an affinity about an order of magnitude higher than the binding affinity of ATP to the inhibitory site. This seemingly paradoxical result is explained using a gating model based on the allosteric mechanism of Monod-Wyman-Changeux (MWC) for protein allostery where the binding of just the first ATP almost completely inhibits the channel. They then go on to use their experimental and analysis methods to determine the effects of two different childhood diabetes mutations and the effect of the SUR1 subunit on gating.This paper describes experiments to investigate the mechanism of nucleotide inhibition of KDecision letter after peer review:+ channel explored with patch-clamp fluorometry" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Richard Aldrich as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Nucleotide inhibition of the pancreatic ATP-sensitive KThe reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:ATP channels important for insulin secretion from the pancreas and neonatal diabetes. KATP channels are heteromers of KIR6.2 pore forming subunits and SUR1 modulatory subunits and contain 3 different classes of ATP binding sites, two excitatory sites on SUR1 and one inhibitory site on KIR6.2. This study uses a set of powerful methods including noncanonical amino acid incorporation, fluorescence resonance energy transfer (FRET), and patch-clamp fluorometry (PCF) to simultaneously measure the binding of a fluorescent derivative of ATP (TNP-ATP) specifically to the inhibitory site in KIR6.2, and the inhibition of the ionic current. These authors found, somewhat surprisingly, that the inhibition of the current occurs with an affinity about an order of magnitude higher than the binding affinity of ATP to the inhibitory site. This seemingly paradoxical result is explained using a gating model based on the allosteric mechanism of Monod-Wyman-Changeux (MWC) for protein allostery where the binding of just the first ATP almost completely inhibits the channel. They then go on to use their experimental and analysis methods to determine the effects of two different childhood diabetes mutations and the effect of the SUR1 subunit on gating.This manuscript describes experiments to investigate the mechanism of nucleotide inhibition of KThe PCF data are beautiful and the paper is very well-written.Essential revisions:1) Please address the discrepancy between your conclusion that inhibition by TNP-ATP is weak and incomplete for the C166S mutation, and previous reports that (unmodified) ATP at 10 mM produces complete inhibition of this mutant . Your raw data in Figure 3D are compatible with strong inhibition at high concentration, but your model results are not . It seems important that you address this, ideally with some new data at higher [TNP-ATP], but at the very least by discussion and perhaps additional modeling to learn how more complete inhibition would affect the conclusions.Related to this, please explain the basis for selecting the prior distribution of the parameters for the modeling. Might it make sense to include information about the WT parameters in the prior distribution when fitting the mutant data?2) The kinetics work is the weakest part of the paper. There are no controls for the minimum time required to exchange the solution by perfusion, there is barely one time point to constrain the on rate at 100 µM, and the assignment of the rates are model dependent. We think that this section should be removed. Essential revisions:1) Please address the discrepancy between your conclusion that inhibition by TNP-ATP is weak and incomplete for the C166S mutation, and previous reports that (unmodified) ATP at 10 mM produces complete inhibition of this mutant . Your raw data in Figure 3D are compatible with strong inhibition at high concentration, but your model results are not . It seems important that you address this, ideally with some new data at higher [TNP-ATP], but at the very least by discussion and perhaps additional modeling to learn how more complete inhibition would affect the conclusions.ATP to nucleotide inhibition. Some report complete inhibition at high ATP concentrations  whereas others only report partial inhibition . The maximal block at high nucleotide concentrations appears to be background dependent.There have been numerous previous studies examining the effect of amino acid substitutions at Kir6.2-C166 on the sensitivity of KTo directly address this issue in our construct, Kir6.2*,C166S-GFP + SUR1, we applied 10 mM ATP to excised patches and observed only partial inhibition. We have added these data to Figure 3D. We did not measure inhibition by TNP-ATP at concentrations > 1 mM as our TNP-ATP was purchased as a triethylammonium salt (of indeterminate molar ratio of TNP-ATP:triethylammonium). We found that triethylammonium inhibits both Kir6.2*-GFP +SUR1 and Kir6.2*,C166S-GFP + SUR1 at millimolar concentrations. Thus, we would expect it to add to the total amount of current inhibition at high TNP-ATP concentrations. We have added a new supplement  to demonstrate this.4L*D the MWC-type model predicts a nucleotide-insensitive plateau of current at higher concentrations rather than a bona fide right shift in the inhibition curve relative to the binding curve. The height of the plateau at saturating nucleotide concentrations is proportional to the unliganded open probability of the channel, which may help to explain the diversity of results from the existing literature. We have included additional discussion in the text (paragraph four subsection “Kir6.2-C166S affects the ability of bound nucleotides to close KATP.”).We also performed additional modelling  to illustrate that at relatively high values for Related to this, please explain the basis for selecting the prior distribution of the parameters for the modeling. Might it make sense to include information about the WT parameters in the prior distribution when fitting the mutant data?L, which gave too much weight to unrealistic values for unliganded open probability (<1%). Thus, we believe we have chosen our priors well. We have included these new fits as Figure 4—figure supplement 4 and added additional discussion in the text (subsection “Bayesian model fitting”).We have now repeated our fits to the mutant data using two additional sets of priors. We used Gaussian fits to the posterior probabilities from the wild-type data as our first set. This resulted in parameter estimates that were very similar to wild-type, but the resulting curves fit the data poorly. We generated an additional set of priors using the centres of the Gaussian fits to the wild-type posterior probabilities, but with a 10-fold larger standard deviation . The resulting fits were similar to those generated from our relatively unbiased priors, with the exception of our estimates for 2) The kinetics work is the weakest part of the paper. There are no controls for the minimum time required to exchange the solution by perfusion, there is barely one time point to constrain the on rate at 100 uM, and the assignment of the rates are model dependent. We think that this section should be removed.We agree with your assessment and have removed this section from the paper and edited the Materials and methods accordingly (subsection “Epifluorescence imaging and spectroscopy.”). We moved the remaining panels from the original Figure 4 to Figure 4—figure supplement 1, and renumbered other figures accordingly.


Streptomyces mobaraensis by inhibition experiments. This is the first case of a butcher with an allergy to transglutaminase. After moving to a small enterprise without enzyme use, his symptoms improved. Sensitization and the course of the symptoms indicate a dominant role of transglutaminase in the patient’s allergic asthma. A 37-year-old butcher developed respiratory symptoms during sausage and chicken production in a large company. In addition to various spices, the enzyme transglutaminase was a possible cause. The lung function test showed mild partial reversible airway obstruction and severe bronchial hyperresponsiveness. The IgE test showed sensitizations to various spice mixtures, coriander (0.74 kU/L), and to the ImmunoCAP-bound transglutaminase preparation from the workplace (7.12 kU/L). The skin prick tests with this transglutaminase were also positive. In the immunoblot of this preparation, a 40-kD protein reacted with the patient’s IgE and was identified as transglutaminase from  In tStreptomyces mobaraensis catalyzes the cross-linking of proteins and thus leads to a more stable texture or joins pieces of meat. It has been increasingly used in the production of meat, fish, and dairy products since mid-2011. With this knowledge, a butcher who had developed respiratory problems while working in a large meat processing company was medically examined in Frankfurt in 2017. He had irritable cough and sneezing attacks in the morning during sausage production and the addition of spice mixtures with paprika and pepper. These symptoms became worse in the afternoon during chicken production, and he also suffered from shortness of breath and wheals on his skin. A possible trigger was the addition of very dusty binding agents which also contained the enzyme transglutaminase. The enzyme transglutaminase from The 37-year-old butcher suffered from pollen allergy and bronchial asthma since his youth. He had been a smoker for 20 years with 10 cigarettes a day. For 8 years he has additionally suffered from season-independent symptoms during his work in the meat factory. These could not be prevented even when wearing a breathing mask, possibly due to insufficient tightness. However, he was only given the breathing mask after the symptoms began, and he did not wear it permanently. He relieved his severe respiratory symptoms in the afternoon during the production of chicken nuggets by double dosing his asthma spray containing beclomethasone dipropionate and formeterol fumarate (200 µg/6 µg per stroke). 1 69% of the target value) was found, which was either not reversible, or could only be reduced by a maximum of 15% with β-mimetics. In the preliminary findings, there were indications of slightly elevated IgE antibodies to a spice mixture with laurel, paprika, green pepper, and mustard (test system unknown). The skin prick tests revealed sensitization to pollen from birch, alder, hazel, mugwort, and house dust mites. However, the total IgE value was within the normal range. In the documented lung function tests, a moderate obstruction  were analyzed. According to Modernist Pantry, Activa TI transglutaminase is an enzyme preparation without added helper protein for stabilization. Two samples of the transglutaminase preparation from the workplace (manufacturer and composition unknown), and a transglutaminase from All samples were first analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining , with 0.In addition, both transglutaminases (15 µg protein each) were transferred to PVDF membranes after SDS-PAGE, which were then cut into strips . One str1 at 74% of target) and a highly hyperreactive bronchial system. The patient’s lung function test in the body plethysmograph revealed a slight obstruction (FEVStreptomyces mobaraensis (7.48 kU/l). The spices mace (f266), nutmeg (f282), cardamom (f267), and bell pepper (f218) were negative in the ImmunoCAP IgE test. The total IgE value was 55.9 kU/l. The skin prick test with transglutaminase from the workplace was positive . In vitro sensitization was shown in the ImmunoCAP IgE test for coriander  as well as for the workplace transglutaminase bound to ImmunoCAPs (7.12 kU/l) and Activa transglutaminase from In the immunoblot, a 40-kD protein was identified as IgE-binding, which was present in the preparation from the workplace as well as in the Activa enzyme preparation. The patient reacted positively in both the nasal and bronchial provocation tests after workplace simulation with paprika powder. Provocation with transglutaminase was not performed for safety reasons, due to the reactivity of this enzyme. Streptomyces mobaraensis and no helper proteins. The sIgE test with this product was also clearly positive (slightly higher than with transglutaminase from the workplace). In the analysis by SDS-PAGE, the two transglutaminases differed only by some additional bands in the product used at the workplace. The IgE immunoblot then identified a 40-kDa protein in both products as the allergen. This fits well with the theoretical molecular weight of 44 kDa of the transglutaminase from Streptomyces mobaraensis [With pre-existing pollen allergy, the butcher presented in this case report developed respiratory problems at work throughout the year. His symptoms were particularly severe in the afternoon while making chicken nuggets. In this process, the enzyme transglutaminase acts as a binder, linking pieces of meat by cross-linking proteins. Both the skin prick test and the specific IgE test showed a clear sensitization to the enzyme product used at the workplace. However, the composition of the enzyme product was unknown. In principle, sensitization to components added for stabilization, such as wheat or milk proteins, would also be possible. Therefore, an enzyme preparation with a known composition was additionally tested. This contained transglutaminase from the bacterium araensis . The ideStreptomyces mobaraensis is used in the production of meat, fish, and dairy products. Two previous cases have been described in the literature: a case of respiratory allergy to microbial transglutaminase in a person working in company where ingredients for the food industry were mixed together [Streptomyces mobaraensis is therefore added to the list of industrially produced enzymes that are triggers of occupational respiratory allergies [The enzyme transglutaminase from together  and a catogether . This isllergies . The study was financed by the German Social Accident Insurance (project IPA-14). The authors state that there is no conflict of interest.


The SIE variability in PAS during the late spring and early summer  plays a key role in determining the SIE during the following fall when SIE is at a minimum. We find that the Pacific Decadal Oscillation (PDO), which is the most dominant variability of sea surface temperature (SST) on the low-frequency timescales, differently influences the SIE in PAS during AMJ before and after the mid-1990s. While a positive phase of PDO during the previous winter acts to increases SIE during AMJ before the mid-1990s, it acts to decrease SIE during AMJ after the mid-1990s. Further analysis indicates that atmospheric circulation associated with PDO differently influences the variability of SIE in the PAS during AMJ by modulating poleward moisture transport across the Alaska or the Far East Asia peninsula. This results in the change in the relationship of PDO and SIE in the PAS before and after the mid-1990s.Characteristics of sea ice extent (SIE) have been rapidly changing in the Pacific Arctic sector (PAS) in recent years As a direct result of the Arctic sea ice loss, voyage across the Arctic Ocean is now possible, which has reduced the time required to cross the ocean and enabled the exploitation of new natural resources. Although it is a controversial issue, the reduction of sea ice cover could alter atmospheric circulation, resulting in severe extreme cold events and significant snowfall in North America and Eurasia during the boreal winter8. Therefore, predicting the variability of SIE in fall when SIE is at a minimum is important for economic benefits, human health, and safety. The variability of SIE in fall could be influenced by many relevant factors including atmospheric moisture, cloud, atmospheric heat transport, oceanic heat transport and sea ice motion15.The record-breaking minimum year of the Arctic sea ice cover has been replaced every several years due to global warming16. That is, preconditions for changes in sea ice in the following fall and early winter are associated with a sea ice loss in previous spring17. While ice-albedo feedback is not yet active until early spring since solar radiation merely reaches the surface, anomalous Arctic cloudiness or moisture convergences are primarily responsible for melting sea ice in early spring via increased downward longwave radiation18. On the other hand, there is a study argued that sea ice motion in previous winter is essential for sea ice thickness variability, which is also an indicator of the SIE in the following fall14. Therefore, it is crucial to understand the physical processes leading to the SIE variability in spring to predict the SIE in fall. In this study, we focus on the SIE variability during the late spring and early summer  by analyzing the reanalysis datasets. In particular, we first highlight the role of Pacific Decadal Oscillation (PDO), which is the most dominant variability of sea surface temperature (SST) in the North Pacific, on the variability of the SIE in the PAS during AMJ. While the PDO is dominant on the low-frequency timescales, it is also characterized by a considerable variability less than decadal timescales   including Beaufort, Chukchi and East Siberian seas from year to year. However, it is well known that the SIE variability in late spring and early summer is a good indicator of SIE variability in September when SIE is at a minimumles Fig. . Such teles Fig. . Our ana19. The importance of sea ice variability in AMJ is highlighted by displaying the seasonal auto lead-lag correlation of SIE. We then studied physical processes associated with the atmospheric circulation related to PDO, which affect SIE variability during late spring and early summer (AMJ) using reanalysis data  from National Oceanic and Atmospheric Administration/National Snow and Ice Data Center (NOAA/NSIDC)ata see “” section21, which acts through the entire season. In particular, anomalous southerly wind also would alter the ice surface properties, thereby strengthening the ice-albedo feedback in the PAS21. In addition, previous studies also suggested the melt-to-growth sea ice reemergence mechanism, where spring sea ice anomalies are stored as upper ocean heat content anomalies and reemerge the following winter23. Simply put, Fig. Figure 24. We also found that the downwelling long wave radiation during JFM is negatively correlated with the SIE during AMJ in the PAS during 1958–2017, i.e., − 0.43, which is statistically significant at the 95% confidence level. An anomalous downward longwave radiation acts persistently to reduce sea ice thickness, resulting in a thinning of sea ice in the same season25  seasonal cycle of radiative fluxes and oceanic heat fluxes and their standard deviations show that the oceanic heat transport and its variability is quite small across the Bering Strait compared to the radiative fluxes including shortwave, longwave, sensible and latent heat fluxes during JFM Fig. . Therefo958–2017 We display the spatial manifestations of the positive phase of PDO during JFM, which are characterized by cool temperatures in the western and central North Pacific with an elliptical shape and are accompanied by anomalously warm temperatures to the east, north, and south, and the opposite is true in the negative PDO phase see Fig. a. Theref29  and after the mid-1990s , respectively. Figure 31, the mid-latitude North Pacific SST variability including PDO is able to modify the intensity and location of oceanic fronts, subsequently, it leads to modify the atmospheric circulation. We calculated the meridional SST gradient associated with the PDO, which is associated with the characteristics of baroclinic instability in the North Pacific, between the two periods  experiments see “” sectionIn this study, we focused on the variability of SIE in the PAS during AMJ due to its significant correlation with the following fall and winter. In particular, we examined the role of the PDO during JFM on the variability of SIE during AMJ. We found that the spatial pattern of PDO during JFM, which is associated with the structural changes in the meridional SST gradient as well as the zonal mean zonal wind in the North Pacific, differs significantly before and after the mid-1990s. This could affect the atmospheric circulation associated with the PDO across the mid-1990s, resulting in the changes in the relationship of PDO during JFM and SIE in the PAS during AMJ via modifying the poleward moisture transport in P1 and P2, respectively.32. We found that the characteristics of anomalous sea ice thickness with sea ice motion associated with the PDO change across the mid-1990s  for the entire analyzed period of 1958–2017 , the correlation coefficient between JFM PDO and OND SIE is 0.35 in P1 and − 0.35 in P2, respectively, which is statistically significant at the 90% confidence level. Therefore, the PDO index could be used to predict the SIE during OND in different periods, which is consistent with the results in the present study. Although the linear trend does not imply the radiative forcing, on the other hand, the overall results with and without a linear trend little change. Therefore, we speculate that the role of radiatively-forced trend on the impact of the PDO on the SIE variability is not significant.33, is obtained from the University of Washington (https://jisao.washington.edu/pdo/PDO.latest). In this study, we mainly use the PDO index averaged during JFM, when the PDO intensity is maximized34. Monthly SST is obtained from Hadley Centre SST (HadISST1) data set with resolution of 1° × 1°36. Monthly sea ice concentration (SIC) at a spatial resolution of 25 km is obtained from National Oceanic and Atmospheric Administration/National Snow and Ice Data Center (NOAA/NSIDC) for the period of 1979–201719. We also used gridded SIC with 0.25° × 0.25°  from37 prior to 1979 and reconstructed the SIC dataset having a spatial resolution of 25 km. Note that the results little change when we used the SIC from the ERA-Interim reanalysis dataset. The entire analyzed period is 1958–2017 and the analysis period starts in 1958 because of data credibility issue38. It is noteworthy that the results in the present study are little changed when the analyzed period is limited after 1979.The PDO index, which is defined as the principal component time series of the first empirical orthogonal function SST anomalies in the North Pacific poleward of 20° N39 available from 1979. The data is gridded to 25 km × 25 km. Monthly precipitable water content; sea level pressure; surface pressure; specific humidity, zonal and meridional winds at 8 pressure levels from 1000 to 300 hPa with horizontal resolution of 2.5° × 2.5° are obtained from Reanalysis 1 of the National Centers for Environmental Prediction-National Center for Atmospheric Research (NCEP-NCAR)40. We also analyzed heat fluxes including shortwave radiation, longwave radiation, latent heat flux and sensible heat flux at surface from NCEP-NCAR Reanalysis 1 with horizontal resolution of T62 (192 × 94). The total heat flux at surface is obtained from the summation of four heat fluxes components as follows,SW, QLW, QSH, and QLH indicate the net shortwave flux, net longwave flux, sensible heat flux, and latent heat flux, respectively. QSW, and QLW are composed of downwelling and upwelling components, respectively. From the viewpoint of the surface, sign of flux is positive when it downwells so that it warms the surface.Monthly sea ice thickness and ice motion vectors are obtained from Pan-Arctic Ice Ocean Modeling and Assimilation System (PIOMAS)41, the vertical integration is performed from surface to 300 hPa. The SIE is defined as the sum of the area of gridcell wherever SIC is at least 15% in PAS . When sea ice is completely covered, the SIE in the PAS is approximately 3.26 million km2, which is equivalent to 1 unit of SIE used in this study. The climatological period is defined as 1958–2017 to obtain the SIE anomaly.Convergence of vertically integrated moisture flux is calculated using specific humidity, zonal and meridional winds at 8 levels from surface pressure to 300 hPa. The vertically integrated moisture flux can be written as,42. Oceanic heat flux across the Bering Strait is calculated from the ORAS4 data using the following equation:-3), Cw is the specific heat capacity of water (3900 J kg-1 K-1).Ocean potential temperature and meridional currents with 1° × 1° horizontal resolution at 42 levels are from European Center for Medium-Range Forecasting’s Ocean Reanalysis System 4 (ORAS4)29. Based on this methodology, the periods before and after the mid-1990s (1958–1994 and 1995–2017) are considered separately to examine the change in the relationship between PDO during JFM and SIE variability in the PAS during AMJ. Note that the least squares linear trends of the analyzed variables are removed in each period.To determine the period when the relationship between PDO and SIE is different, we employed the regime shift analysis using Rodinov’s sequential t-test43 and HadISST1, respectively.Global Ocean Global Atmosphere (GOGA) simulation using the Community Earth System Model version 1 (CESM) is used for the analyzed period of 1958–2015. Total 10 ensemble members are integrated from the same initial condition with a small change in 2 m temperature. The historical SST and sea ice, which is used to force the CESM, is obtained from Extended Reconstructed Sea Surface Temperature version 4 (ERSSTv4)https://nsidc.org/data/seaice_index and the SST data is available from the UK Meteorological Office Hadley Centre at https://www.metoffice.gov.uk/hadobs/. The SIT and ice motion vectors are available from from the PIOMAS at https://psc.apl.uw.edu/research/projects/arctic-sea-ice-volume-anomaly/data/model_grid. NCEP-NCAR reanalysis data is available at https://www.esrl.noaa.gov/psd/. Finally, ORAS4 ocean potential temperature and meridional currents are available at https://www.ecmwf.int/en/research/climatereanalysis/ocean-reanalys. The GOGA simulation data is downloaded via CESM website.The data that support the findings of this study are openly available. The SIC data used in this study is from NOAA/NSIDC available at Supplementary Information 1.


The retinoblastoma tumor suppressor protein (RB) plays an important role in biological processes such as cell cycle control, DNA damage repair, epigenetic regulation, and genome stability. The canonical model of RB regulation is that cyclin-CDKs phosphorylate and render RB inactive in late G1/S, promoting entry into S phase. Recently, monophosphorylated RB species were described to have distinct cell-cycle-independent functions, suggesting that a phosphorylation code dictates diversity of RB function. However, a biologically relevant, functional role of RB phosphorylation at non-CDK sites has remained elusive. Here, we investigated S838/T841 dual phosphorylation, its upstream stimulus, and downstream functional output. We found that mimicking T-cell receptor activation in Jurkat leukemia cells induced sequential activation of downstream kinases including p38 MAPK and RB S838/T841 phosphorylation. This signaling pathway disrupts RB and condensin II interaction with chromatin. Using cells expressing a WT or S838A/T841A mutant RB fragment, we present evidence that deficiency for this phosphorylation event prevents condensin II release from chromatin. The retinoblastoma tumor suppressor protein (RB) is well characterized for its role in regulating entry into the cell cycle, and this is its canonical function. In G1, RB is bound to E2F transcription factors, repressing their ability to express cell cycle target genes . This leThis model, however, represents only one facet of RB function, and CDK-independent roles are emerging. These noncanonical functions are ascribed to a pool of chromatin-bound RB that localizes to various parts of the genome. For example, RB is required for recruiting DNA repair machinery for nonhomologous end joining (NHEJ) and homology-directed repair (HR) pathways at double-strand breaks (DSBs) , 8. In aMechanistically, posttranslational modifications regulate RB’s diverse functions . NotablyHere, we characterize RB phosphorylation on S838 and T841 (S838/T841) as a mechanistic link between T-cell receptor (TCR) activation and chromatin decondensation in Jurkat T-cells. Prompted by existing phospho-proteomic data, we generated an antibody specific to phosphorylation at S838/T841. We show that p38 MAPK is an upstream kinase that induces RB phosphorylation at these sites upon stimulus that mimics TCR activation in culture. Finally, we demonstrate that inability to phosphorylate these sites leads to defective condensin II release from chromatin.Multiple residues located throughout RB are phosphorylated, and the functional roles of CDK sites have been extensively characterized. Many of these modifications serve to inactivate its canonical function of negatively regulating cell cycle entry through E2F transcription factor binding. Interestingly, RB monophosphorylation of CDK sites has been shown to activate specific molecular functions of RB that may extend beyond cell cycle control . We sougA depicts the open reading frame of RB, its known CDK phosphorylation sites (top), and potential phospho-acceptor residues that are uncharacterized (bottom). We focused on RB C-terminus (RBC) as it forms an α-helix, and any phosphorylation within this region is likely to disrupt protein–protein interactions, leading to a novel functional outcome. To identify candidate phosphorylation sites for our investigation, we curated predicted phosphorylation events within tryptic fragments corresponding to RBC from available phospho-proteomic data in different cells or tissues upon various treatments (B)  , 28, 29.The phospho-proteomic data reveal that various combinations of serine and threonine residues within RBC are readily phosphorylated, supporting the idea that the RB phosphorylation “code” is yet incomplete. There is significant variability between the data sets on which exact sites are phosphorylated. This may indicate that complex, differential phosphorylation events occur in RBC depending on the cell type and stimulus, or that whole cell proteomics cannot accurately discern between neighboring phosphorylation events. While all of these potential modifications can be found in untreated cells, we focused on S838 and T841 phosphorylation because it is inducible upon TCR activation, a relevant signaling pathway in Jurkat leukemic T-cells.A). Next, we sought to find evidence for RB phosphorylation on S838/T841 in vitro as predicted by phospho-proteomic data. We incubated glutathione S-transferase-tagged RBC (GST-RBC) with whole cell extract (Ext) from Jurkat cells treated with phosphatase inhibitors pervanadate and calyculin A (PVA/CA) to globally activate kinases that may phosphorylate RB. We found a time-dependent increase in RB S838/T841 phosphorylation in the presence of ATP (B). To account for the possibility that our antibody cross-reacts with other phosphorylation events in RBC (amino acids 792–928), we repeated the experiment with GST-RBC where S838 and T841 were substituted to alanine either together or as single mutants. This reveals that this antibody requires either S838 or T841 phosphorylation, but not both (C). For this reason, we describe the antibodies and detected phosphorylation events throughout this paper as pS838/pT841 because phosphorylation of either site alone, or both in combination, is possible. With the specificity of our antibody established, we determined if endogenous RB is phosphorylated on S838/T841. We treated Jurkat cells with PVA/CA to activate kinases and RB phosphorylation, prepared nuclear extracts (NE), and immunoblotted with our antibody. RB phosphorylation on S838/T841 was increased upon PVA/CA treatment, but it was also evident that our antibody cross-reacted with approximately six or seven proteins other than RB (D). To ensure we were certain of RB’s identity, we repeated the cell treatment and immunoprecipitated total RB from Jurkat NE and performed an immunoblot with anti-RB pS838/pT841antibodies. Once again, we confirmed that RB phosphorylation on S838/T841 was induced upon PVA/CA treatment (E). In all subsequent experiments, we first immunoprecipitated RB, then probed with the antibody to assess S838/T841 phosphorylation.In order to study S838 and T841 phosphorylation on RB further, we sought to generate a specific antibody for these modifications. We generated rabbit antiserum against an RB-derived peptide corresponding to amino acids 834 to 845, phosphorylated on S838 and T841. An ELISA was performed to confirm the specificity of our antibodies following purification from the antiserum. We found that anti-RB pS838/pT841antibodies had a tenfold higher affinity for the S838/T841 phosphorylated RB peptide than its unphosphorylated counterpart A. Next, e of ATP B. To accnot both C. For th than RB D. To ensreatment E. In allbona fide RB posttranslational modification that has not yet been studied functionally.These data demonstrate that our antibody is highly specific for phosphorylation within RBC, namely on S838/T841. Although it cross-reacts with a small number of nuclear proteins, first enriching for RB by immunoprecipitation is a robust and specific means of detecting phosphorylation of these sites. More importantly, we have shown the first line of evidence that S838/T841 phosphorylation is a A). As a positive control for p38 inhibition, we also measured phosphorylation of a known p38 target, MAPKAPK-2, and found that phosphorylation of its activation loop at T334 was also reduced. Next, we determined if immunoprecipitated p38 can phosphorylate RB at S838/T841 in vitro. We first isolated p38 by immunoprecipitating the active form with anti-p38 pT180/pY182 phosphorylation-specific antibodies. It was then incubated with GST-RBC in the presence or absence of its inhibitor. p38 phosphorylation of RB increased over time, and its inhibitor largely abrogated this effect .As a first step to understanding the functional role for RB S838/T841 phosphorylation, we sought to identify the kinase that was responsible for this modification. p38 MAPK has been shown to phosphorylate RB even when CDK sites are mutated . TherefoFig. 3in vitro suggesting it may do so directly.These experiments strongly suggest that RB phosphorylation at S838/T841 is dependent on p38. PVA/CA treatment indiscriminately activates kinases, yet selective inhibition of p38 was sufficient to reduce RB phosphorylation to background level. Furthermore, partially purified, active p38 was necessary and sufficient to induce RB phosphorylation A). TCR activation reproducibly induced p38 activation within the first 30 min . At 30 min we immunoprecipitated RB and found strong S838/T841 phosphorylation . In addition, TCR activation did not induce additional phosphorylation of neighboring CDK phosphorylation sites, S807 and S811 consistent with these cells already being in a proliferative state. S838/T841 phosphorylated RB also retained its interaction with E2F1 and E2F2 as they were comparably coimmunoprecipitated relative to untreated cells. The shift in E2F2 migration upon TCR activation is consistent with previous proteomic data that report S117/S123 phosphorylation in Jurkat cells upon PVA/CA or TCR activation  signaling, and Jurkat cells are leukemic T-cells, we hypothesized that TCR activation phosphorylates RB through p38. We mimicked TCR activation by treating cells with an antibody cocktail that physically aggregates TCR and its costimulatory receptor together, as previously reported , 31. We Fig. 4tivation , 29.FiguThese experiments implicate TCR activation as a relevant mechanism that induces RB S838/T841 phosphorylation and suggests that RB S838/T841 phosphorylation may regulate its noncanonical functions. Our antibody cocktail treatment appears to closely mimic TCR activation as both ZAP70 and p38 were activated sequentially in the anticipated order. A slight temporal delay in peak phosphorylation of ZAP70, p38, and ultimately RB also suggests that this signaling cascade is biologically relevant to TCR activation. Furthermore, because S838/T841 phosphorylation in response to TCR signaling is unlinked to CDK phosphorylation, its function is unlikely to be related to cell cycle entry or control of E2F-dependent transcription.nessy mutant allele of CAPH2, one of the subunits of the complex, results in defective condensation of T-cell chromatin and development . However, TCR cross-linking for 30 min induced release of RB, E2F1, and CAPH2, but not SMC1 from chromatin . Using densitometric analysis of these experiments, we found that TCR cross-linking released 40% of CAPH2 from chromatin (B). To examine the precise role of S838/T841 phosphorylation in this process, we created a phospho-acceptor mutation in RB with a S838A/T841A double alanine substitution. To stably express this mutant in cells, we generated lentiviral vectors coding for HA-tagged large pocket fragment of RB . Jurkat cells were transduced with lentiviruses expressing either the WT RBLP (RBLP-WT) or S838A/T841A (RBLP-AA) construct (C). We confirmed equivalent expression of both constructs by immunoblotting for the HA-tag (D). We cross-linked TCRs as before and investigated CAPH2 and SMC1 chromatin levels (E). This revealed that RBLP-AA expressing cells retained similar levels of chromatin-bound CAPH2 as unstimulated cells and that RBLP-WT expressing cells released CAPH2 similarly to untransduced Jurkat cells (E). We normalized CAPH2 release to SMC1-bound chromatin levels to quantitate the inhibition of CAPH2 release by RBLP-AA (F). These experiments confirm that the inability to phosphorylate RB at S838/T841 dominantly blocks condensin II release from chromatin.In order to compare chromatin occupancy upon TCR cross-linking, we first sought to establish that this stimulus did not affect total expression of the proteins of interest. Indeed, we confirmed that expression of CAPH2, RB, E2F1, and SMC1, a cohesin subunit, was not changed in whole cell extracts A, left. hromatin A, right.hromatin B. To exaonstruct C. We cone HA-tag D. We cron levels E. This rat cells E. We nor RBLP-AA F. These A) . We repeated this TCR cross-linking experiment using cells that express RBLP-WT or the -AA mutant. This revealed that chromatin isolated from RBLP-AA expressing cells was sheared less effectively compared with that from RBLP-WT expressing cells (D–E). This suggests that the RB S838A/T841A mutant disrupts TCR cross-link-mediated chromatin decondensation and suggests that the phosphorylation of RB on these sites plays a key role in chromatin decondensation by releasing RB and condensin II from chromatin.Next, we sought to describe a biochemical consequence of defective CAPH2 unloading. To assess changes in chromatin structure upon TCR cross-linking, we used sensitivity to sonication-induced chromatin shearing as a readout for chromatin compaction as previously reported A 33). U. UA)  localize to histone clusters in mouse embryonic stem cells and serve to maintain topologically associated domains that regulate transcription . The peptide immunogen corresponded to the sequence 834-SIGE(pS)FG(pT)SEKF-845 of pRB. Peptides were immobilized using the Sulfo-link coupling reagent (Thermo Fisher) following the manufacturer’s recommendations. The phospho-specific antibody was purified using standard procedures by passing serum first over the nonphosphorylated peptide column and secondly over the phosphorylated-peptide column to remove any antibodies recognizing the nonphosphorylated species. Anti-RB pS838/pT841 antibody was used in primary antibody solution at 0.5 mg/ml for Western blotting. Anti-MAPKAPK-2 pT334 (27B7), p38 pT180/pY182 (9211), total p38 (9212), α-tubulin (11H10), and RB pS807/pS811 (9308) antibodies were obtained from Cell Signaling Technology. Anti-HA antibody (3F10) was from Sigma. Anti-pRB monoclonal antibody (G3-245) was from BD Pharmingen, pRB rabbit polyclonal antibodies and E2F1 antibody (C-20) were from Santa Cruz, CAP-H2 antibody (A302-275A) was from Bethyl Laboratories. Anti-rabbit or anti-mouse IgG goat antibody conjugated to HRP was from GE Healthcare. For TCR activation studies, anti-CD3ε (OKT3) and CD28 (CD28.2) antibodies were from BioLegend, and light chain-specific goat anti-mouse IgG (115-005-174) was from Jackson ImmunoResearch Labs.2. For SB203580 inhibitor pretreatment, Jurkat cells (107) were suspended in 5 ml enriched DMEM and SB203580  was added from 10 mM stock to a final concentration of 10 μM. Equal volume of DMSO was added to control samples. The cells were then incubated at 37 °C for 2 h until treatment with pervanadate and calyculin (PVA/CA). Cells were centrifuged at 300g for 5 min at 4 °C and washed twice with PBS. After the last wash, the pellet was resuspended in 5 ml enriched DMEM, and PVA/CA was added to a final concentration of 100 and 0.1 μM, respectively. Control samples received equal volumes of PBS and DMSO vehicles, respectively. Both groups were incubated at 37 °C for 20 min. TCR activation was achieved as previously described with modifications (6) were resuspended in 1 ml enriched media and treated with 5 μg/ml anti-CD3ε and CD28 antibodies and 30 μg/ml goat anti-mouse IgG to cross-link the primary antibodies. The cells were then incubated for indicated times at 37 °C, after which they were harvested.Suspension Jurkat cells were grown in DMEM enriched with 10% v/v FBS, L-glutamine, penicillin, and streptomycin at 37 °C in 5% COications . Jurkat 3VO4, 5 mM NaF, 1 mM PMSF, 5 μg/L aprotinin, and leupeptin immediately before use. For whole cell extract (Ext), cells were washed twice with cold PBS and resuspended in lysis buffer containing 25 mM Tris-Cl pH 7.5, 100 mM NaCl, 5% (v/v) glycerol, and 1% (v/v) NP-40 and incubated on ice for 10 min. Insoluble material was cleared by centrifugation at 12,000g at 4 °C for 10 min. For NE, cells were washed twice with cold PBS, resuspended in hypotonic lysis buffer containing 10 mM Tris-Cl pH 7.5, 10 mM KCl, 3 mM MgCl2, 1 mM EDTA, and 0.05% (v/v) NP-40, and incubated on ice for 5 min. Nuclei were isolated by centrifuging the suspension at 1300g at 4 °C for 5 min. The pelleted nuclei were washed twice in the same buffer. The nuclei were lysed in buffer containing 20 mM Tris-Cl pH 7.5, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% (v/v) glycerol, 0.1% (v/v) NP-40 and incubated on ice for 10 min. Insoluble material was cleared by centrifugation at 12,000g at 4 °C for 10 min. For chromatin fractionation, PBS washed cells were incubated for 10 min on ice in buffer containing 10 mM Tris-Cl pH 8.0, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% (v/v) glycerol, and 0.1% (v/v) Triton X-100 to lyse outer membrane. Nuclei were isolated by centrifuging the suspension at 1300g at 4 °C for 5 min. The pelleted nuclei were washed twice in the same buffer without Triton X-100. The nuclei were lysed in buffer containing 3 mM EDTA and 0.2 mM EGTA for 30 min on ice with occasional mixing. Nucleoplasmic fraction was removed by centrifuging the lysate at 4000g at 4 °C for 5 min. Remaining chromatin fraction was solubilized with DNaseI treatment at 37 °C for 10 min in buffer containing 10 mM Tris-Cl, 2.5 mM MgCl2, 0.5 mM CaCl2. Protein concentration was quantified by Bradford assay following standard procedures.All of the following lysis buffers were supplemented with 250 μM NaAntibodies to RB or pp38 T180/Y182 were diluted 1:50 in Ext containing 1 to 2 mg total protein. The lysates were incubated at 4 °C overnight with gentle agitation. In total, 50 μl Dynabeads Protein G (Thermo Fisher) was added and incubated at 4 °C for 2 h with gentle agitation. A magnet was used to immobilize the beads and to discard the supernatant.2, 0.25 mM Na3VO4, and 5 mM NaF. Each of the time points consisted of a 40 μl reaction containing 5 μl of immunoprecipitated kinase or WCE (where indicated), 10 μg of GST-RBC, 1 mM ATP, and 50 μM SB203580 (where indicated) in kinase buffer. GST fusion proteins were expressed in E. coli BL21 (DE3) and purified with Glutathione Sepharose 4B (Sigma) following the manufacturer's recommendation. The reactions were prepared on ice, then transferred to 37 °C to start the assay. After the given incubation times, the immunoprecipitated proteins were immobilized with a magnet, and the remaining supernatant was saved.Immunoprecipitated pp38 T180/Y182-Dynabeads complexes were used in kinase assays similar to previous reports. The complexes were resuspended in 50 μl kinase buffer containing 25 mM Tris-Cl pH 7.5, 5 mM β-glycerophosphate, 10 mM MgClProtein extracts, supernatants, and immunoprecipitated beads were diluted in SDS-PAGE sample buffer to a final concentration of 62.5 mM Tris-Cl pH 6.8, 10% glycerol, 2% SDS, 72.5 mM β-mercaptoethanol, and 0.005% bromophenol blue. SDS-PAGE was performed following standard procedures. After gel electrophoresis, proteins were transferred to PVDF membrane and incubated with primary antibodies diluted in 5% milk or BSA in TBS-T over night at 4 °C with gentle agitation. Membranes were washed five times in tris buffered saline-Tween 20 (TBS-T) for 5 min and incubated with horseradish peroxidase (HRP)-conjugated goat secondary antibody for 1 h at room temperature. Membranes were washed again, incubated in SuperSignal WestDura (Thermo Fisher) for 5 min protected from light, and developed on a ChemiDoc (Bio-rad). For Coomassie staining, gels were washed three times in water and incubated in GelCode Blue (Thermo Fisher) over night at room temperature with gentle agitation. Stained gels were washed again twice in water. Bands were imaged on a ChemiDoc.6) were transduced with the filtrate containing 8 μg/ml polybrene. Transduced Jurkat cells were maintained in 0.3 μg/ml puromycin.Briefly, pSicoR-Ef1a-mCh-Puro (Addgene #31845) was double-digested with AfeI and SmaI to replace mCherry coding sequence with that of RBLP with matching sticky ends in-frame. S838A/T841A substituted plasmid was made with QuikChange Site-directed mutagenesis kit (Agilent), following manufacturer’s recommendations and mutagenic primers TTAGTATCAATTGGTGAAGCATTCGGGGCTTCTGAGAAGTTCCAGAAA and TTTCTGGAACTTCTCAGAAGCCCCGAATGCTTCACCAATTGATACTAA. HEK293 T cells at 70% confluency on 10-cm plates were transfected with 12 μg expression vector, 9 μg pMD2.G (Addgene #12259), and 3 μg psPAX2 (Addgene #12260) using Lipofectamine 3000 (Life Technologies) following manufacturer’s recommendations. Two days later, the cell media was harvested and passed through a 0.45 μm filter. Jurkat cells . All of the above lysis buffers were supplemented with 250 μM Na3VO4, 5 mM NaF, 1 mM PMSF, 5 μg/L aprotinin, and leupeptin immediately before use. Chromatin was sonicated in Bioruptor Pico (Diagenode) at 4 °C with one to three cycles. Each cycle consisted of 15 s ON and 30 s OFF. After sonication, chromatin–protein complexes were reverse-cross-linked by adding NaCl to a final concentration of 200 mM to each sample and incubating overnight at 65 °C. RNA and protein were removed by RNase A and proteinase K digestion, respectively. DNA was isolated by phenol-chloroform extraction and ethanol precipitation and analyzed on 3% agarose gels. DNA was stained with ethidium bromide and visualized on a Chemi Doc. High-molecular-weight DNA bands were quantified as a percentage of the total lane intensity with lower threshold cutoff at 100 bp using Image Lab 5.2.Jurkat cells were washed twice in PBS and fixed in 1% formaldehyde/PBS for 10 min at room temperature with gentle agitation. Fixation was quenched with 0.125 M glycine for 5 min at room temperature. Cells were washed twice in cold PBS and incubated for 10 min on ice in buffer containing 10 mM HEPES pH 6.5, 10 mM EDTA, 0.5 mM EGTA, and 0.25% Triton X-100. Nuclei were isolated by centrifuging the suspension at 600t-test. That of HA-RBLP WT or AA expressing cells was compared using two-way ANOVA and Sidak’s multiple comparisons test. For sonicated chromatin densitometry, the mean of triplicate values was compared between treatment conditions, or cell lines, as indicated by two-way ANOVA. Sidak’s multiple comparison test was used.All statistical analyses were performed on Prism 8. CAPH2 chromatin loading relative to SMC1 in untransduced cells was compared using Student’s All data are contained within the manuscript.The authors declare that they have no conflicts of interest with the contents of this article.


The Dawn mission found that the dominant colour variation on the surface of dwarf planet Ceres is a change of the visible spectral slope, where fresh impact craters are surrounded by blue  ejecta. The origin of this colour variation is still a mystery. Here we investigate a scenario in which an impact mixes the phyllosilicates present on the surface of Ceres with the water ice just below. In our experiment, Ceres analogue material is suspended in liquid water to create intimately mixed ice particles, which are sublimated under conditions approximating those on Ceres. The sublimation residue has a highly porous, foam-like structure made of phyllosilicates that scattered light in similar blue fashion as the Ceres surface. Our experiment provides a mechanism for the blue colour of fresh craters that can naturally emerge from the Ceres environment. The origin of blue ejecta around the fresh craters of dwarf planet Ceres is unknown. Here, the authors show that the blue color results from high porosity of the surface, induced by sublimation of ice-phyllosilicate mixture produced by impacts. Material that we refer to as blue is characterized by a negative spectral slope, both in absolute and relative sense. The average spectrum of Ceres is almost flat and featureless from the visible up to 2.7 μm, where a complex of bands beyond 2.7 μm indicates the presence of ammoniated phyllosilicates5, likely of the smectite group6. The widespread distribution of these aqueous alteration products points to a global distribution of liquid water in the interior of Ceres in the past7. Presently, water ice appears to be abundant just below the surface10, and the crust may hold up to 25% of water ice by volume11. Blue material on Ceres is uniquely associated with fresh features, predominantly young impact craters13. Some of the strongest blue signatures are associated with Haulani, a 34 km diameter crater located at 10. 8°E, 5. 8°N, which can be clearly recognized at the centre of the global map in Fig. 4). Haulani is one of the youngest craters on Ceres, with an estimated age of 2 Ma14, and both its interior and ejecta are blue. Figure 15. The plot shows the reflectance spectra at two locations, one in the blue interior and the other outside the crater at a location about two crater radii distant, where bluing is not evident4. The ratio spectrum reveals that the blue nature of Haulani is characterized by the introduction of a negative spectral slope over the visible and near-IR range, up to 3 μm. Beyond 3 μm the spectrum is increasingly dominated by thermal emission. The blue trend in the ratio spectrum in the thermal IR reflects the fact that Haulani is colder than its surroundings16.When Dawn arrived at dwarf planet 1) Ceres, it quickly became clear that the dominant colour variation over the surface is a variation of the spectral slope from the visible to the near-infrared (near-IR) Ceres, i17, but water ice is not stable on the surface of Ceres18, apart from permanently shadowed regions on the poles19. Schröder et al.4 proposed an explanation on the basis of an experiment by Poch et al.20, in which sublimation of a mixture of montmorillonite and water ice resulted in an extremely porous sublimation residue that was bluer than the original material. When smectite phyllosilicates like montmorillonite are mixed with liquid water, hydration of the inter-layer space will lead to partial delamination, resulting in suspended nano-sized platelets and aggregates thereof. Freezing the suspension leads to the growth of water ice crystals, with platelets concentrating at the crystal interfaces21. Subsequent sublimation replaces the ice crystals with voids, and only the phyllosilicate platelets remain to form a porous network, or foam. Poch et al. tentatively attributed the bluing in their sublimation residue to Rayleigh scattering by sub-μm-sized scattering centres in the foam.The origin of the blue colour is not yet understood. On comet 67P/Churyumov-Gerasimenko, a bluing of the spectral slope is interpreted as meaning enriched in water ice4. Stephan et al.13 instead argued that it is the impacts themselves that pulverize the phyllosilicates into dust, out of which aggregates spontaneously form to scatter light in Rayleigh fashion. The latter model is based on grinding experiments with antigorite by Bishop et al.22. In both explanations, the bluing is attributed to Rayleigh scattering by fine phyllosilicate structures, but they differ in how these structures are formed. Bluing due to the presence of salts, which have been identified on the surface of Ceres23, is problematic because it would be accompanied by a strong increase in brightness. Rayleigh scattering in a particulate surface was first proposed by Clark et al.24 to explain the blue reflectance spectrum of the icy surface of Dione. Models incorporating Rayleigh scattering have since been developed to interpret observations of bluing on other planetary surfaces26. The question remains how structures of ultra-fine phyllosilicate particles come into being. It is the goal of this paper to demonstrate that such structures can form naturally and in large quantities under Ceres conditions.On Ceres, impacts would mix the phyllosilicates on the surface with water ice below to create ejecta of icy mud, which sublimation residue could be similarly porous and blue4. We create ice particles with a compositional analogue of Ceres surface material and sublimated the sample under pressure and temperature conditions representative for Ceres. After completion, we determine the physical state and mechanical properties of the sublimation residue. Reflectance spectra of the sample are acquired throughout the experiment.Here, we show experiments in support of the hypothesis that the blue colour results from a high porosity of the surface, induced by sublimation of an ice–phyllosilicate mixture produced by impactsAt the start of the sublimation experiment, the icy sample of the Ceres analogue material appeared pale grey to the eye Fig. a. In theFigure 21.The sublimation residue had greatly increased in volume compared to the original material (estimated by a factor 5–10), and was lightweight with a foam-like texture Fig.  and 13. Micrographs in Fig. 26. For Rayleigh scattering to occur, the scattering centres need to be smaller than the wavelength (≪1 μm) and scatter independently, i.e., be separated by a distance larger than the wavelength25. The filaments in Fig. 27. The bluing is less strong for the Ceres analogue residue than the nontronite residue, which is probably related to the presence of the other mineral particles. Beyond 3 μm, the spectral shape is similar for both materials, with the residue being 10% darker, perhaps because of the presence of pits on the surface. The 2.7 μm absorption band is pronounced in the ratio spectrum, indicating that it is deeper for the residue. However, the residue spectrum is affected by absorption bands associated with water vapour present in the optical path . The residue in our experiment was extremely porous, as experimental constraints limited the concentration of phyllosilicate in the ice to 1% by weight. On Ceres, the residue may be of higher density and thereby less porous, but still offer increased (vacuum) insulation through its low thermal conductivity21. Depending on the thickness of the porous layer, water ice could be stable at lower depths on Ceres than previously assumed.On Ceres, any sufficiently large impact would intimately mix the phyllosilicates present on the surface25. A porous network is required, because a surface made of sub-μm-sized particles in close contact would merely reflect like a slab25. Apart from their blue colour and higher reflectance, there is some evidence that the ejecta of fresh craters are in a different physical state than their surroundings. The blue ejecta of Azacca crater possess an unusually narrow opposition effect32. If it is due to shadow hiding, expected for a material this dark, Hapke’s theory predicts a high porosity33. A similarly narrow opposition peak for the moon Europa was interpreted in terms of a very high porosity, assuming shadow hiding34.Our experiment suggests that sub-μm-sized filaments of phyllosilicate composition inside the residue scatter light in a similarly blue fashion as observed on Ceres, with the foam-like structure providing the necessary distance between the scattering filaments for Rayleigh scattering to occur12. Our experimental results suggest that the reflected light mostly originates in the upper 1 mm of the residue surface. How would space weathering affect a top layer of porous phyllosilicate? The Ceres analogue residue was flexible and had an internal cohesion that made it resilient21. The mechanical properties of the much thicker nontronite residue layer were easier to study, and we found it to be cohesive, sticky, and spongy . We produced and sublimated ice particles by following the protocol of Poch et al.20. The experimental procedure is outlined in Fig. The experiment was performed in April 2019 at the Institut de Planétologie et d’Astrophysique de Grenoble in Grenoble, France, using the Cold Surface Spectroscopy (CSS) facility  or high vacuum (~1.5 × 10−6 mbar). It has a removable lid on top with a transparent, sapphire window, through which the reflectance of the sample was measured by the spectro-gonio radiometer SHINE (SpectropHotometer with variable INcidence and Emergence)43. The instrument operated in the special Gognito mode, optimized for dark targets, in which the sample illumination and observation areas are disks with a diameter of 7.1 and 20 mm, respectively44. The monochromatic light used for illuminating the sample is modulated and the detection synchronous. Thereby, SHINE measures light reflected by the sample only, and is neither sensitive to scattered light from external sources nor thermal emission. The accuracy of its absolute radiometric calibration is better than 0.1% over the 0.4–2.5 μm range. The spectra were recorded over the 0.4–4.2 μm range with a fixed sampling of 20 nm and a spectral resolution of 3.2 nm below 0.68 μm, 6.3 nm below 1.4 μm, 13 nm below 3.0 μm, and 26 nm above 3.0 μm. For each wavelength interval, the intensity was recorded by a 300 ms integration repeated 10 times. The calibrated reflectance factor was derived from the average of these 10 values, and their standard deviation  was adopted as the associated uncertainty. The time needed to acquire a single spectrum was about 50 min, during which time the spectrometer progressed along the wavelengths. Prior measurements of reference surfaces allowed calibration of the measurements to reflectance factor  = , with incidence angle ι, emission angle ϵ, and phase angle α. For this geometry, the reflectance factor (REFF) is identical to the radiance factor (I/F). The presence of the sapphire window over the sample induces secondary reflections between sample and window, which reduces the measured reflectance and contrast of absorption features. The calibration includes a correction for this effect45. We verified the accuracy of the SHINE spectra of the original sample and sublimation residue by repeating the measurements with SHADOWS , which is a similar spectro-gonio radiometer optimized for dark surfaces44.The experimental setup with spectro-gonio radiometer and cryogenic chamber is shown in Fig. 3O4), 35 vol% (22 wt%) of NH4-bearing nontronite, 13 vol% (9 wt%) of antigorite 3Si2O5(OH)4), and 8 vol% (6 wt%) of dolomite (CaMg(CO3)2). The NH4-bearing nontronite was produced according to Ferrari et al.46, starting from the NH4-free nontronite (NAu-1)47. SEM images of both samples are provided in Supplementary Figs. 3, with a reflectance of around 0.07 at the standard geometry  at the Istituto di Astrofisica e Planetologia Spaziali-INAF in Rome, Italy. This analogue material was a mixture composed of 44 vol% (63 wt%) of magnetite  was weighed at 0.468 g, meaning that only ~20 wt% of the analogue material had been incorporated in the ice particles. The similarity of this number to the abundance of nontronite in the original sample (22 wt%) suggests that only nontronite was incorporated into the ice. We mixed the residue with the ice particles in an aluminium bottle, pre-cooled with liquid nitrogen, using a vortex shaker. In all, 0.178 g of residue was combined with 22.31 g of ice particles to meet our original goal of a 1 wt% solid-to-ice mixture. Thus, the final analogue ice sample was a combination of an intra- and inter-mixture, having the same mineral weight proportions as the original analogue material. Inside the freezer, we sieved the ice mixture (mesh size 400 μm) to fill the 2-cm-deep sample holder to the rim, smoothing the surface with a spatula. The holder and spatula had been pre-cooled with liquid nitrogen. A spectrum of the empty sample holder had been acquired at 173 K and high vacuum to verify that the sample was optically thick over the course of the experiment. The full sample holder was quickly transferred from the freezer to the CarboNIR cryogenic chamber inside a polystyrene box cooled with liquid nitrogen.We aimed to make an icy intra-mixture, in which all components of the analogue material are present as inclusions in the water ice grains 18. During the experiment, SHINE acquired one spectrum every hour and we visually inspected the sample through the window twice per day. The first and last spectrum in the series were acquired on 23 April, 19:40 h and 29 April, 08:40 h, respectively. After 6 days, the hourly spectra showed no perceptible changes and the ice absorption bands had disappeared completely. We inferred the absence of water ice below the surface from the duration of the sublimation experiment (by comparison with previous sublimation experiments) and the absence of any motions on the surface of the residue caused by the passing flow of water vapour. We removed the sample from the CarboNIR chamber while it was still at 173 K. To keep it cold and dry, we temporarily kept it in a box with liquid nitrogen for an initial characterization. We measured the sample thickness to be slightly less than 1 mm using a ruler. When lifting the dust mantle, we found no water ice below. The sample was then warmed to room temperature for further characterization.We put the icy sample in the CarboNIR chamber around 18:00 h on Tuesday 23 April. Initially, we held it under medium vacuum in order to record a spectrum of the mixture under stable conditions. We proceeded to high vacuum after about 2 h to start the sublimation of the ice. The chamber interior was kept at a temperature of 173 ± 1 K throughout the experiment, which is in the range of the mean surface temperatures experienced on Ceres at lower latitudesSupplementary InformationPeer Review File


Regarded as constitutively active enzymes, known to participate in many, diverse biological processes, the intracellular regulation bestowed on the CK1 family of serine/threonine protein kinases is critically important, yet poorly understood. Here, we provide an overview of the known CK1-dependent cellular functions and review the emerging roles of CK1-regulating proteins in these processes. We go on to discuss the advances, limitations and pitfalls that CK1 researchers encounter when attempting to define relationships between CK1 isoforms and their substrates, and the challenges associated with ascertaining the correct physiological CK1 isoform for the substrate of interest. With increasing interest in CK1 isoforms as therapeutic targets, methods of selectively inhibiting CK1 isoform-specific processes is warranted, yet challenging to achieve given their participation in such a vast plethora of signalling pathways. Here, we discuss how one might shut down CK1-specific processes, without impacting other aspects of CK1 biology. The post-translational modification of proteins offers multiple and diverse ways of controlling protein function. Of the post-translational modifications that proteins can undergo in cells, phosphorylation is perhaps one of the best studied to date. Protein kinases, the enzymes that catalyse the phosphorylation of primarily serine, threonine, and tyrosine residues on protein substrates ,2, play in vitro [In humans, the kinase superfamily has been classified into two principle groups, comprising the eukaryotic protein kinases, of which there are 497, and the atypical protein kinases, of which there are 58 . The eukin vitro . Howeverin vitro ,11. The in vitro . Due to Seven mammalian CK1 isoforms and their associated splice variants have been reported . These iin vitro with purified catalytic domains, and as such fail to capture any potential regulation from accessory proteins or other enzymes. It is now becoming clear that certain CK1 isoforms can become activated through direct association with cellular proteins [Xenopus laevis orthologue of CK1α was shown to phosphorylate Tyr residue in synthetic peptide substrates [All CK1 isoforms have been reported to act as monomeric enzymes and are classically thought to exist in a constitutively active state ,20,21. Hproteins . A comprproteins ,26. Thatproteins , and thebstrates . As ubiqbstrates ,20,21,29bstrates ,30–35.Early attempts aimed at elucidating the substrate specificity of CK1 identified the requirement for a priming phosphorylation event in the -3 position of the CK1 phosphorylation site. This consensus sequence of pS/pT-X-X-S*/T*, where X is any amino acid and S*/T* denotes CK1-phosphorylation residues, has long since been suggested as the optimal CK1 phosphorylation motif ,36,37. Gin vitro, and each can phosphorylate substrate residues with identical motifs [in vitro is thought to be largely different from that observed in vivo, and different isoforms are known to impact distinct biological processes, suggesting tight regulation of distinct isoforms in cells [in vitro versus in vivo substrate specificity is attributed to intracellular regulatory mechanisms involved in modulating CK1 isoforms, including functional binding partners and post-translational modifications. Furthermore, as the kinase domain of CK1 isoforms constitutes the vast majority of the protein sequence . Added in cells ,20,21. Tsequence , regulatsequence . NonetheOne prominent mode of regulation known to modulate CK1 function in cells involves the use of scaffold or anchoring proteins, which function to direct and/or co-ordinate the correct positioning of protein complexes. Scaffolds can also act as signalosome assembly platforms, bringing functional enzymes, such as kinases, in close proximity to their substrate proteins, thereby increasing reaction efficiency through spatial control . In someFAM83G pathogenic palmoplantar keratoderma mutations, which encode A34E and R52P substitutions within the conserved DUF1669 domain respectively, both led to the inhibition of CK1 binding, suggesting that the interaction between the DUF1669 domain of FAM83 proteins and CK1 appears to be more complicated than a simple binding motif [More recently, the Family of sequence similarity 83 (FAM83) family of proteins have been shown to interact and co-localise with unique sets of CK1α, δ and ɛ isoforms, and in doing so, act to localise the interacting CK1 isoforms to distinct subcellular compartments to streamline CK1-dependent signalling ,57 Figu. For exang motif . As suchAdditionally, post-translational modifications of CK1 isoforms are known to be involved in the regulation of CK1 activity. These are largely thought to occur through phosphorylation, either by CK1 itself through autophosphorylation, or by other kinases ,65,66. IGiven the reported phosphorylation of numerous substrates by CK1 family isoforms, it is perhaps unsurprising that CK1 kinases have been implicated in a plethora of biological processes ,20,21. TTo date, the canonical Wnt signalling pathway is perhaps the best characterised CK1-regulated process in cells . In shorXenopus embryos, through direct association with CK1α [FAM83G severely impeded canonical Wnt signalling, and its overexpression in Xenopus presented with axis duplication phenotypes [FAM83G mutations [Despite this seemingly resolved model, the exact CK1 isoform that mediates phosphorylation of β-catenin on Ser45 has not been irrefutably identified. This is largely due to the non-selective nature of CK1 inhibitors, and the fact that CK1 isoforms are largely essential for cell viability, thereby making genetic ablation experiments very challenging, if not impossible. Indeed, virtually all CK1 isoforms have been implicated in Wnt signalling ,78,79, mith CK1α . Knockouenotypes . FAM83G,enotypes . As descenotypes . Intriguutations ,81,82. Mutations . Thus, iin vitro, with phosphorylation by CK1α only occurring at high CK1α concentrations [The tumour suppressor protein p53 functions as a key regulator of many aspects of biology, including the cell cycle, apoptosis and DNA damage responses . p53 andtrations . Phosphotrations . Additiotrations . CK1δ-metrations . Thus, tHowever, and as seen in Wnt signalling, different CK1 isoforms can have contrasting effects on p53 signalling, depending on the experimental conditions used and the cell type being investigated. For example, in contrast with the stimulatory effects of p53 phosphorylation by CK1 δ and ɛ, knockdown of CK1α has been shown to activate p53 , suggestDuring development, Hippo signalling contributes to correct organ maturation through regulation of cell proliferation and apoptosis . As suchCK1 isoforms have been suggested to regulate Hippo signalling . Indeed,Hedgehog (Hh) signalling is critical for both correct development, and maintenance of healthy adult cells, although the activity of the Hh signalling pathway is vastly reduced in adulthood . Hh signIn addition to the role of CK1 in promoting GLI transcription factor proteolysis, CK1 was also shown to have a positive regulatory role on the Hh pathway. Mammalian SMO was shown to be phosphorylated by CK1α and G Protein-coupled Receptor Kinase 2 (GRK2), to promote full SMO activation and release of GLI transcription factors from the inhibitory multiprotein complex  Figure . Thus, aThe cell cycle in eukaryotes is divided into two main phases: interphase, in which the cell replicates its DNA in preparation for division; and M phase, in which the duplicated DNA is segregated and cell division takes places, creating two genetically identical daughter cells in the process . ThroughFAM83D, or those harbouring a CK1-binding deficient F283A knockin mutation generated with CRISPR/Cas9 gene editing, failed to recruit CK1α to the spindle apparatus [In addition to CK1δ, CK1α has long been suggested to have a role in mitosis. Early immunostaining efforts identified CK1α on mitotic spindles , and injpparatus . Concomipparatus . Furtherpparatus . This appparatus , suggestDrosophila, the CK1 orthologue Doubletime (dbt) phosphorylates the transcription factor Period (PER), which acts to modulate the oscillatory circadian cycle [in vivo by introducing a PER2 S478A knockin mutation into mice. CK1-dependent phosphorylation of Ser478 on PER2 triggers its association with β-TrCP, leading to PER2 proteolysis. By mutating Ser478 to alanine, these knockin mice presented with longer circadian rhythms and accumulated PER1 and CRY1/2 proteins in the nucleus, likely as a consequence of increased PER2 stability [Circadian rhythms are biological processes that oscillate in a predictable, entrainable manner, and are regulated by an endogenous time keeper, or circadian clock . CK1 wasan cycle . In mamman cycle ,116. Folan cycle ,116. In an cycle   responsible for PER1/2 phosphorylation is a subject of debate, but CK1δ and ɛ have both been implicated, and the vast majority of data support roles for both isoforms in the regulation of circadian rhythm. Perhaps the most striking evidence for the involvement of CK1ɛ in the regulation of circadian rhythm stems from evidence gathered in ent mice . In thesent mice . Further rhythms . Howeverl period .CSNK1E-deficient mice are viable, whereas CSNK1D-deficient mice are not [Collectively, there is strong evidence supporting roles for both CK1δ and ɛ in circadian rhythm regulation, suggesting that there may be redundancy between these two enzymes. As CK1δ and ɛ are very closely related proteins, it is feasible that they may act redundantly in some contexts. The fact that  are not , perhapsCSNK1A1, sensitises tumour cells to TNF-related Apoptosis-inducing Ligand (TRAIL)-induced apoptosis [Programmed cell death, or apoptosis, is an evolutionarily conserved mechanism whereby cells act to terminate themselves in an effort to limit the proliferation of faulty and disease-prone cell populations . In shorpoptosis . Recentlpoptosis . Furtherpoptosis . In thispoptosis . Howeverpoptosis , whilst poptosis .Thus, the exact contributions of each CK1 isoform in the various cell death pathways, and their underlying regulatory mechanisms, are not well established, and more selective tools are required in order to ascertain which CK1 isoform acts where in the distinct cell death pathways.Given the involvement of CK1 isoforms in many, key signalling pathways involved in regulating proliferation, cell growth and viability, both during development and in tissue homeostasis, it is unsurprising that dysfunctional CK1 activity has been linked to many human diseases. Of note, neurodegeneration and cancer are particularly prominent diseases that CK1 isoforms have been implicated in ,67,130. The role of CK1 isoforms in neurodegenerative diseases is perhaps best showcased in Alzheimer's disease (AD). CK1δ transcripts are up-regulated 30-fold in the hippocampus of AD brains , and it FAM83G were shown to disrupt the interaction between FAM83G and CK1α [Xenopus embryos harbouring these mutations, presented with dysfunctional Wnt signalling [FAM83H are associated with the dental disease amelogenesis imperfecta (AI) [FAM83H mutations result in N-terminal truncations of the FAM83H protein, generating FAM83H species that maintain CK1-binding, but lack the C-terminus, which presumably regulates FAM83H localisation [In some cases, the pathogenicity of dysfunctional CK1 signalling can be attributed to mutations occurring in regulatory CK1 binding partners. This is perhaps best showcased in FAM83G. Recently, palmoplantar keratoderma-associated mutations within and CK1α . Both hugnalling , suggestcta (AI) . FAM83H cta (AI) ,141–143.lisation ,57. ThesGiven such pleiotropy in the cellular roles of CK1 kinases, they have long been deemed undruggable enzymes. Thus, methods to selectively inhibit CK1-specific functions, in a temporally and spatially controlled manner, are highly warranted, yet extremely challenging to generate. That said, recent years have seen increasing interest in CK1 isoforms as drug targets . HoweverCSNK1A1 gene leading to haploinsufficiency of the CK1α protein [As alternative approaches to kinase inhibition, the thalidomide-related drug lenalidomide, of the immunomodulatory imide drug (iMiD) family, was recently found to induce the degradation of CK1α through recruiting CK1α to the Cereblon-containing Cullin 4 ubiquitin E3 ligase complex, for its subsequent ubiquitination and proteolytic degradation . Such tr protein . Moreove protein ,146. In  protein . These b protein . Such ap protein .Interestingly, targeted proteolysis of FAM83D through the Affinity-directed PROtein Missile (AdPROM) system ,149 was Despite such extensive research into CK1 biology over the last few decades, the study of CK1 isoforms within their endogenous settings has proved difficult. Given such a high degree of homology within their kinase domains, the generation of selective antibodies to enable immunofluorescence-based localisation and immunoprecipitation-based interactome studies have proved challenging. This problem is particularly apparent for CK1δ/ɛ, which have highly similar sequences even outside their kinase domains . Thus, iCSNK1A1 and CSNK1E loci allowed robust assessment of CK1α and ɛ localisation during mitosis [CSNK1D and CSNK1E identified a vesicle-localised CK1δ/ɛ substrate, GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1), required for endocytosis [However, recent advances in genome editing may overcome some of these limitations, and allow selective and specific research into individual CK1 isoforms. Through knocking in epitope tags into individual CK1 loci, the true subcellular distribution and changes in interactomes of individual CK1 isoforms upon modulation of individual signalling pathways can be assessed, assuming the tags themselves do not interfere with CK1 biology in the first place. Indeed, knockin of an mCherry tag into the  mitosis . More reocytosis . Expansiocytosis . Such anocytosis . ConsequGiven the diverse range of CK1 functions within the cell, and their regulatory roles within many signalling pathways, CK1 isoforms are key players in cell biology. However, and as discussed above, the seemingly contradictory roles of CK1 isoforms within the same signalling pathways make CK1 isoforms exceptionally difficult therapeutic targets. Future efforts aimed at researching CK1 functional binding partners, such as FAM83 proteins, and proceeding to disrupt their interaction with the aim of selectively inhibiting a CK1-specific process, may provide an attractive alternative means of targeting CK1 kinases, to facilitate basic research and develop potential therapeutic interventions. In order for such approaches to be successful, there is a drastic need to improve the research design, data analysis and interpretation of findings in the field of CK1 research. Impeding factors such as cross-reacting antibodies , and non-isoform selective inhibitors, can add confusion to an already complicated biological problem. Through using CK1 isoform-specific antibodies and other reagents, correct kinase-substrate relationships can be established, and one could quickly and confidently assay which CK1 kinase is participating in the biological event in question. In cases where CK1 isoform-specific antibodies cannot be used reliably, CRISPR/Cas9-based gene editing strategies, aimed at targeting the endogenous CK1 loci with epitope tags, will undoubtedly provide a means of assessing the true localisation profiles of these dynamic kinases. Once regarded as promiscuous kinases, the emerging roles and regulation of these distinct CK1 isoforms has highlighted the diversity of fundamental cellular functions that these enzymes co-ordinate. Armed with better tools, reagents and novel strategies to modulate specific CK1 functions, accurate determination of the physiological substrates for each CK1 member can be achieved.