Phaeodactylum tricornutum. Conditionally phosphorylated peptides were detected at low iron (n = 108), during the diel cycle (n = 149), and due to nitrogen availability (n = 137). Through a multi-omic comparison of transcript, protein, phosphorylation, and protein homology, we identify numerous proteins and key cellular processes that are likely under control of phospho-regulation. We show that phosphorylation regulates: (1) carbon retrenchment and reallocation during growth under low iron, (2) carbon flux towards lipid biosynthesis after the lights turn on, (3) coordination of transcription and translation over the diel cycle and (4) in response to nitrogen depletion. We also uncover phosphorylation sites for proteins that play major roles in diatom Fe sensing and utilization, including flavodoxin and phytotransferrin (ISIP2A), as well as identify phospho-regulated stress proteins and kinases. These findings provide much needed insight into the roles of protein phosphorylation in diel cycling and nutrient sensing in diatoms.Diatoms are major contributors to global primary production and their populations in the modern oceans are affected by availability of iron, nitrogen, phosphate, silica, and other trace metals, vitamins, and infochemicals. However, little is known about the role of phosphorylation in diatoms and its role in regulation and signaling. We report a total of 2759 phosphorylation sites on 1502 proteins detected in  Widely distributed across the world’s aquatic environments, diatoms are important unicellular eukaryotic phytoplankton that are responsible for approximately 20% of annual global primary production and play a significant role in biogeochemical cycles . The ava3 and increasing NH4 assimilation pathways while also increasing Fe scavenging proteins and reducing Fe-containing proteins  of (2) ammonium, (3) nitrite, or (4) nitrate at a cell density of ~7.5 × 105 cells mL−1. Treatments were then sampled at t = 15 min, 45 min, and 18 h in duplicate for transcriptome analysis . Th. ThP. tr3+-NTA-agarose beads were prepared using Ni-NTA-agarose beads [Protein samples taken for each experiment were digested, labeled, and analyzed with iTRAQ-based mass spectrometry (MS) at the Pacific Northwest National Laboratory as described in Smith et al. . Samplesse beads . PeptideProteome and phosphoproteome peptides were identified using SEQUEST  as descrData was further manually curated  accordinp-values, based on the probability density function, between phosphoproteome and proteome to determine significantly enriched or depleted KOG groups and classes for phosphorylation  to each protein to determine phosphorylation loci and number of p-sites per protein. This was then compared to the p-sites detected by Chen et al. . Phosphohttps://software.broadinstitute.org/morpheus), identifying 5 diel clusters. For the Nshort-P subset . To overcome this and identify conditionally phosphorylated proteins, we compared patterns of phosphopeptide detection against the patterns of their respective proteins by calculating Pearson correlation coefficient (PCC). However, since the dataset was not robust enough to produce a normal distribution, PCC values could not be used as a cut off for analysis purposes and were instead only used to broadly indicate “anti-correlated” (PCC < −0.7) or “uncorrelated” (−0.2 < PCC < 0.2) phosphoproteins and proteins.P. tricornutum genome, ~7700 proteins were detected in this study, which is more than double the number identified in previous P. tricornutum studies [p = 1.9−11) and transcription (p = 2.9−5) relative to the total proteome (p = 5.9−4), and replication recombination and repair (p = 6.6−4) were proportionally depleted for phosphorylation. Phosphorylation on serine residues was most common (81%), followed by threonine (16%) and tyrosine (3%). The proportion of phosphoproteins with one and two p-sites were 55% and 21% respectively, and proteins with greater than five p-sites consisted of 5.6% of the detected phosphoproteins  was performed d. SampleProteins that are conditionally phosphorylated as a function of nutrient status are good candidates for regulatory components of the cellular response to varying conditions. Of the 3059 phosphopeptides detected in this study, 76, 50 and 101 phosphopeptides were uniquely detected at low, medium and high Fe treatments respectively, while 712 phosphopeptides were detected in the Nshort experiments e. Pattern = 108), we found 12 phosphoproteins, including flavodoxin (Phatr3_J23658), E3 ubiquitin protein ligase , ferrocytochrome oxygenase  and 9 hypothetical proteins, that were previously characterized as transcriptionally responsive to low Fe [Of the LowFe-P consistently detected in the proteome  and CCR-associated factor 1  could have implications for transcriptional control under low Fe. The FACT complex binds to DNA, destabilizing one histone dimer from the nucleosome, facilitating transcription. In humans, when SSRP1 of the FACT complex is phosphorylated on Ser-510 by casein kinase 2, DNA binding is inhibited . Both huPhosphorylation of ribosomal proteins (RP) S3 and L24 , may influence translation or trigger signaling at low Fe. Ribosome phosphorylation results in conformational change and reduced protein synthesis . HoweverA. thaliana, a phytochrome-interacting transcription factor is phosphorylated in response to red and far-red light resulting in its degradation to promote photomorphogenesis [Spinacea oleracea, the plant-specific thylakoid soluble phosphoprotein was phosphorylated during the light period and was ultimately hypothesized to control gene expression of photosynthesis genes through redox-mediated signal cascade [n = 149) highlighted 5 clusters of phosphoproteins that changed over the 12h light:dark diel cycle  of the TCA cycle is also phosphorylated pre-light (6AM) and dephosphorylated post-light (10AM). In radation . AlthougTo further emphasize the potential impact of phosphorylation on cellular C allocation, we highlight that three of the five phosphoproteins discussed in the pre- and post-light cluster  have key roles in the PEP-pyruvate-oxaloacetate node. The PEP-pyruvate-oxaloacetate node is responsible for partitioning and directing C flux to catabolic, anabolic, and energy production pathways . Tight pA. thaliana CPSB or human CAD. It was interesting to note that pgCPS transcript expression was upregulated immediately after light transitions (10 AM/10 PM) while protein accumulates after the dark [A. thaliana DUR3 (53.5% identity). Although no phosphorylation sites are currently known for this protein, the transcript is upregulated at night and is preferentially phosphorylated in the day despite protein abundance remaining relatively stable. Our hypothesis is that phosphorylation inhibits urea transport by DUR3 during the day when urease expression is downregulated and the catabolic component of urea cycle activity is low in diatoms [C and N flux are also tightly coupled within the cell and N assimilation genes, such as nitrate and nitrite transporters, nitrate reductase and glutamine synthetase, were shown to be transcriptionally upregulated during the day . Althougthe dark  when pho diatoms ,71.A. thaliana VPS35 (29.6% identity). A. thaliana VPS has been shown to interact with aquaporins, localize to the vacuolar membrane and is a candidate for facilitating N transport across the vacuolar membrane [The other phosphoprotein with a potentially direct role in N metabolism is a vacuolar sorting-associated protein . VPS was found to be phosphorylated on Ser-193, at an un-conserved region upstream of the Vps35 functional domain when aligned to membrane ,73. PhosA. thaliana [Over the course of the 12 h light:dark diel cycle, 6 samples were collected  and phosphorylation of different translation related proteins oscillated, possibly affecting translation rates and ribosomal binding to mRNA. Two such phosphoproteins were small ribosomal subunit 40 S9  and 60S acidic ribosomal protein P0 . Protein abundance and phosphorylation of Pt_S9 was well correlated  and relathaliana ,75. As sthaliana . In contthaliana . P0 protthaliana . Thus, wthaliana .P. tricornutum and human eEF2K. While it is difficult to extrapolate conclusions about the relationship between phosphorylation of eEF2K and its activity, it is noted that the phosphorylation profile of Pt_eEF2K is very similar to the protein abundance profile of Pt_EF3A  and eukaryotic elongation factor 2 kinase , which were both part of the pre- and post-light cluster. EF3A interacts with the small ribosomal subunit to control translational accuracy in yeast in a mechanism that is similar to its bacterial homolog ,80. Yeas Pt_EF3A . Taken tIn addition to ribosome abundance and translation elongation, translation initiation, which enables ribosomal binding to mRNA, is also essential. Two eukaryotic translation initiation factors, eIF4F  and eIF4G3 , were identified to have phosphorylation patterns in response to diel light cycling. eIF4F phosphorylation peaks during the day (2 PM to 6 PM) while eIF4G3 phosphorylation was highest post-dark (10PM) with a secondary peak post-light (10 AM). To date, eIF phosphorylation has not been extensively investigated. We found four p-sites for eIF4F and eight p-sites for eIF4G3 in our dataset. However, only phosphopeptide abundance of pSer-9 of eIF4F and pSer-574 of eIF4G3 seem to be affected by the diel cycle unlike the other p-sites detected on these eIFs. eIF4F and 3IF4G3 are both part of the “cap building complex” and are important for translation. Specifically, eIF4F allows for mRNA discrimination enabling variation in mRNA translation rates  and eIF4Novel phosphorylation of two important iron-sensitive proteins, flavodoxin and phytotransferrin , was detected in the phosphoproteome. Transcriptional levels for each of these gene products are known to increase in response to low Fe conditions (20 pM). mRNA expression levels for flavodoxin and pTF are commonly used as molecular markers of Fe stress in marine phytoplankton communities ,87,88. CTwo N replete timepoints (15 min and 45 min) and one early N deplete timepoint (18 h) were sampled and log2 fold change (log2FC) of phosphopeptide abundance was calculated to identify phosphoproteins that respond to fluctuating N . Since sn = 17, 43 and 69 respectively; Based on hierarchical clustering of these 137 phosphoproteins, three clusters were apparent: N transition, N replete, and N deplete clusters  is phosphorylated on Ser-246, by nitrogen permease reactivator 1, as a response to suboptimal nitrate levels [P. tricornutum and Ser-578 of Pt-NRT2 is not conserved in H. polymorpha. Furthermore, despite protein abundance increasing during nitrogen depletion, Pt_NRT2 was dephosphorylated, which is opposite of YNT1. Phylogenetically, diatom NRT2s appear to be derived from the secondary exosymbiont and should function more similarly to yeast NRT2 [N transporters and N metabolism proteins, especially mitochondrial proteins, were phosphorylated in response to changes in cellular N status. Phosphorylated transporters included plasma membrane urea symporter DUR3, ammonium transporter , and nitrate/nitrite transporter . Interestingly, the p-site of DUR3 was at Thr-69 instead of the diel responsive Ser-70. Transcript and protein profiles of DUR3 both increase during N limitation while phosphopeptide abundance of Thr-69 decreases significantly  during both low Fe and low N conditions. Transcript and protein levels for GDH2 were downregulated during N deplete conditions , GDH2, ammonium-dependent ornithine urea cycle (OUC) type carbamoyl-phosphate synthetase  and pgCPS. PEPCK is responsible for the conversion of oxaloacetate to PEP and in activity ,99. The  10-fold . The p-snditions , suggestThalassiosira pseudonana to diel light cycling conditions [P. tricornutum. Hence, further supporting the hypothesis that pgCPS phosphorylation at Ser-970 inhibits protein activity. During N deplete conditions, although pgCPS transcript and protein levels are decreased, dephosphorylation may still allow for small amounts of pyrimidine biosynthesis.Phosphorylation of unCPS is anti-correlated while phosphorylation of pgCPS is relatively well correlated to respective transcript and protein levels . unCPS inditions , we mainnditions ,102, thiShewanella oneidensis results in transcription derepression of HnoC [Neurospora crassa, phosphorylation of eIF2a was found to be controlled by the circadian clock. It is phosphorylated during the day, leading to reduced translation during the day [P. tricornutum may impact protein synthesis and TOR signaling as a response to N depletion.Translation is also affected by phosphorylation in response to changes in N status. We noted that phosphorylation of 40S small ribosomal subunit 20  and translation initiation factor eIF2 gamma  correlates well with transcript abundance instead of protein abundance. Previous research has shown that phosphorylation of a protein could directly impact its transcript expression in a signaling feedback loop. For example, phosphorylation of HnoC in  of HnoC . Thus, 4 of HnoC . In yeas of HnoC . mTOR si of HnoC . In the  the day . AlthougFinally, we identified two nucleotide binding proteins that had significant decreases in log2FC in phosphopeptide abundance between N replete and N deplete conditions, CAF1 and transcription initiation factor TFIID subunit BDF1 (Phatr3_J44399). In yeast and mammalian cells, the CCR4-CAF1-NOT complex associates with microRNAs, destabilizing them and resulting in translational repression of target mRNAs ,108. SinP. tricornutum, only phosphorylation of threonine was found to be affected by N depletion (Thr-601). Since P. tricornutum HSP90 protein abundance increases in response to N depletion, it appears to be dephosphorylated and active during N depletion  and manganese superoxide dismutase  are affected by N. HSP90 is dephosphorylated as cellular N is depleted. In yeast, phosphorylation of HSP90 has been shown to inhibit HSP90 conformational change, affecting DNA repair. Although phosphorylated on both serine and threonine residues, 4 serine residues were identified to be important phosphorylation sites in yeast and dephosphorylation occurred through Ser/Thr phosphatase Ppt1 . Howeverepletion , similarnditions , it is pPhosphorylation and ubiquitination have been shown to overlap, resulting in “crosstalk” in post-translational modification . PhosphoP. tricornutum, phosphorylated InsP6/PP-IP5K is anti-correlated to both transcript and total protein abundance , a Ca2+/calmodulin-dependent protein kinase , an inositol hexakisphosphate/ diphosphoinositol-pentakisphosphate kinase , and a serine/threonine kinase . Phosphopeptide abundance of CDPK and CaMK was correlated with total protein abundance and they were both part of the N deplete cluster . Since tbundance , indicatbundance . Thus, wResearch on diatoms have included the cellular response to nutrient limitation  in the context of carbon sequestration, global biogeochemical cycling, and diatom bloom dynamics. From a bioengineering perspective, diatoms have been used to study lipid accumulation for purposes related to fuels, food, and production of high value compounds . We beliP. tricornutum observed in our experiment [C. reinhardtii, could be used for future studies.Most of the diel light cycling related protein phosphorylation we have identified in this study occur around the transitions between light and dark period. In response to the onset of the light period, under diel light cycling conditions, we observed phosphorylation patterns for key proteins involved in controlling C flux in diatoms. These data suggest an important role of phospho-regulation in shuttling C towards fatty acid biosynthesis. Additional studies that quantitatively track the PEP, pyruvate, and oxaloacetate metabolite pools over the diel cycle in conjunction with the abundance and phosphorylation status of proteins such as Pt_ACC, Pt_PPDK, Pt_MDH, will be helpful. Generation of p-site point mutations for such proteins are an important next step for examining the importance of phospho-regulation for controlling key aspects of C flux within the cell. Moreover, to further grasp the dynamics of global phosphorylation as cells transition between light regimes, a shorter sampling interval would be essential for future studies. Additionally, phosphorylation events related to cell cycle dynamics were not detected, which could be due to minimal growth synchronization of periment . Thus, a3 availability [Phosphorylation of the key Fe metabolism proteins, flavodoxin and pTF, was shown to differ based on Fe status of the cell. Additional biochemical studies on the role of phosphorylation in regulating these proteins will result in better understanding of the mechanisms by which diatoms sense, scavenge and assimilate Fe in the environment. Additionally, phosphorylation changes between N replete and deplete cellular states discussed in this study provide a new perspective to the mechanisms behind the early diatom response to N depletion. Studies on the transcriptional response of diatoms to the sudden onset NOlability  have sholability ,121, funlability ,96,122 alability , phosphoP. tricornutum under low Fe, diel light cycling, and across a transition of N availability. We have shown that C and N flux, stress responses and ubiquitination/deubiquitination are likely under the control of phospho-regulation. Additionally, overall transcriptional output appears to be governed by phosphoproteins that affect binding to TATA regions, histone stability, and chromatin accessibility. Furthermore, our data suggest that phospho-regulation likely plays a role in dictating the diversity and quantity of the cellular protein pool through phosphorylation of translation initiation, translation accuracy and translation elongation proteins. Overall, phosphorylation appears to play a major role in regulating cellular processes and signal cascades. When considered in conjunction with quantitative transcriptomics, metabolomics, and proteomics, additional studies into phosphorylation patterns in response to acute shifts in environmental conditions will likely result in new insights into diatom signaling and metabolic mechanisms that are key for controlling diatom physiological acclimation and growth.Our results serve to establish new insight into phospho-regulation in the model diatom


Aberrant hedgehog (HH) signaling is implicated in the development of various cancer entities such as medulloblastoma. Activation of GLI transcription factors was revealed as the driving force upon pathway activation. Increased phosphorylation of essential effectors such as Smoothened (SMO) and GLI proteins by kinases including Protein Kinase A, Casein Kinase 1, and Glycogen Synthase Kinase 3 β controls effector activity, stability and processing. However, a deeper and more comprehensive understanding of phosphorylation in the signal transduction remains unclear, particularly during early response processes involved in SMO activation and preceding GLI target gene regulation.We applied temporal quantitative phosphoproteomics to reveal phosphorylation dynamics underlying the short-term chemical activation and inhibition of early hedgehog signaling in HH responsive human medulloblastoma cells. Medulloblastoma cells were treated for 5.0 and 15 min with Smoothened Agonist (SAG) to induce and with vismodegib to inhibit the HH pathway.Our phosphoproteomic profiling resulted in the quantification of 7700 and 10,000 phosphosites after 5.0 and 15 min treatment, respectively. The data suggest a central role of phosphorylation in the regulation of ciliary assembly, trafficking, and signal transduction already after 5.0 min treatment. ERK/MAPK signaling, besides Protein Kinase A signaling and mTOR signaling, were differentially regulated after short-term treatment. Activation of Polo-like Kinase 1 and inhibition of Casein Kinase 2A1 were characteristic for vismodegib treatment, while SAG treatment induced Aurora Kinase A activity. Distinctive phosphorylation of central players of HH signaling such as SMO, SUFU, GLI2 and GLI3 was observed only after 15 min treatment.This study provides evidence that phosphorylation triggered in response to SMO modulation dictates the localization of hedgehog pathway components within the primary cilium and affects the regulation of the SMO-SUFU-GLI axis. The data are relevant for the development of targeted therapies of HH-associated cancers including sonic HH-type medulloblastoma. A deeper understanding of the mechanisms of action of SMO inhibitors such as vismodegib may lead to the development of compounds causing fewer adverse effects and lower frequencies of drug resistance.Video Abstract The role of protein phosphorylation in the control of cellular behavior has been well appreciated and intensely studied for many years. Phosphorylation is one of the most important post translational modifications (PTMs) and regulated through a well-controlled interplay of kinases and phosphatases to regulate cellular signaling . Thus, pIn vertebrates, the hedgehog (HH) pathway plays a central role in the control of embryonic development, cell proliferation, and survival. The HH pathway is induced by the release of HH ligands such as sonic HH. Upon binding of HH protein to Patched 1 (PTCH-1), the G-protein coupled-like receptor Smoothened (SMO) is released from inhibition by PTCH1, allowing its translocation to and accumulation in the primary cilium. The primary cilium is a microtubule-based surface projection essential for canonical HH signal transduction in vertebrates , 18. AftPersistent and irreversible activation of HH signaling has been implicated in the development of various cancers such as basal cell carcinoma and medulloblastoma –23. In oPhosphatases are the major counterparts of kinases and represent an important class of enzymes involved in the regulation of the HH pathway, and their role in sonic HH signaling has been reviewed recently . HoweverDespite the examples discussed above, the complex role of protein phosphorylation in the early phase of HH pathway activation and inhibition has not been elucidated so far. It is essential to better understand SMO modifying drugs, particularly in light of resistance development and the severe adverse effects such as muscle spasms caused by a number of, but not all, SMO inhibitors . Hence, In order to systematically study the role of phosphorylation in hedgehog-driven cancer signaling, we used the human medulloblastoma cell line DAOY for our studies, which we have previously shown to allow manipulation of canonical pathway activity by  agonist and antagonist treatment . We conf(A) Three biological replicates of human medulloblastoma cells were treated with DMSO, SAG, vismodegib or EGF in starving medium. (B) Analytical workflow: After treatment, sample preparation, and TMT 10-plex labeling, peptides of each time point and treatment were pooled. Aliquots were utilized for pH 8 fractionation and deep proteome profiling or phosphopeptide enrichment by metal oxide affinity chromatography. Measurements of peptides and phosphopeptides were conducted with reversed-phase HPLC coupled to high-resolution mass spectrometry.2) beads. Following enrichment, phosphopeptides were fractionated using hydrophilic interaction liquid chromatography (HILIC) prior to nano-HPLC-MS/MS analysis for detection and relative quantification of phosphopeptides  or protein , the average of the three normalized intensity values of each treatment or control was used to calculate the ratio of each particular treatment versus control. All three biological replicates were considered for ratio calculation. To determine p-values and statistical significance of more than 7000 phosphopeptides in an unbiased manner, LIMMA (Linear Models for Microarray) statistical testing was applied.After cell lysis, reduction and alkylation, tryptic digestion, and TMT labeling for relative quantification, phosphopeptides were enriched by metal oxide affinity chromatography (MOAC) using titanium dioxide , 200 mM guanidine hydrochloride and trypsin   for 14 h at 37 °C. Later, spin filters were transferred to new 2 mL protein LoBind Eppendorf tubes , centrifuged at 13,500 g for 25 min, and followed by two other 15 min centrifugation steps with the addition of 100 μL 50 mM TEAB and 50 μL water on top of the membrane. To control digestion efficiency, 1.0 μg of each digest was injected into a monolithic HPLC system as described in Burkhart et al. [Hyperconfluent HH responsive DAOY cells   (dissolved in 41 μL acetonitrile) for 60 min at 25 °C with slight agitation. Reaction was stopped upon incubating for 15 min at 25 °C with 0.27% hydroxylamine (v/v). Samples were pooled together in a ratio of 1:1 and dried under vacuum.TMT pooled samples were solubilized in 300 μL 0.10% aqueous TFA and desalted with SPEC C18 AR 15 mg cartridge . The cartridge was first washed with 400 μL 100% acetonitrile (ACN), equilibrated twice with 400 μL 0.10% (v/v) TFA, loaded twice with TMT sample, washed twice with 400 μL 0.10% (v/v) aqueous TFA, and the sample was eluted twice with 200 μL 70% (v/v) ACN into a protein LoBind Eppendorf tube . After vortexing, 50 μg of each pool were removed for high pH reversed-phase fractionation. Samples were dried under vacuum.The aliquots were solubilized in 15 μL10 mM ammonium acetate, pH 8.0 (buffer A) and fractionated on an HPLC System . Peptides were separated on a 150 mm × 1.0 mm i.d. C18 column  with a 55 min gradient ranging from 2.5 to 42% ACN in 10 mM ammonium acetate, pH 8.0, at a flow rate of 12.5 μL/min. In total, 20 fractions were collected in 60 s intervals in concatenation mode. Each fraction was dried under vacuum and resuspended in 15 μL of 0.10% (v/v) aqueous TFA for nano-HPLC–MS/MS analysis.TMT pooled samples were solubilized in 80% (v/v) ACN, 5.0% (v/v) TFA and 1.0 M glycolic acid. Based on Engholm-Keller’s et al.  protocol5 and the maximum injection time 120 ms. For MS/MS, precursors were selected using the top 15 ions with 30 s dynamic exclusion. Peptides were fragmented using higher-energy collisional dissociation (HCD) at 33% relative collision energy, and fragment ion spectra were acquired in the Orbitrap with a resolution of 60,000, an AGC target value of 2 × 105 ions, a maximum injection time of 200 ms, and an isolation window of 0.4 m/z for the high pH fractions and isolation window of 0.8 m/z for the HILIC fractions (phosphoproteome). A Table with an overview of applied gradients is added in the supplementary section in Table S-Samples were analyzed using a nano HPLC system  coupled to a quadrupole-Orbitrap mass spectrometer  in data dependent acquisition mode. All samples were preconcentrated on a 20 mm × 75 μm i.d. trapping column  in 0.1% aqueous TFA for 10 min using a flow rate of 20 μL/min, followed by separation in a 50 cm × 75 μm i.d. separation column  at a flow rate of 250 nL/min using a binary gradient  as indicated in Table MS raw files were converted to the open standard format mzML and centroided using msconvert (3.0.11781). Further processing was performed using the KNIME Analytics platform (Version 3.7.0)  and OpenIdentification was performed using the MSGFPlusAdapter with MS-GF+  . ProteinThe normalization of the phosphoproteome was performed as described by Shema et al. . Median casecpb.shinyapps.io/ksea/) [https://apps.ingenuity.com/).LIMMA statistical testing (R package limma v. 3.36.5)  was applo/ksea/)  using thFollowing the analytical strategy described above, we identified and quantified a total of 7696 highly confident phosphopeptides after 5.0 min and 9976 phosphopeptides after 15 min treatment with a false localization rate (FLR) below 0.05. We further quantified 7547 protein groups after 5.0 min and 7763 protein groups after 15 min using extensive pH 8 fractionation. As expected, the changes in the global proteome after short-term treatment were negligible. We found a normal and narrow distribution of the ratios SAG/DMSO, Vismo/DMSO and SAG/Vismo, similar to the distribution of the positive control EGF as depicted in Figure S-Strikingly, there were several distinctive features in the distributions of the phosphopeptide ratios SAG/DMSO, Vismo/DMSO and EGF/DMSO. Figure Ingenuity pathway analysis (IPA) was performed with all quantified phosphopeptides for both time points. All proteome-normalized phosphopeptide ratios of respective treatment relative to DMSO as control treatment were uploaded and analyzed using the phosphorylation analysis tool in the IPA software platform. Notably, differences in global phosphorylation dynamics between activation and inhibition of the HH pathway were more pronounced after the short-time treatment time point of 5.0 min as depicted in Fig. Protein phosphorylation is a fast and highly dynamic process, which can transmit intracellular signaling within minutes and even seconds , 61, 62.The most significantly enriched pathways after 5.0 min treatment were Protein Kinase A signaling, Insulin Receptor signaling, as well as ERK/MAPK signaling Fig. b. IntereProtein Kinase A has been recognized to negatively modulate the hedgehog pathway and thus influence cell fate and proliferation . In accoAdditionally, we find ERK/MAPK signaling both enriched and activated after 5.0 min vismodegib treatment Fig. . ExtensiResearch on the crosstalk of sonic HH signaling and the mTOR/S6K1 pathway revealed an activation of GLI1 in a SMO-independent manner indicating a positive crosstalk via non-canonical HH pathway activation . In contMoreover, our data indicates that SAG and vismodegib treatment differentially influences JAK/STAT signaling Fig. a. A posiInterestingly, PTEN signaling was strongly inhibited after 5.0 min of vismodegib treatment. In contrast to our results, Hartmann et al. reported a link between PTEN loss and proliferation of medulloblastoma cells on genomic and epigenetic levels . HoweverThese time-dependent dynamics in pathway activation and inhibition explain that the differences between SAG and vismodegib treatment on pathway level found after 5.0 min were not observable after 15 min treatment Figure S-A. VismodP-values were determined to evaluate the statistical significance of up- and downregulated phosphopeptides after 5.0 and 15 min treatment. Statistical significance was evaluated for each treatment relative to DMSO as control treatment and between both treatments SAG and vismodegib. Whereas only one phosphopeptide was found to be significantly upregulated after 5.0 min SAG treatment compared to DMSO as control  at serine 1280 was significantly upregulated after HH pathway inhibition Fig. a. BRCA1 Furthermore, Intraflagellar transport protein 172 homolog (IFT172) was significantly higher phosphorylated at serine 273 after vismodegib treatment compared to SAG treatment. IFT172 is part of Intraflagellar transport complex B (IFT B) and important for ciliary assembly and essential for brain development . Our poiKinase substrate enrichment analysis of phosphopeptides identified after 5.0 min treatment revealed activation of Polo-like kinase 1 (PLK1) after vismodegib treatment along with CDK5, MAP 3 K8 and BRAF Fig. c. Like AFurthermore, Casein Kinase 2A1 (CSNK2A1) was predicted to be significantly downregulated after 5.0 min vismodegib treatment Fig. c. CaseinWe filtered the phosphoproteome of each time point for proteins, which are involved in or linked to HH signaling. Interestingly, the quantitative changes in the phosphoproteome of HH pathway components were rather low. However, various differences in the phosphorylation pattern of particular components of the Hedgehog pathway were observable. Pathway maps relying on identified phosphopeptides and the corresponding heatmap of HH pathway phosphoproteins are presented in Fig. In total, we identified 51 phosphopeptides involved in HH signaling after 5.0 min treatment, of which 12 phosphopeptides were significantly different between SAG and vismodegib treatment  was found to be significantly higher phosphorylated after 15 min of vismodegib treatment compared to SAG treatment Fig. . A dual In sum, we come up with the following effects on the phosphorylation dynamics within the HH pathway after 15 min SAG and vismodegib treatment as depicted in Fig. Monitoring of close to 10,000 phosphopeptides of human medulloblastoma upon short-term stimulation with SMO activators and inhibitors, respectively, allowed us to investigate the complex early phosphosignaling during HH pathway regulation. Changes in the phosphoproteome in response to activation or inhibition of the HH pathway are already observed after 5.0 min of treatment and mainly affect signaling pathways involved in cellular growth and proliferation. Of note, rapid changes in phosphorylation patterns of HH pathway components mainly affect components of the primary cilium as well as signaling via the SUFU-GLI axis. Intraflagellar transport proteins such as IFT172 were differentially phosphorylated indicating a crucial role of phosphorylation in the regulation of the intraflagellar transport machinery and hence the transport of HH pathway components in the primary cilium. Furthermore, phosphorylation influences Protein Kinase A signaling besides PI3K/AKT/mTOR and PTEN signaling as we find these pathways differentially regulated by phosphorylation dynamics upon short-term activation and inhibition of the HH pathway.Our data suggests that several kinases play important roles in the regulation of short-term activation or inhibition of HH signaling. Aurora Kinase A may be involved in the activation of HH signaling upon SAG treatment. El-Sheikh et al. already showed that inhibition of Aurora Kinase A enhances the chemosensitivity of medulloblastoma , and tarElucidation of these deep phosphorylation cascades in HH signaling paves the way to a profound understanding of the basis of the immediate events of HH pathway activation important for the development of targeted therapies of sonic HH-type medulloblastoma patients. The new insights into changes in the phosphorylation landscape upon vismodegib treatment clears up its mechanism of action and will be beneficial for the prevention of adverse effects and therapeutic resistance.1The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.Additional file 1: Table S-1. Eluent B (B) gradient range and gradient duration for the different analyzed fractions. Table S-2. Applied R-packages. Responsiveness of DAOY cells to SAG and vismodegib treatment. RNA isolation and quantitative PCR (qPCR). Table S-3. Primer Sequences used for qPCR. Figure S-1. Hedgehog target gene expression in SAG and vismodegib treated DAOY cells. Figure S-2. Comparable Hedgehog pathway induction by natural and synthetic HH pathway activators. Figure S-3. Distribution of protein groups identified after 5.0 min treatment (A) and 15 min treatment (B). Figure S-4. Ingenuity pathway analysis of all phosphopeptides identified after 15 min using a phosphorylation analysis. Figure S-5. Time dependent Ingenuity pathway analysis of cancer associated pathways. Figure S-6. Western blot validation of PKA activity modulation in response to SMO agonist and antagonists. Table S-4. Antibodies used for Western blot analysis. Figure S-7. Overlap of quantified phosphopeptides after 5.0 and 15 min. Figure S-8. Volcano plots of phosphopeptides identified after 5.0 min treatment. Figure S-9. Kinase substrate enrichment analysis (KSEA) was performed for phosphopeptides identified after 5.0 min treatment for the ratio SAG/Vismo using the online platform KSEA App (https://casecpb.shinyapps.io/ksea/). Figure S-10. Volcano plots and kinase set enrichment analysis for phosphopeptides identified after 15 min treatment. Figure S-11. IFT172 phosphorylation and expression after 5.0 and 15 min.


When exposed to bright light, living mice increase the rate of GTP and ATP synthesis in their retinas; concomitant with IMPDH1 aggregate formation at the outer segment layer. Inhibiting IMPDH activity in living mice delays rod mass recovery. We unveil a novel mechanism of regulation of IMPDH1 in vivo, important for understanding GTP homeostasis in the retina and the pathogenesis of adRP10 IMPDH1 mutations.We report the in vivo regulation of Inosine-5´-monophosphate dehydrogenase 1 (IMPDH1) in the retina. IMPDH1 catalyzes the rate-limiting step in the de novo synthesis of guanine nucleotides, impacting the cellular pools of GMP, GDP and GTP. Guanine nucleotide homeostasis is central to photoreceptor cells, where cGMP is the signal transducing molecule in the light response. Mutations in IMPDH1 lead to inherited blindness. We unveil a light-dependent phosphorylation of retinal IMPDH1 at Thr IMPDH1 mutations have also been associated to rare autosomal dominant Lebers Congenital Amaurosis (adLCA), characterized by nearly complete blindness from an early age: R105W and N198K , the enzyme responsible for the first and rate-limiting step in GTP synthesis, are associated to severe forms of inherited blindness. At least nine mutations have been associated to the RP10 form of autosomal dominant retinitis pigmentosa, that primarily manifests as night blindness and gradually progresses to loss of central vision: R224P ; D226N ; R231P ; T116M, nd N198K . DespiteRP10 mutations are allegedly ‘gain-of-function’ mutations, given that IMPDH1 knock-out mice present only a mild retinopathy . MutatioThere are two IMPDH isozymes, IMPDH1 and IMPDH2, that are 84% identical. While most human tissues show only basal IMPDH1 and high IMPDH2 expression, the retina is one of a few exceptions where IMPDH1 predominates . In the de salvage pathways, nucleotides are produced from recycled purine bases  in a NAD dependent manner t manner . GMP actt manner . The regt manner . In eukat manner .IMPDH1 mutations associated to RP10 overlap with GDP/GTP binding sites at the CBS domain and result in diminished GDP/GTP negative allosteric control of human IMPDH1 in vitro  under conditions that require higher IMPDH activity to keep with GTP demand , like hiIMPDH1-inherited retinal dystrophies.Little is known about the relevance of IMPDH1 catalytic activity in photoreceptor cells of the retina or its physiological regulation in vivo. We here report the first posttranslational modifications of IMPDH1 in native retinal tissue, associated to dark/light states. We show that the predominant retinal splice form of IMPDH1 is phosphorylated at the Bateman domain (T159/S160) in response to light in vivo; and that this phosphorylation desensitizes the enzyme to GDP/GTP allosteric inhibition of catalytic activity in vitro. Together, these results reveal a novel light-dependent regulatory mechanism that impedes IMPDH allosteric inhibition, facilitating the elevated GTP levels required for phototransduction. Supporting the proposed mechanism, we show that exposure of living mice to bright light results in an increase in the global flux through de novo purine nucleotide synthesis in the retina, which correlates with a progressive accumulation of IMPDH1 aggregates at the outer segment layer. Furthermore, inhibition of IMPDH activity in living mice by intravitreal injection of IMPDH inhibitors has a moderate but consistent effect on mass rod response recovery measured by electroretinogram. Thereby, our results point to the de novo GTP synthesis being relevant to sustain the GTP pool during constant light exposure, likely to withstand the increased cGMP turn-over. This study gains insight into the complex in vivo regulation of IMPDH1 to maintain GTP homeostasis with changing illumination, central to rod dark/light physiology and relevant to understand 159 or Ser160 with a 50:50 probability . This numbering correlates with the canonical human IMPDH1β. The peak areas of the precursor ions for the monophosphorylated peptide 154–169 in 3 dark and 3 light biological replicates revealed a light preference of the observed phosphorylation event (light/dark log2 fold change = 1.67 with p=0.03). The light/dark log2 fold change of phosphopeptides representative of well characterized light- or dark-dependent phosphorylation events in rhodopsin  revealed three phosphorylation sites in IMPDH1 . One phohodopsin , phosduchodopsin  and GRK1hodopsin  is shown159 and Ser160 map within the CBS motif 1 of the Bateman domain, in close proximity to Asn198, Arg224 and Asp226 mutated in adLCA or adRP10 . This residue maps at the mobile flap in the catalytic domain . Ser477 maps to a region that has not been involved in the catalytic process. Remarkably, all the phosphorylated residues are conserved in mammalian IMPDHs . This antibody immunoprecipitated all IMPDH1 isoforms in the murine retina , To obtain insight into the predominant phosphorylated species in dark and light, immunoprecipitated IMPDH1 from 32P atoms. Therefore, a similar intensity of the mono- and di-phosphorylated IMPDH1 bands actually reflects that the di-phosphorylated form is half as abundant as the mono-phosphorylated form.The same species IMPDH1-0P, −1P and −2P were observed by IEF separation of retinal homogenates from living mice that were either dark-adapted or exposed to 2000 lux light for 5, 20 and 60 m. Monophosphorylated forms are the predominant species in both dark and light conditions . Please Measured isoelectric points (IP) of IMPDH1-603/604aa-0P, −1P and −2P correlated well with IP predicted values, Taken together, our results show that the most abundant isoform of IMPDH1 in the retina, which is a retina-specific spliced form, is phosphorylated to a high extent in vivo. Nearly two thirds of the protein are phosphorylated in vivo, with mono- and di-phosphorylated forms being present but monophosphorylated species predominating under dark or physiological light conditions.m or Vmax of the enzyme. However, the S416D-hIMPDH1α (546aa) mutant showed a slower reaction  and IMPDH1γ (595aa) . In ordereaction . This rereaction .416 phosphorylation by measuring the effect on kinetics and IMP dependence of the S416D substitution in the canonical and in both prevalent hIMPDH1 retinal isoforms: hIMPDH1α (546aa) and hIMPDH1γ (595aa) .We confirmed the effect of Ser (595aa) . Figure It has recently been established that GDP and GTP allosterically inhibit the catalytic activity of eukaryotic IMPDH enzymes in vitro . Therebymax values (Vmaxapp in the presence of 2 mM GTP or GDP divided by Vmax in the absence of nucleotide) for all the hIMPDH1β (514aa) phosphomimetic mutants as well as for the wild-type IMPDH1 enzyme. As expected, phosphorylation at Thr159/Ser160 had an obvious effect on the allosteric inhibition of IMPDH1 in vitro  in dark/light retinas by high pressure liquid chromatography analysis using murine retinas obtained from dark-adapted mice, and kept in situ for 5 m in darkness or exposed to bright light (2000 lux light). It must be noted that although our nucleotide determinations were done in whole retinal extracts, guanine nucleotide levels largely reflect nucleotide content in photoreceptor cells, as cGMP and GTP levels are reduced to less than 20% their values in retinas that lack the photoreceptor cell layer , following the intravitreal injection of a stable isotope form of labeled glycine. Because the amino acid glycine contributes carbon and nitrogen atoms to the scaffold on which the purine ring assembles, labeled glycine allowed us to analyze whether the incorporation of labeled Gly atoms into IMP, AMP and GMP increased with light in vivo.The nucleotide determination by HPLC showed that GMP and GDP levels increased with 4 hr of light exposure, while GTP levels were maintained ,Table 2.13C2;15N-Glycine was injected intravitreally in dark-adapted mice. Mice were then kept in the dark for 4 hr, or exposed to 1600 lux light for 4 hr. Whole retinal extracts were analyzed by mass spectrometry, to determine the levels of labeled and total cGMP, IMP, AMP, GMP, ATP and GTP, see Methods for experimental details.For the metabolic flux analysis in vivo, Taken together, our results show that the global flux through the de novo synthesis of purine nucleotides increases with light. This increased flux could serve to maintain the ATP and GTP pools as their consumption increases in the phototransduction process.Two processes that increase GTP consumption upon light exposure in photoreceptor cells would be GTP hydrolysis by the GTPase transducin at the activation step, and GTP conversion to cGMP synthesis during recovery of the light response .To assess whether IMPDH1 catalytic activity was required to sustain the light response or rod mass response recovery, we recorded electroretinogram (ERG) responses simultaneously from both eyes of mice that were injected intravitreally with an IMPDH1 inhibitor (right eye) or control physiological saline buffer (left eye). The result of transiently inhibiting IMPDH activity on the rate of mass rod recovery after a saturating flash was tested using a paired-flash ERG paradigm , and is 2] in the high intensity range of mixed responses  contributes to mass rod recovery by sustaining the GTP pool upon light exposure.G/S160G/S416G/S477G) or with individual phosphorylation sites restored in the 4KO  were purified to perform in vitro phosphorylation assays with the kinases PKA, PKCα, PKG, CaCM-Kinase, CK II, PKB-Akt and AMPK. 159 and Ser160 residues. No other kinase among the kinases tested showed specificity towards Thr159/Ser160; or towards Ser416 or Ser477 residues.In order to identify the kinase/s responsible for the phosphorylation of the described residues of IMPDH1, the human IMPDH1α (546aa) isoform, and mutant forms with all of these 4 residues mutated to Gly  isoform carrying individual mutations associated to RP10  or LCA , and used them as substrates for phosphorylation reactions with PKCα in vitro , to direct IMP towards ATP synthesis. In this respect, S416 maps at a region that resembles a consensus site for AMPK , althougActually, isoelectrofocusing separation of retinal extracts from dark-adapted or light-adapted living mice revealed that >60% of the protein is phosphorylated under physiological conditions; with the predominant monophosphorylated form corresponding to phosphorylated T159/S160 in the light state and S477 in the dark state (based on the mass spectrometry data); and di-phosphorylated forms likely reflecting slow dephosphorylation kinetics of the reciprocal site.S477D substitution did not have a direct effect on enzymatic activity neither on GTP allosteric regulation of the enzyme. Thereby, we speculate that S477 phosphorylation might be involved in other aspects of enzyme regulation in vivo: for example inducing filament disassembly. To this respect, a recently reported structure of IMPDH2 polymers has revealed details of the interface that mediates cytoophidia assembly: the 12 amino-terminal residues of the canonical IMPDH2 isoform extend from the catalytic domain to bind into the adjacent molecule, in a shallow surface groove formed by a short helix (476-485), two beta strands (51-63), and two short loops , . Strikin1/2 for GDP/GTP inhibitory allosteric regulation of the enzyme in vitro, suggesting that light-dependent phosphorylation of these residues in vivo would effectively desensitize the enzyme to GDP/GTP allosteric control and promote enzyme activation.We here demonstrate that the T159D and S160D (or T159E and S160E) substitutions increased about 5-fold the KLight-dependent activation of IMPDH1 might seem at odds with the assumption in the field that GMP, generated from cGMP hydrolysis during light exposure, would act as a negative feedback regulator of purine nucleotide synthesis by inhibiting IMPDH1, 5-phosphoribosyl 1-pyrophosphate synthetase (PRS), and the first enzyme of the purinosome Glutamine phosphoribosyl amidotransferase (GPA). However, by performing an in vivo metabolic flux analysis of purine nucleotide synthesis in dark/light conditions following an intravitreal injection of labeled glycine, we found that the purinosome activity was substantially increased with light, with IMP levels nearly doubling in light conditions . We founConsidering these results, we believe that most of the GMP increase observed upon light exposure by HPLC actually reflects the light-driven overall increase in de novo purine nucleotide synthesis, rather than the GMP coming from cGMP hydrolysis alone. It is likely that the fraction of GMP coming from the hydrolysis of cGMP would be under detection by HPLC, given that cGMP itself is under detection by HPLC in retinal extracts. cGMP levels in retinal extracts have been classically determined by radioimmunoassay .By aligning our results with recent IMPDH structure/function studies , we propIn the dark, IMPDH1 would be sensitive to GDP/GTP inhibitory allosteric control , like in Du et al. study  and thisAccording to our model it might seem surprising that Impdh1-/- mice display only a slowly progressive retinal degeneration. However, despite maintenance of the retinal structure, these mice present gradually diminished ERG responses from normal at 5 m to nearly extinguished at 13 m of age . FurtherWe here demonstrate that the light-dependent phosphorylated residues T159 and S160 in retinal hIMPDH1 were selective in vitro substrates for PKCα kinase; but not for the other kinases tested . We also show that treatment of retinas with the PKC inhibitor bisindolylmaleimide in situ led to a noticeable decrease of GTP retinal levels upon light exposure ; and itsOur proposed model that GDP/GTP allosteric regulation of IMPDH1 is controlled by phosphorylation in vivo to adapt GTP synthesis to the illumination conditions supports our previous hypothesis that IMPDH1 mutations would cause the pathology by resulting in higher than normal IMPDH1 activity; and/or by forming irreversible filaments. Abnormally high IMPDH1 activity could conceivably result in abnormally high cGMP synthesis, which is one of the well-known causes of photoreceptor cell damage ; or coulIMPDH1 mutations in adRP10 and adLCA, including the mutations N198K, R224P and D226N analyzed in this study, desensitize the enzyme to GDP/GTP allosteric inhibition  of Catalonia; and approved by the ethics committee on animal experiments of the University of Barcelona .3, 112.5 mM NaCl, 3.6 mM KCl, 2.4 mM MgCl2, 1.2 mM CaCl2, 0.1 mM EDTA and 10 mM glucose pH7.4) for 1 hr of dark-adaptation. Retinas were then excised under dim red light. Three retinas were kept for 5 m at room temperature in the dark; while three retinas were exposed to light of 3000 lux for 5 m. Following dark/light exposure retinas were homogenized in homogenization buffer (HB) . Retinal homogenates were kept at −80°C until further analysis. Subsequently, a basic fractionation protocol was performed to obtain fractions corresponding to soluble, peripheral and membrane proteins from each sample.Fresh calf eyes were obtained immediately postmortem and processed at the slaughterhouse premises. Upon collection, eyes were processed in a dark room by excising the cornea and submerging eyecups in chilled oxygenated Locke’s buffer  or Filter Aided Sample Prep (FASP) digestion  membran. SamplesPhosphopeptide mixes were analyzed in an Orbitrap Fusion Lumos mass spectrometer  coupled to an EasyLC (Thermo Scientific (Proxeon), Odense, Denmark). Peptides were directly loaded onto the analytical column and separated by reversed-phase chromatography using a 50 cm column with an inner diameter of 75 μm, packed with 2 μm C18 particles spectrometer . Chromatographic gradients started at 95% buffer A and 5% buffer B with a flow rate of 300 nl/min and gradually increased to 22% buffer B in 79 m and then to 35% buffer B in 11 m. After each analysis, the column was washed for 10 m with 5% buffer A and 95% buffer B. Buffer A: 0.1% formic acid in water. Buffer B: 0.1% formic acid in acetonitrile.The mass spectrometer was operated in DDA mode and full MS scans with 1 micro scans at resolution of 120.000 were used over a mass range of m/z 350–1500 with detection in the Orbitrap. Auto gain control (AGC) was set to 2E5 and dynamic exclusion to 60 s. In each cycle of DDA analysis, following each survey scan Top Speed ions with charged 2 to 7 above a threshold ion count of 1e4 were selected for fragmentation at normalized collision energy of 28%. Fragment ion spectra produced via high-energy collision dissociation (HCD) were acquired in the Ion Trap, AGC was set to 3e4, isolation window of 1.6 m/z and a maximum injection time of 40 ms was used. All data were acquired with Xcalibur software v3.0.63.Proteome Discoverer software suite  and the Mascot search engine  protein was amplified from total RNA from fresh bovine retinas and cloned into pET15b bacterial expression plasmid  for expression in E. coli BL21 (DE3) cells. The expression of recombinant protein was induced in bacterial cultures at OD600 = 0.6 for 5 hr at 37°C. Cells were then collected and bacterial inclusion bodies were obtained and solubilized in 6M guanidinium hydrochloride buffer as previously described . Bovine escribed . bIMPDH1Purified bIMPDH1(514aa).His was used to immunize two New Zealand White rabbits following a standard 84 day protocol: initial injection of protein (0.5 mg) emulsified in Freund’s complete adjuvant, and three boosts of protein (0.25 mg) in incomplete adjuvant at three-week intervals. At exsanguination, blood serum was affinity purified with bIMPDH1-crosslinked Aminolink coupling resin ; dialyzed against 0.1M sodium phosphate, 0.15M NaCl, pH 7.2, and concentrated with 10K Amicon devices.3, 112.5 mM NaCl, 3.6 mM KCl, 2.4 mM MgCl2, 1.2 mM CaCl2, 0.1 mM EDTA, 10 mM glucose, sodium succinate, sodium glutamate, vitamin and amino acid supplement, pH7.4] containing 1mCi/ml 32P-orthophosphoric acid . Retinas were incubated for 90 m at 37°C in a 5% CO2 incubator in the dark, to allow incorporation of the 32P radionuclide into the ATP pool of retinal cells. One multiwell dish was then placed over the bench at room temperature for 5 m in darkness (dark retinas); and the other was exposed to 2000 lux white light (light retinas) for 5 m. Pools of two retinas were homogenized in HB. Samples were then centrifuged at 14.000 rpm for 20 m, 4°C, to obtain supernatant and pellet fractions. Pellets were resuspended in HB with 1% TritonX100. Immunoprecipitation of IMPDH1 was carried out with 10 μg of anti-IMPDH1 pAb and 50 μl of Dynabeads-protein G . Samples were resolved by SDS-PAGE and transferred to nitrocellulose membranes, that were directly exposed to an X-ray film  and subsequently immunoblotted for IMPDH1 . An immunoprecipitation control was carried with an anti-IgG isotype control.Eight six-week-old C57BL6 mice were dark-adapted overnight. Retinas were dissected under dim red light using a night vision dissecting scope, and placed in individual wells of two 12-well dishes –eight retinas per dish- in 600 μl of Locke’s buffer . Samples were loaded into prehydrated pH-gradient IEF gel strips , and run in an Ettan IPGphor3 IEF system . Gel strips were incubated for 10 m in transfer buffer  and proteins transferred to nitrocellulose membranes by capillary action. Membranes were first exposed to an X-ray film and subsequently immunoblotted for IMPDH1 as indicated above.For isoelectric focusing analysis of IMPDH1 from retinas of living mice after dark/light adaptation, mice were dark-adapted for 16 hr and exposed to 2000 lux fluorescent light after pupil dilation for 5, 20 or 60 m. Retinas were dissected and homogenized in HB. Retinal homogenates were centrifuged at 13000 rpm for 20 m 4°C and supernatant fractions were kept. Protein was precipitated with 3 volumes of ice-chilled TCA-acetone buffer , and samples were cooled at −20°C for 16 hr, and centrifuged at 13200 rpm for 1 hr at 4°C. Protein pellets were washed in acetone, air-dried, resuspended in IEF buffer and resolved by IEF as described.Human IMPDH1 (1–514) ORF was obtained from a cDNA library and cloned into an in house modified pET15b expression vector . A PCR-bPhosphomimetic mutants for enzymatic analysis were obtained by introducing mutations that created individual substitutions T159D, S160D, S416D, S477D into the pETEV15b_HsIMPDH1(546) vector; or in the pETEV15b_HsIMPDH1(514), using the QuickChange II Site-directed mutagenesis Kit (Agilent Technologies).Phospho-knockout mutants for in vitro phosphorylation assays were obtained by introducing mutations that created triple substitutions that left only one phosphorylation site S160G/S416G/S477G (T159-only); T159G/S416G/S477G (S160-only); T159G/S160G/S477G (S416-only) and T159G/S160G/S416G (S477-only) into the pETEV15b_HsIMPDH1(546). A mutant was also generated with the 4 phosphorylation sites mutated to Gly (4KO mutant).Blindness associated mutations R105W, N198K, R224P, D226N, V268I and H372P were introduced by site-directed mutagenesis in the pETEV15b_HsIMPDH1(546) vector.Site-directed mutagenesis was confirmed by sequencing both DNA strands in all generated constructs.Escherichia coli strain BL21 (DE3) and purified by nickel-chelating affinity chromatography as described before  as a function of the nucleotide concentration. Normalized Vmax values were calculated by dividing the Vmaxapp value in the presence of GTP/GDP nucleotides by Vmax in the absence of nucleotides.IMPDH activity was studied by monitoring the increase in absorbance at 340 nm upon reduction of NADescribed . The exp2; 0.05 mM phorbol 12-myristate 13-acetate (PMA); 0.2 mM CaCl2; 0.2 mM ATP]; 1 μl of 32P-ATPγ 3000 Ci/mmol  and 1 μl of recombinant hPKCα . Samples were incubated for 15 m at 30°C, and reactions were stopped by addition of 4x SDS loading buffer. Sample fractions corresponding to 2.5 μg of HsIMPDH1(546) were resolved by 12% SDS-PAGE, and proteins transferred to 0.2 μm nitrocellulose membranes (Bio-rad). The membranes were first exposed to X-ray films  for 2 hr; and subsequently immunoblotted for IMPDH1. Membranes were scanned at an Odyssey Scan System (LI-COR) and quantified with the Fiji (ImageJ) software.For in vitro phosphorylation assays with PKC, 5 μg of recombinant protein HsIMPDH1(546) or its mutants were diluted in 12 μl of dilution buffer , mixed with an equal volume of 2x kinase buffer  in phosphate saline buffer. The injection was performed under dim red light. Mice were kept in darkness, or immediately transferred to a bright light setting (1600 lux white light) for 4 hr. Mouse recovery from anesthesia in the dark or light setting was promoted by keeping the mouse body temperature with a heating pad. After 4 hr, retinas were collected, frozen in dry ice and kept at −80°C until mass spectrometry analysis.A group of 5 week-old C57Bl mice was dark-adapted overnight. They were anesthesized with an intraperitoneal injection of ketamine  and xylazine  in saline solution (NaCl 0.9%), and injected intravitreally with 1 μl of a 20 mM solution of Metabolites were extracted from the retina by vortexing retinas in cold acetonitrile: methanol: water (5:4:1 v:v:v) in 1.5 ml tubes protected from light. After vortex, samples were subjected to four cycles of freezing/thawing by immersion in liquid nitrogen and sonication. Samples were kept in ice for 1 hr and then clarified by centrifugation .13C2; 15N-Glycine into purine nucleotides is in line with the reported range of incorporation  in other tissues  with electrospray ion source (LC-ESI-QqQ) working in positive mode. 10 µl of extract were injected to the liquid chromatographic system. Metabolites were separated using an InfinityLab Poroshell 120 HILIC-Z, 2.1 × 100 mm, 2.7 μm (PEEK lined) column (Agilent Technologies). Mobile phase A was water with 50 mM ammonium acetate and 5 μM medronic acid, and mobile phase B was acetonitrile. Separation of the samples was conducted under the following gradient: 0–0.5 min 80% of B; 0.5–7.5 min decrease to 70% of B; 7.5–8.5 min decrease to 50% of B; 50% B was maintained for 30 s; 9–9.5 min raise to 80% of B; 9.5–11 min 80% of B. The flow was 0.7 ml/min. The mass spectrometer parameters were: drying and sheath gas temperatures 270°C and 400°C, respectively; source and sheath gas flows 12 and 12 l/min, respectively; nebulizer flow 20 psi; capillary voltage 3000V; nozzle voltage 1500V; and iFunnel HRF and LRF 70 and 80V, respectively. QqQ worked in multiple reaction monitoring (MRM) mode using two transitions (quantitation and confirmation transition) for each compound. The transitions for unlabelled metabolites and the collision energy (CE(V)) were: AMP 348→136(16), 348→119(56); ATP 508→136(32), 508→410(12); GMP 364→152(8), 364→135(52); GDP 444→152(32), 444→135(60); GTP 524→152(32), 524→135(56); cGMP 346→152(12), 346→135(54); IMP 349→137(16), 349→110(60); Gly 76→30(8),76→58(20). For labelled metabolites were: AMP 351→139(16), 351→122(56); ATP 511→139(32), 511→413(12); GMP 367→155(8), 367→138(52); GDP 447→155(32), 447→138(60); GTP 527→155(32), 527→138(56); cGMP 349→155(12), 349→138(54); IMP 352→140(16), 352→113(60); Gly 79→32(8), 79→61(20), as stated in  tissues , given tA group of four mice was used for benzamide (BZM) injection, and a group of 7 mice for mycophenolate mofetyl (MMF) injection. A group of four mice was used for injection of bisindolylmaleimide PKC inhibitor. Dark-adapted (>12 hr) animals were anaesthetized with an intraperitoneal injection of ketamine  and xylazine  in saline solution (NaCl 0.9%) and pupils were dilated with one drop of 1% tropicamide. For each mouse the right eye was injected with the drug of interest, and the left eye with the physiological saline buffer used for drug dilution. MMF was injected intravitreously to an effective concentration of 200 nM (1 μl injection of 1 μM stock); benzamide riboside was injected to an effective concentration of 80 μM (1 μl injection of 400 μM stock); and bisindolylmaleimide was injected intravitreously to an effective concentration of 250 nM .2) followed by a probe flash of identical intensity at increasing inter-stimulus (IST) intervals: 400, 600, 800, 1200, 1500 and 2000 ms (double flash). The rate of recovery for the a- and b-waves at each IST was obtained by calculating the ratio of a- or b-wave of the probe flash to the a- or b-wave of the test flash. Cone responses were recorded following 5 m of light-adaptation with background white light (50 Cd/m2) to a flash light of 3 Cd·s/m2. Recorded electrophysiological responses were amplified; filtered , and digitalized . The recording process was controlled with Scope version 3.8.1 software . The stimulation protocols were designed according to the International Society for Clinical Electrophysiology of Vision.Two corneal electrodes were used to record simultaneous ERGs from both eyes . Electrodes were placed in the visual axis 2–3 mm from the cornea, with a drop of 2% methyl-cellulose  between corneas and electrodes. Mice were maintained for >10 m in absolute darkness before the recordings, initiated 20 m after drug injection. Mouse temperature during the recording was maintained at 37°C with a heating pad . Full-field flash ERG was performed, with the retina illuminated with a Ganzfeld dome. To test the effect of the drug under study on rod mass recovery after a saturating light flash, we applied the paired-flash paradigm , by trigFor the analysis of spicula formation in response to mycophenolic acid (MPA) treatment, retinas from 16 hr dark-adapted mice were dissected under dim red light and placed in DMEM-high glucose in the presence or absence of 10 μM MPA, protected from light in a 5% CO2 incubator for 1 or 2 hr. Retinas were then fixed and embedded in acrylamide as described . CryosecFor analysis of the effect of living mice exposure to constant bright light, 16 hr dark-adapted mice were exposed to 1600 lux bright light for 20 m, 2 hr or 4 hr after pupil dilation with a drop of phenylephrine and tropicamide. Retinas were processed and IMPDH1 immunolocalization assays were performed as indicated above.For 15d constant-dark or 15d constant-light rearing, 4wk-old mice were either housed in a dark cabinet or in a set of constant bright light exposure (1600 lux) for 15 days. Mice were sacrificed, retinas were processed and IMPDH1 immunolocalization was analyzed.Confocal microscopy images were acquired with a confocal laser scanning microscope Leica SP5 equipped with two HyD detectors. The spectral bands were set to 491–546 nm and 566–663 nm for 488 nm and 555 nm, respectively. The used objective was a HC PL APO 100x/1.4NA Oil and the samples were mounted on type 1.5 coverglasses (170 mm thick). The stacks covered approximately 2 μm in the z-axis with a 0.13 μm step size. The settings of the image acquisition provided a pixel size of about 74.8 nm x 74.8 nm.The image analysis of the immunofluorescence data was performed using Fiji (ImageJ). In order to maximize the signal to noise ratio, we used the average projection of groups of 3 planes from large z-stacks. For the particle quantification, we selected a region of interest (e.g. magnified frames of rod outer segment layer) and we applied a light Gaussian filter (radius of one pixel). To determine the size of the areas with concentration of fluorescence we selected the pixels from the image that had a signal beyond a certain value. This value was determined from the statistics of intensity, taking only the 8% of the brighter pixels. Then, the areas of the spots were analyzed by using the plugin ‘analyze particles’ from Fiji. Counted spots were plotted according to its size and the statistical analysis were performed with Graph Pad Prism (unpaired t-tests). In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Photoreceptors in the vertebrate retina rely upon cGMP to gate a cation channel, causing it to be open in the dark. The phototransduction pathway results in the destruction of cGMP, and thus channel closure, resulting in hyperpolarization and a reduction in the release of glutamate. The production of GMP, GDP, and GTP is via the enzyme, IMPDH1. The importance of this enzyme to vision is illustrated by the effects of mutant alleles in humans, where retinitis pigmentosa, or loss of night vision followed by loss of daylight vision, occurs with changes at specific amino acids. The regulation of the activity of this enzyme is thus of interest and has been shown to be at least in part regulated by negative, allosteric feedback by GDP/GTP. The study by Plana-Bonamaiso et al. reports that a PKC-dependent phosphorylation event that is regulated by light relieves the feedback inhibition. This mechanism is part of a regulatory pathway that help to keep the levels of cGMP high during light exposure. The data are novel and are part of an emerging story of the regulation of IMPDH enzymes, which are regulated in several ways, including by formation of larger structures, or cytoophidia, and now phosphorylation, and allostery.Decision letter after peer review:[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]eLife. Your article has been reviewed by a Senior Editor, a Reviewing Editor, and three reviewers. The following individuals involved in review of your submission have agreed to reveal their identity: Robert Molday (Reviewer #2).Thank you for submitting your work entitled "Phosphorylation-controlled GDP/GTP allosteric regulation sets IMPDH1 activity in the retina." for consideration by eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife for future submissions.Three experts carefully reviewed the manuscript. While the reviewers found the work interesting, the number of substantive questions raised was such that we feel we must reject it. We hope that the reviewers' comments will be useful to you. We apologize for not being able to deliver better news, and we hope that you will continue to consider The reviewers' comments are appended.Reviewer #1:The authors have discovered three phosphorylation sites in retinal IMPDH1: (1) S416 phosphorylation changes catalytic activity directly but is light independent; (2) S477 accumulates in the dark and has no apparent effect on function. (3) T159/S160 changes allosteric regulation by GDP/GTP and increases in the light. Poor quality figures and missing experimental details make it difficult to assess these findings. Assuming these issues are oversights, these findings are interesting and significant new insights into the pathological mechanism of IMPDH1 mutations that cause retinitis pigmentosa. The authors also show that IMPDH forms higher order structures in rod cells in response to light and inhibitors. The authors propose an intriguing model for the regulation of IMPDH activity by phosphorylation: light induced phosphorylation of T159/S160 relieves GDP/GTP inhibition, activating IMPDH1 to replenish the guanine nucleotide pool. Unfortunately, the supporting evidence is weak. The paper is also marred by shifting nomenclature and overstatements.1) The phosphorylation experiments are not consistent: the mass spec experiments suggest that T159/S160 phosphorylation increases with light exposure, but other experiments show that the overall phosphorylation status does not change. This conundrum should be resolved with additional experiments to monitor the phosphorylation status of individual sites. .2) The critical panel B of Figure 2 is illegible and the colors are not explained.3) The retinal guanine nucleotide levels do not appear to change significantly with light. Perhaps this is because the authors are looking at the entire retina, which contains other kinds of cells in addition to photoreceptor cells. Of course, this also means that the phosphorylated IMPDH1 can be in the other cells.max/Vmaxapp? It is not defined in the text or figure legend.4) Important details are missing in the allosteric regulation experiments – what exactly is V1/2 were more complex sigmoidal inhibition is observed. Note that the text sometimes uses the term Ki for all, other times uses IC50. The nomenclature should be consistent. The title should reflect that both kinds of measurements are reported, as should the column headings. Which substrates were varied, which were held constant? Are the units really mM? The values of Ki for GMP and XMP are 1-2 orders of magnitude greater than expected. The data are not provided. The authors need to report the concentration of NAD and enzyme in the experiment, as well as the temperature and buffer, in the figure and table legends.5) Table 1 appears to report a mixture of different constants – some are Ki determined for competitive inhibitors and some are K6) The phosphomimetic mutations at T159/S160 do have a significant effect on allosteric inhibition, but "abolish" is too strong a term – it is just a 5-fold increase. How exactly is the IC50 for GTP determined? The sigmoidal dependence on GTP concentration is very steep – what is the value of the Hill coefficient? What equation was used?7) Why does Figure 8 report nucleotide levels in picomole/retina? Isn't picomole/mg protein the better measure? Also, how much of the nucleotides come from photoreceptor cells as opposed to the other cells in the retina?8) In vitro kinase assays are of questionable physiological relevance.9) Is the S477 phosphorylation likely to affect formation of rods and rings?Reviewer #2:This manuscript aims to elucidate the mechanisms that regulate IMPDH1, an enzyme that catalyzes the rate-limiting step in de novo synthesis of guanine nucleotides, in the retina. Using a range of proteomic, biochemical, physiological and imaging techniques, they show that IMPDH1 isoform in the retina is regulated by light-dependent phosphorylation of T159/S160 residues in the Bateman domain by PKC. This abolishes the inhibition of IMPDH1 by GDP/GTP enabling GTP levels to be maintained during the light response.This is an interesting study that provides new insight into the allosteric regulation of the enzyme IMPDH1 in the retina. IMPDH1 plays a central role in maintaining guanine nucleotides, key components of the visual response in photoreceptors. The importance of IMPDH1 is highlighted by the finding that mutations in IMPDH1 are known to cause severe retinal degenerative diseases including retinitis pigmentosa (RP10) and Lebers Congenital Amaurosis. Accordingly, insight into the regulatory mechanisms underlying IMPDH1 activity is important for understanding the visual response and molecular mechanisms underlying retinal degenerative diseases. The studies are well done with sufficient statistical analysis and controls. They incorporate both in vitro and in vivo experiments to examine the role of phosphorylation in controlling the GDP/GTP regulation of IMPDH1 activity and provide support for importance of the GDP/GTP allosteric regulation of IMPDH1 as the principle mode of in modulation of this enzyme as earlier proposed by Buey et al. The experiments and analysis also argue against the competitive inhibition by GMP as a primary mode of regulation of IMPDH1. As part of this study, the authors have also determined the effect of several disease-linked mutations on the PKC catalyzed phosphorylation of IMPDH1 at the T159/S160 sites and showed that two mutants (N198K and R224P) have statistically reduced phosphorylation. Although these studies are incomplete since enzymatic activities were not examined, nonetheless they serve as a basis for a more thorough analysis of how these mutations may affect the control of guanine nucleotide levels and lead to disease.Essential revisions:Further discussion on the following points would enhance this manuscript.It is unclear what the underlying mechanism is for light dependent phosphorylation of IMPDH1 at the T159/S160 residues. The authors nicely show that PKC is involved in the phosphorylation. Is PKC activated directly or indirectly in the light or does IMPDH1 undergo a conformational change in the light to enable phosphorylation by PKC? Discussion or speculation on this point would be useful.The mechanism appears to be mainly involved in controlling guanine nucleotide levels in rods. Is a similar mechanism proposed for cones?The authors show that the N198K and R224P disease-causing mutations display a decreased level of phosphorylation . Do these mutations affect GDP/GTP interaction and/or the enzymatic activity of IMPDH1 or just its phosphorylation by PKC? Further discussion would be useful.Reviewer #3:IMPDH1 mutations cause retinal degeneration but its mechanisms and regulation are still poorly understood. Plana-Bonamaiso et al. identified four phosphorylation sites, T159/S160, S416 and S477 and found T159/S160 phosphorylation was light-sensitive. Additionally, they measured the activities of phosphorylation-mimic mutants in response to GTP and GDP and identified the upstream kinase for T159/S160. By using ERGs and immunofluorescence with or without IMPDH1 inhibitor in live mice, they conclude that phosphorylation controls GDP/GTP allosteric regulation of IMPDH activity in response to light.Essential revisions:1) Light-regulated phosphorylation of T159/S160 is not solid. The difference between light vs dark in Figure 2 is from bovine retinas. The splicing isoforms of IMPDH1 in bovine retina are still unknown although the phosphorylation sites are conservative for known isoforms. As the authors pointed out in the discussion, they were also under nutritional deficit. Additionally, Figure 3C in comparing phosphorylation in mouse isoform is not statistically significant. It will be important to know whether T159/S160 is the major site of phosphorylation in 603/604 aa IMPDH1. It should be easily done by proteomics or PKC inhibitor.2) The GMP is significantly increased but GTP/GDP remains unchanged by light. However, the authors proposed that GTP/GDP but not GMP regulates IMPDH1 by light. One argument is that Ki of GMP is much higher (1.94 mM) for hIMPDH1-514 than physiological mouse retina. HIMPDH1-514 is not the major isoform in the human retina. (PMID: 16936083) (546 and 595 are the major ones). It is unknown whether hIMPDH1-514 is phosphorylated in vivo . Furthermore, Ki of GMP varies in big ranges with different IMPDH1 proteins (PMID: 26558346). The second argument is GTP/GMP ratio (GTP is constant but GMP is increased) is more important than GMP in regulating IMPDH1. Is there data that support this hypothesis?3) The author proposed that light activates IMPDH activity through phosphorylation. Using PKC inhibitor to block the phosphorylation will give a strong evidence. Will the IMDPH1 inhibitor affect GTP, GMP and IMP levels?4) In the Discussion section "IMPDH1 mutations would cause the pathology by resulting in higher than normal constitutive IMPDH1 activity… Abnormally high IMPDH1 activity could conceivably result in abnormally high cGMP synthesis.,Consequently, every clue would lead to unregulated, constitutive IMPDH1 activity as the underlying cause of the adRP10 phenotype." How would the author explain the discrepancy by other reports: (1) IMPDH1 human mutants have similar IMPDH activity including D226N (PMID: 1829559); (2) Supplement with GTP improves ERGs from IMPDH1 -/- mice (PMID: 19822744). [Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]Reviewer #1:[…]1) The phosphorylation experiments are not consistent: the mass spec experiments suggest that T159/S160 phosphorylation increases with light exposure, but other experiments show that the overall phosphorylation status does not change.32P incorporation into endogenous ATP. The protein is substantially phosphorylated both in dark and light conditions. This is consistent with mass spectrometry results, that revealed that there is one site that is phosphorylated preferentially in darkness, while another is preferentially phosphorylated in light. Therefore, there is 32P incorporation at IMPDH1 both in dark and light conditions.The overall phosphorylation status does not change when assayed by in situ metabolic labeling with What we wanted to assess by in situ metabolic labeling is that the phosphorylation events detected by LC-MS/MS were happening to a high extent in vivo, to an extent that they would exert an overall effect on the protein. The in situ metabolic labeling revealed something we could not extract from the mass spectrometry result: that >60% of the protein is phosphorylated at any light condition. The electrofocusing experiments confirmed that two sites are phosphorylated.This conundrum should be resolved with additional experiments to monitor the phosphorylation status of individual sites.The phosphorylation status of individual sites has been monitored by LC-MS/MS, on dark/light bovine retinas – biological triplicates – , and the statistical results are provided.The analysis of phosphopeptides by mass spectrometry typically require large amounts of starting material, because the process entails a step of phosphopeptide enrichment (phosphopeptides are much harder to ionize and therefore harder to detect than peptides by mass spectrometry). We used about 0,75 mg total protein in each homogenate (18 samples) in the phosphoproteomic analysis. Because this corresponded to fractionated tissue, we estimate that we would have needed pools of at least 6 murine retinas per sample . That is one of the reasons why we performed the phosphoproteomic analysis with bovine retinas. The cost of the LC-MS/MS analysis was over 10,000€, which is why it is not straight-forward to repeat it now in the murine system, with a number of samples that would allow us to perform robust statistics again.Previous to the phosphoproteomic analysis we had performed IMPDH1 immunoprecipitation in dark/light bovine samples, followed by LC-MS/MS. We had detected peptide A0JNA3[154-169] LVGIVTSRDIDFLAEK with a phosphate assigned to either Thr159 or Ser160 (50:50 probability) in the light sample; and the same peptide unphosphorylated in the dark sample. However, this simple experiment allows the detection of the phosphopeptide, but not a quantitative comparison between samples.Another attempt at doing quantitative phosphoproteomic analysis in dark/light bovine retinas by iTRAQ  and LC-MS/MS failed to reach the depth of analysis required for IMPDH1 detection. These type of phosphoproteomic analysis are not simple, and are very expensive.In brief, we have demonstrated by in situ metabolic labeling and isoelectrofocusing experiments that the major isoform of IMPDH1 in the murine retina (603/604aa) is phosphorylated at two sites. We have based the identity of those two sites on the mass spectrometry analysis performed with bovine retinas. This is not uncommon in the field. Rhodopsin phosphorylation sites were originally characterized in the bovine system, and were assumed and later shown to be conserved in the murine system. The same happened with phosducin phosphorylation, and with practically any well-established phosphorylation event in signaling proteins in response to dark/light.Besides, we are providing a supplementary figure that shows that we detected previously reported, well-established phosphorylation sites in rhodopsin, rhodopsin kinase and phosducin with the proper dark/light-preference in our phosphoproteomic analysis..The statement “monophosphorylated species predominate” is true and accurate in vivo, at any light condition. It is obvious in Figure 3E (arrows at the left added for clarity). Monophosphorylated species (1P arrow) in retinal extracts obtained from living mice are much more abundant than di-phosphorylated species (2P arrow) in any lane.32P atoms, and that is why it reflects twice its abundance. This is now explained in Results text, and in the figure legend. Arrows have been introduced in the figure, to make this clear.In Figure 3D, autoradiograph, the di-phosphorylated form of the protein (2P) has incorporated 2 It is true that IP values are not accurate to 4 significant figures. Corrected.2) The critical panel B of Figure 2 is illegible and the colors are not explained.This oversight has been corrected.3) The retinal guanine nucleotide levels do not appear to change significantly with light. Perhaps this is because the authors are looking at the entire retina, which contains other kinds of cells in addition to photoreceptor cells. Of course, this also means that the phosphorylated IMPDH1 can be in the other cells.In this new manuscript retinal guanine nucleotide levels are provided from extracts from whole retinas obtained from living mice that were dark- or bright light-adapted, determined by HPLC or by LC-MS/MS. We have consistently observed that GMP increases with light, while GTP levels are maintained. We have also observed that AMP levels increase with light while ATP levels are maintained.It must be noted that although our nucleotide determinations were done in whole retinal extracts, guanine nucleotide levels largely reflect nucleotide content in photoreceptor cells, as cGMP and GTP levels are reduced to less than 20% their values in retinas that lack the photoreceptor cell layer [rd1 mouse model ]. This is now mentioned in this Results section.There are numerous studies since the 70s that have determined guanine nucleotides levels in whole retinas, in rd1 mice lacking the photoreceptor cell layer ; and in the different retinal layers obtained by tangential sectioning . It is well established that cGMP and guanine nucleotide levels are much higher in the photoreceptor cell layer than at inner retinal layers.IMPDH1 is expressed to a much higher extent in the photoreceptor cell layer of the retina than at the inner retina. If the gain at the confocal microscope is adjusted to avoid saturation of the IMPDH1 signal at the photoreceptor cell layer, then the signal at the inner retina is negligible. Representative image shown .max/Vmaxapp? It is not defined in the text or figure legend.4) Important details are missing in the allosteric regulation experiments – what exactly is Vmax are the values derived from the non-linear fitting to a Michaelis-Menten or sigmoidal graph of enzyme velocity vs. substrate equations. Vmax,appvalues refer to the apparent Vmax in the presence of modulators, such as GTP or GDP. This information has now been clarified in the new manuscript.V1/2 were more complex sigmoidal inhibition is observed. Note that the text sometimes uses the term Ki for all, other times uses IC50. The nomenclature should be consistent. The title should reflect that both kinds of measurements are reported, as should the column headings. Which substrates were varied, which were held constant? Are the units really mM? The values of Ki for GMP and XMP are 1-2 orders of magnitude greater than expected. The data are not provided. The authors need to report the concentration of NAD and enzyme in the experiment, as well as the temperature and buffer, in the figure and table legends.5) Table 1 appears to report a mixture of different constants – some are Ki determined for competitive inhibitors and some are Kmax values, determined at high substrate concentration (up to 5 millimolar IMP), but the Km values, which are strongly affected by the low substrate concentrations. The lowest IMP concentrations used for these experiments were 19 microM and the enzyme concentration was 1.8 microM. Thereby, these are not Michaelis-Menten conditions, and the Km values for the lowest IMP experiments might have been not reliably estimated. Consequently, the Ki values might be biased by the wrong Km values, since Km is used to calculate Ki.The reviewer is right. We have thought about this issue and have concluded that our competitive Ki constants might be biased by the use of a relatively large concentration of enzyme  for the enzyme kinetic experiments. This should not significantly influence the VTherefore, we repeated the enzyme kinetic experiments using a much lower enzyme concentration (50 nM) and monitored the reaction progress using the NADH fluorescence, which is more sensitive than monitoring NADH absorbance, as we used in our previous experimental setup.m and competitive Ki,GMP values are now orders of magnitude lower, in accordance to the reviewer´s comment and the previously published literature.The new KHowever, the demonstration that de novo synthesis of IMP, GMP and AMP increases with light in vivo, overcomes the discussion on the kinetic constants of inhibition for GMP and GDP/GTP (that has been removed). The ultimate question for our model is whether IMPDH1 activity increases with light in vivo.According to our HPLC and LC-MS/MS data, light increases the levels of GMP in vivo (as it does increase the levels of AMP and IMP). The overall flux through de novo purine nucleotides increases with light, including IMPDH1 activity.We have added detailed information about the enzyme kinetics experiments, as requested by the reviewer.6) The phosphomimetic mutations at T159/S160 do have a significant effect on allosteric inhibition, but "abolish" is too strong a term – it is just a 5-fold increase. How exactly is the IC50 for GTP determined? The sigmoidal dependence on GTP concentration is very steep – what is the value of the Hill coefficient? What equation was used?50 for GTP can only be roughly estimated because higher concentrations of GTP would be needed. This issue has now been clarified in the main text, where we have substituted the “IC50 = 6 mM” by “IC50 > 6 mM”.The reviewer is right: “abolish” is too strong. We have substituted this term by “decreases” or “desensitizes”. IC7) Why does Figure 8 report nucleotide levels in picomole/retina? Isn't picomole/mg protein the better measure? Also, how much of the nucleotides come from photoreceptor cells as opposed to the other cells in the retina?Landmark papers in the field, e.g.  have expressed nucleotide levels both ways…it may facilitate the contrast of information among different laboratories. We have now restricted values in Table 2 to those expressed in picomole/mg protein.8) in vitrokinase assays are of questionable physiological relevance.We are now providing new evidence that supports PKC is very likely the kinase responsible for light-dependent IMPDH1 phosphorylation in vivo.Treatment of retinas in situ with PKC selective inhibitor bisindolylmaleimide results in a drop in GTP levels with light exposure; and treatment of retinas with this drug in living mice causes a delay in mass rod recovery . By determining the kinase, we can now explore the pathway with selective inhibitor drugs.Results point to light setting in motion a signaling pathway that activates PKC, which impacts main metabolic pathways in photoreceptor cells.9) Is the S477 phosphorylation likely to affect formation of rods and rings?eLife Johnson and Kollman, (2020) we now speculate in the discussion section that S477 phosphorylation might be involved in inducing filament disassembly. In Johnson´s et al., reported structure of IMPDH2 polymers, details are revealed of the interface that mediates cytoophidia assembly: the 12 amino-terminal residues of the canonical IMPDH2 isoform extend from the catalytic domain to bind into the adjacent molecule, in a shallow surface groove formed by a short helix (476-485), two beta strands (51-63), and two short loops  (51). Strikingly, S477 maps into the short helix at the IMPDH2 cytoophidia longitudinal contact interface, allowing us to speculate that phosphorylation of S477 in IMPDH1 might disrupt cytoophidia assembly.Based on the study just published in Reviewer #2:[…]Essential revisions:Further discussion on the following points would enhance this manuscript.It is unclear what the underlying mechanism is for light dependent phosphorylation of IMPDH1 at the T159/S160 residues. The authors nicely show that PKC is involved in the phosphorylation. Is PKC activated directly or indirectly in the light or does IMPDH1 undergo a conformational change in the light to enable phosphorylation by PKC? Discussion or speculation on this point would be useful.This is indeed a very interesting point. T159/S160 residues are exposed to the bulk solvent in the IMPDH1 tetramers, so PKC can easily access them. These residues only get buried when a nucleotide (ATP and or GTP) induces IMPDH octamerization. Thereby, we think it is rather PKC that gets activated in light and phosphorylates IMPDH1.We have observed in our phosphoproteomic analysis that the phosphorylation sites that prime PKC for activation occur in the retina in the light condition. Thereby, we think it is rather PKC that gets activated in light and phosphorylates IMPDH1.The mechanism appears to be mainly involved in controlling guanine nucleotide levels in rods. Is a similar mechanism proposed for cones?This is a very interesting question for which we do not have an answer at this point. Our overall phosphoproteomic analysis reveals that light changes the metabolic fluxes in retinal cells (likely rod photoreceptors) by controlling the phosphorylation of key metabolic switches. We believe that key phosphorylation events rapidly re-structure the metabolic roadmap in photoreceptor cells in dark/light. These metabolic adaptive changes might be different in rods and cones.The authors show that the N198K and R224P disease-causing mutations display a decreased level of phosphorylation . Do these mutations affect GDP/GTP interaction and/or the enzymatic activity of IMPDH1 or just its phosphorylation by PKC? Further discussion would be useful.Both mutations N198K and R224P affect GTP/GDP interaction, but they do not affect enzymatic activity . N198K is involved in the direct binding of GTP/GDP to the third non-canonical site, which is needed for GTP/GDP-mediated allosteric inhibition. On the other hand, R224P is necessary for GTP/GDP-induced IMPDH octamerization, which is also needed for allosteric inhibition.We do not know at this point the mechanism by which these mutants display a decrease level of phosphorylation.Reviewer #3:[…]Essential revisions:1) Light-regulated phosphorylation of T159/S160 is not solid. The difference between light vs dark in Figure 2 is from bovine retinas. The splicing isoforms of IMPDH1 in bovine retina are still unknown although the phosphorylation sites are conservative for known isoforms. As the authors pointed out in the discussion, they were also under nutritional deficit. Additionally, Figure 3C in comparing phosphorylation in mouse isoform is not statistically significant. It will be important to know whether T159/S160 is the major site of phosphorylation in 603/604 aa IMPDH1. It should be easily done by proteomics or PKC inhibitor.We have demonstrated by in situ metabolic labeling that the major isoform of IMPDH1 in the murine retina (603/604aa) is phosphorylated at two sites. We base the identity of those two sites on the mass spectrometry analysis performed with bovine retinas, that is correct. This is not uncommon in the field. Rhodopsin phosphorylation sites were originally characterized in the bovine system, and are conserved in the murine system. The same happens with phosducin phosphorylation, and with practically any well-established phosphorylation event that happens in signaling proteins in response to dark/light.Please see the answer provided to reviewer #1 on the sensitivity limitations and the cost of phosphoproteomic experiments.2) The GMP is significantly increased but GTP/GDP remains unchanged by light. However, the authors proposed that GTP/GDP but not GMP regulates IMPDH1 by light. One argument is that Ki of GMP is much higher (1.94 mM) for hIMPDH1-514 than physiological mouse retina. HIMPDH1-514 is not the major isoform in the human retina. (PMID: 16936083) (546 and 595 are the major ones). It is unknown whether hIMPDH1-514 is phosphorylated in vivo . Furthermore, Ki of GMP varies in big ranges with different IMPDH1 proteins (PMID: 26558346). The second argument is GTP/GMP ratio (GTP is constant but GMP is increased) is more important than GMP in regulating IMPDH1. Is there data that support this hypothesis?Regarding GMP Ki values, please see the answer provided to reviewer #1.Is IMPDH1 activity increased or decreased with light exposure? The answer, new Figure 7, is that in vivo the flux through de novo purine nucleotide synthesis increases with light, and that includes IMPDH1 activity .Whether GDP/GTP allosteric regulation of IMPDH1 or GMP competitive inhibition of IMPDH1 prevails in vivo… the ultimate question for us is: – 3) The author proposed that light activates IMPDH activity through phosphorylation. Using PKC inhibitor to block the phosphorylation will give a strong evidence. Will the IMDPH1 inhibitor affect GTP, GMP and IMP levels?Yes, we are now providing a new panel in the PKC figure  that shows just that.Furthermore, we also show that bisindolylmaleimide injected intravitreally delays mass rod recovery by ERG (paired flash paradigm) in living mice, having a very similar effect to that of inhibiting IMPDH activity.4) In the Discussion section "IMPDH1 mutations would cause the pathology by resulting in higher than normal constitutive IMPDH1 activity… Abnormally high IMPDH1 activity could conceivably result in abnormally high cGMP synthesis., Consequently, every clue would lead to unregulated, constitutive IMPDH1 activity as the underlying cause of the adRP10 phenotype." How would the author explain the discrepancy by other reports: (1) IMPDH1 human mutants have similar IMPDH activity including D226N (PMID: 1829559); (2) Supplement with GTP improves ERGs from IMPDH1 -/- mice (PMID: 19822744).1) IMPDH1 human mutants have similar IMPDH1 activity (as the wildtype protein) in in vitro assays, in studies from several laboratories, including ours. Mutations decrease the allosteric inhibition by GDP/GTP. .2) That a supplement with GTP improves ERGs from IMPDH1-/- mice is exactly what we would expect and is perfectly consistent with our model. .