Regulatory Phosphorylation Sites Research Articles

The mTOR pathway controls phosphorylation of BRAF at T401

BRAF serves as a gatekeeper of the RAS/RAF/MEK/ERK pathway, which plays a crucial role in homeostasis. Since aberrant signalling of this axis contributes to cancer and other diseases, it is tightly regulated by crosstalk with the PI3K/AKT/mTOR pathway and ERK mediated feedback loops. For example, ERK limits BRAF signalling through phosphorylation of multiple residues. One of these, T401, is widely considered as an ERK substrate following acute pathway activation by growth factors. Here, we demonstrate that prominent T401 phosphorylation (pT401) of endogenous BRAF is already observed in the absence of acute stimulation in various cell lines of murine and human origin. Importantly, the BRAF/RAF1 inhibitor naporafenib, the MEK inhibitor trametinib and the ERK inhibitor ulixertinib failed to reduce pT401 levels in these settings, supporting an alternative ERK-independent pathway to T401 phosphorylation. In contrast, the mTOR inhibitor torin1 and the dual-specific PI3K/mTOR inhibitor dactolisib significantly suppressed pT401 levels in all investigated cell types, in both a time and concentration dependent manner. Conversely, genetic mTOR pathway activation by oncogenic RHEB (Q64L) and mTOR (S2215Y and R2505P) mutants substantially increased pT401, an effect that was reverted by dactolisib and torin1 but not by trametinib. We also show that shRNAmir mediated depletion of the mTORC1 complex subunit Raptor significantly enhanced the suppression of T401 phosphorylation by a low torin1 dose, while knockdown of the mTORC2 complex subunit Rictor was less effective. Using mass spectrometry, we provide further evidence that torin1 suppresses the phosphorylation of T401, S405 and S409 but not of other important regulatory phosphorylation sites such as S446, S729 and S750. In summary, our data identify the mTOR axis and its inhibitors of (pre)clinical relevance as novel modulators of BRAF phosphorylation at T401.

An improved SNAP-ADAR tool enables efficient RNA base editing to interfere with post-translational protein modification

RNA base editing relies on the introduction of adenosine-to-inosine changes into target RNAs in a highly programmable manner in order to repair disease-causing mutations. Here, we propose that RNA base editing could be broadly applied to perturb protein function by removal of regulatory phosphorylation and acetylation sites. We demonstrate the feasibility on more than 70 sites in various signaling proteins and identify key determinants for high editing efficiency and potent down-stream effects. For the JAK/STAT pathway, we demonstrate both, negative and positive regulation. To achieve high editing efficiency over a broad codon scope, we applied an improved version of the SNAP-ADAR tool. The transient nature of RNA base editing enables the comparably fast (hours to days), dose-dependent (thus partial) and reversible manipulation of regulatory sites, which is a key advantage over DNA (base) editing approaches. In summary, PTM interference might become a valuable field of application of RNA base editing.

SHP2-EGFR States in Dephosphorylation Can Inform Selective SHP2 Inhibitors, Dampening RasGAP Action.

SHP2 is a positive regulator of the EGFR-dependent Ras/MAPK pathway. It dephosphorylates a regulatory phosphorylation site in EGFR that serves as the binding site to RasGAP (RASA1 or p120RasGAP). RASA1 is activated by binding to the EGFR phosphate group. Active RASA1 deactivates Ras by hydrolyzing Ras-bound GTP to GDP. Thus, SHP2 dephosphorylation of EGFR effectively prevents RASA1-mediated deactivation of Ras, thereby stimulating proliferation. Despite knowledge of this vital regulation in cell life, mechanistic in-depth structural understanding of the involvement of SHP2, EGFR, and RASA1 in the Ras/MAPK pathway has largely remained elusive. Here we elucidate the interactions, the factors influencing EGFR's recruitment of RASA1, and SHP2's recognition of the substrate site in EGFR. We reveal that RASA1 specifically interacts with the DEpY992LIP motif in EGFR featuring a proline residue at the +3 position C-terminal to pY primarily through its nSH2 domain. This interaction is strengthened by the robust attraction of two acidic residues, E991 and D990, of EGFR to two basic residues in the BC-loop near the pY-binding pocket of RASA1's nSH2. In the stable precatalytic state of SHP2 with EGFR (DADEpY992LIPQ), the E-loop of SHP2's active site favors the interaction with the (-2)-position D990 and (-4)-position D988 N-terminal to pY992 in EGFR, while the pY-loop constrains the (+4)-position Q996 C-terminal to pY992. These specific interactions not only provide a structural basis for identifying negative regulatory sites in other RTKs but can inform selective, high-affinity active-site SHP2 inhibitors tailored for SHP2 mutants.

Delivery of AKT1 phospho-forms to human cells reveals differential substrate selectivity.

Protein kinase B (AKT1) is a serine/threonine kinase that regulates fundamental cellular processes, including cell survival, proliferation, and metabolism. AKT1 activity is controlled by two regulatory phosphorylation sites (Thr308, Ser473) that stimulate a downstream signaling cascade through phosphorylation of many target proteins. At either or both regulatory sites, hyperphosphorylation is associated with poor survival outcomes in many human cancers. Our previous biochemical and chemoproteomic studies showed that the phosphorylated forms of AKT1 have differential selectivity toward peptide substrates. Here, we investigated AKT1-dependent activity in human cells, using a cell-penetrating peptide (transactivator of transcription, TAT) to deliver inactive AKT1 or active phospho-variants to cells. We used enzyme engineering and genetic code expansion relying on a phosphoseryl-transfer RNA (tRNA) synthetase (SepRS) and tRNASep pair to produce TAT-tagged AKT1 with programmed phosphorylation at one or both key regulatory sites. We found that all TAT-tagged AKT1 variants were efficiently delivered into human embryonic kidney (HEK 293T) cells and that only the phosphorylated AKT1 (pAKT1) variants stimulated downstream signaling. All TAT-pAKT1 variants induced glycogen synthase kinase (GSK)-3α phosphorylation, as well as phosphorylation of ribosomal protein S6 at Ser240/244, demonstrating stimulation of downstream AKT1 signaling. Fascinatingly, only the AKT1 variants phosphorylated at S473 (TAT-pAKT1S473 or TAT-pAKT1T308,S473) were able to increase phospho-GSK-3β levels. Although each TAT-pAKT1 variant significantly stimulated cell proliferation, cells transduced with TAT-pAKT1T308 grew significantly faster than with the other pAKT1 variants. The data demonstrate differential activity of the AKT1 phospho-forms in modulating downstream signaling and proliferation in human cells.

Role of Protein Phosphatase 1 (PP1) Upon KCCs Regulation by WNK3 Kinase During Cell Volume Regulation and Ion Transport

Background: The K-Cl cotransporters (KCCs) are SLC12A cation/chloride cotransporters (CCCs) that mediate epithelial transport, maintain cellular volume, and regulate GABA neurotransmission. These mechanisms are complex, and contain multiple sensors, transducers, and effectors, often operating in parallel pathways. It has been shown that KCCs activity is affected by direct phosphorylation and dephosphorylation. In particular dephosphorylation of important regulatory sites on KCCs is necessary to become fully activated, whereas phosphorylation by SPAK/OSR1 kinases will result in KCCs inhibition. Protein phosphatases (PP) inhibitors reduce KCCs function and so, it is generally accepted the presence of phosphatases on the KCCs signaling pathway. Several studies have proposed protein phosphatase 1 (PP1) as the major PP responsible for KCCs dephosphorylation. On the other hand, WNK3 kinases have been shown to directly regulate KCCs activity bypassing their osmotic regulation. That is wild type (WT) WNK3 phosphorylates KCCs under hypotonic conditions where they are normally active, whereas kinase dead WNK3 (DA) activates KCCs function under isotonic conditions where they are normally inhibited. On the present study we analyze the role of protein phosphatase 1 (PP1) upon KCCs regulation by WNK3 kinase during cell volume regulation and ion transport. Methods: HEK 293 cells and Xenopus laevis oocytes were either transfected or microinjected with KCC3 or KCC4 and one of the following PP1 plasmids: WT, H248K (inactive) or T320A (constitutive active), and/or WT WNK3 /DA WNK3. KCCs dephosphorylation in response to cell swelling, hypotonic conditions, was evaluated by Western blot analysis of KCCs regulatory phosphorylation site (pThr1048 in KCC3a). Phosphatase/kinase interaction and the possible formation of a complex was analyzed by transfecting cells or microinjecting oocytes with WNK3 PP1 putative binding sites mutants (WNK3 PP1a y PP1b). Phosphorylation of SPAK/OSR1 activating regulatory site Ser373 was analyzed in parallel under the same conditions. Results: WT or constitutive active mutant PP1 T320A induced KCCs Thr1048 dephosphorylation in HEK cells and X. laevis oocytes even under isotonic conditions, where KCCs are normally phosphorylated. WT WNK3 prevented KCCs T1048 dephosphorylation under hypotonic conditions, ablating their response to cell swelling. Nevertheless, when co-expressed with PP1 T320A, KCCs became dephosphorylated in both osmotic conditions eliminating WT WNK3 inhibitory effect. PP1 inactivating mutation H248K prevented KCCs Thr1048 dephosphorylation in response to hypotonic conditions. However, when co-expressed with DA WNK3, this KCCs regulatory site, became dephosphorylated under hypotonic conditions. SPAK Ser373 regulation was unaffected in any off the studied conditions. Conclusions: Our results show a direct role for PP1 on KCCs function. PP1WT and PP1T320A regulate KCCs activity by its dephosphorylation. In presence of WT WNK3, PP1 activity predominates over WNK3 surpassing the inhibitory phosphorylation of the regulatory Thr residue. KCCs are not dephosphorylated by PP1H248K under hypotonic conditions. Coexpression of kinase dead WNK3 overcomes this effect, supporting the hypothesis of a phosphatase/kinase complex regulating KCCs cell volume regulatory response. AM CF-2023-I-532, INCar 22-1345 PdlH Papiit IN228123. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lipidome remodeling in response to nutrient replenishment requires the tRNA modifier Deg1/Pus3 in yeast.

In the yeast Saccharomyces cerevisiae, the absence of the pseudouridine synthase Pus3/Deg1, which modifies tRNA positions 38 and 39, results in increased lipid droplet (LD) content and translational defects. In addition, starvation-like transcriptome alterations and induced protein aggregation were observed. In this study, we show that the deg1 mutant increases specific misreading errors. This could lead to altered expression of the main regulators of neutral lipid synthesis which are the acetyl-CoA carboxylase (Acc1), an enzyme that catalyzes a key step in fatty acid synthesis, and its regulator, the Snf1/AMPK kinase. We demonstrate that upregulation of the neutral lipid content of LD in the deg1 mutant is achieved by a mechanism operating in parallel to the known Snf1/AMPK kinase-dependent phosphoregulation of Acc1. While in wild-type cells removal of the regulatory phosphorylation site (Ser-1157) in Acc1 results in strong upregulation of triacylglycerol (TG), but not steryl esters (SE), the deg1 mutation more specifically upregulates SE levels. In order to elucidate if other lipid species are affected, we compared the lipidomes of wild type and deg1 mutants, revealing multiple altered lipid species. In particular, in the exponential phase of growth, the deg1 mutant shows a reduction in the pool of phospholipids, indicating a compromised capacity to mobilize acyl-CoA from storage lipids. We conclude that Deg1 plays a key role in the coordination of lipid storage and mobilization, which in turn influences lipid homeostasis. The lipidomic effects in the deg1 mutant may be indirect outcomes of the activation of various stress responses resulting from protein aggregation.

FRI289 It Takes A Village: Musashi And Its Associated Proteins In The Adenohypophysis

Abstract Disclosure: K.L. Bronson: None. L. Hardy: None. A. Lagasse: None. A.M. MacNicol: None. Musashi is an RNA-binding protein that regulates stem and progenitor cell fates. Interestingly, we have reported expression of Musashi pituitary stem cells as expected but also in the differentiated anterior pituitary hormone-producing cell lineages as well as. The adult anterior pituitary relies on the functional cellular plasticity of its hormone-producing cell populations to maintain endocrine homeostasis when physiological demands change. Both Musashi isoforms (Musashi1 and Musashi2) are highly conserved across species and have two N-terminal RNA-recognition motifs (RRMs) and a disordered C-terminus containing two regulatory phosphorylation sites. Musashi can selectively activate or repress the translation of mRNAs depending on the transcript and cellular context. While Musashi phosphorylation is required for translational activation, Musashi does not possess inherent enzymatic activity; therefore, co-associated proteins must mediate Musashi-directed translational control. Musashi immunoprecipitation studies initially identified 50 co-associated proteins in Xenopus oocytes, but only a subset of these were directly implicated in Musashi-dependent translational activation (e.g. Elavl1 and Pabp4). The identity of Musashi-associated proteins in the anterior pituitary and their requirement for Musashi function are not known. To this end, we have immunoprecipitated Musashi from whole male mouse pituitaries for mass spectrometry analysis. 206 proteins significantly (adj p-value < 0.05) associated with Musashi1 and Musashi2, and seven of these overlapped with the Xenopus dataset: Elavl1, Pabp4, Hnrnpc, Mov10, Pura, Rps27, and Upf1. Three proteins from the mouse mass spectrometry dataset were selected for further analyses. Sam68 is a multi-functional protein that sensitizes ovaries to FSH and LH, as well as mediates the activation of leptin and insulin signaling in a variety of tissues. Ago2 is a component of the RNA-induced silencing complex that is necessary for miRNA-directed translational control and is known to associate with Musashi1. The AU-rich element binding protein Elavl1 (HuR) contributes to Musashi-dependent activation of target mRNAs in Xenopus and also stabilizes Gnrhr mRNA in LβT2 cells and sensitizes them to GnRH signaling. We will present our ongoing studies on the requirement for these protein interactions for Musashi-directed mRNA translational regulation. Presentation: Friday, June 16, 2023

Hybrid-DIA: intelligent data acquisition integrates targeted and discovery proteomics to analyze phospho-signaling in single spheroids

Achieving sufficient coverage of regulatory phosphorylation sites by mass spectrometry (MS)-based phosphoproteomics for signaling pathway reconstitution is challenging, especially when analyzing tiny sample amounts. To address this, we present a hybrid data-independent acquisition (DIA) strategy (hybrid-DIA) that combines targeted and discovery proteomics through an Application Programming Interface (API) to dynamically intercalate DIA scans with accurate triggering of multiplexed tandem mass spectrometry (MSx) scans of predefined (phospho)peptide targets. By spiking-in heavy stable isotope labeled phosphopeptide standards covering seven major signaling pathways, we benchmark hybrid-DIA against state-of-the-art targeted MS methods (i.e., SureQuant) using EGF-stimulated HeLa cells and find the quantitative accuracy and sensitivity to be comparable while hybrid-DIA also profiles the global phosphoproteome. To demonstrate the robustness, sensitivity, and biomedical potential of hybrid-DIA, we profile chemotherapeutic agents in single colon carcinoma multicellular spheroids and evaluate the phospho-signaling difference of cancer cells in 2D vs 3D culture.

Human KCC2a Protein Expression and Phosphorylation is Altered by Elimination of the Second Cryo-EM Identified Chloride Binding Site

Background: Potassium-chloride cotransporters (KCCs) are responsible for the symport of Cl- with K+ in an electroneutral manner. They play important roles in numerous crucial physiological processes, from cell volume regulation to transepithelial solute transport, intracellular ion homeostasis and regulation of GABA neurotransmission. Under isotonicity, except for KCC2, KCCs remain inhibited due to phosphorylation of residues located in cytoplasmic domains, whereas in response to hypotonic cell swelling, dephosphorylation of these residues stimulates transport activity. After several decades of studies, new breakthroughs with the cryo-EM structural determination of KCCs have deepened our understanding of their molecular basis and transport function, including architecture, ion binding sites, and coupled ion transport. In the present study, we mutated and analyzed the two chloride-binding sites in human KCC2a recently reported by cryo-EM to determine whether the phosphorylation status is modified upon their removal. Methods: Xenopus laevis oocytes and HEK-293 cells were either microinjected or transfected with human KCC2a wild type (WT) or chloride-binding site mutants, G136/V137/I138A (S1-Cl), and G436/I437/M438/Y592A (S2-Cl). KCC2a phosphorylation status in response to isotonic and hypotonic conditions was evaluated by immunoblot analysis of regulatory phosphorylation sites pThr926 and pThr1030. In parallel, phosphorylation status of SPAK/OSR1 activating regulatory site pSer373/pSer325 was analyzed under the same conditions. Results: Elimination of either chloride-binding site induced changes in total expression of KCC2a. Removal of S1-Cl slightly increased total KCC2a expression, whereas lack of S2-Cl showed a significant reduction of total protein in Xenopus laevis oocytes and HEK-293 cells. Phosphorylation of regulatory Thr1030 in S2-Cl mutant is significatively decreased on both biological models, even under isotonic inhibitory (phosphorylated) conditions. pThr1030 on S1-Cl mutant behaves similarly to WT KCC2a. Phosphorylation of regulatory SPAK kinase is not affected on both mutants. Conclusions: Our results suggest that while both sites affect KCC2a total expression, only the S2-Cl appears to be affecting KCC2a activating response. It appears S2-Cl mutation dysregulates SPAK signaling pathway, since the kinase osmotic response remains unchanged on presence of KCC2a chloride binding mutants. AM INCar 22-1345 and PdlH UNAM Papiit IN222320 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mutation of regulatory phosphorylation sites in PFKFB2 does not affect the anti-fibrotic effect of metformin in the kidney.

The anti-fibrotic effect of metformin has been widely demonstrated. Fibrosis in the kidney after injury is associated with reduced expression of genes involved in both fatty acid and glycolytic energy metabolism. We have previously reported that the anti-fibrotic effect of metformin requires phosphoregulation of fatty acid oxidation by AMP-activated protein kinase (AMPK). To determine whether metformin also acts via regulation of glycolysis, we mutated regulatory phosphosites in the PFKFB2 isoform of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB2), a key regulator of glycolysis in the kidney. Mice with inactivating knockin (KI) mutations of the phosphorylation sites in PFKFB2 (PFKFB2 KI mice), which reduces the ability to increase the rate of glycolysis following stimulation, were used. Metformin was administered via drinking water to mice with a unilateral ureteric obstruction (UUO) model of renal fibrosis. In the PFKFB2 KI mice treated with metformin, there was decreased fibrosis and macrophage infiltration following UUO as assessed by Western blot for fibronectin and RT-PCR for α-smooth muscle actin, collagen 3, and F4.80, and confirmed by histology. Expression of the inducible PFKFB3 isoform was increased with metformin in UUO in both WT and PFKFB2 KI mice. There was no significant difference between WT and PFKFB2 KI mice treated with metformin in the degree of fibrosis following UUO in any of the Western blot or RT-PCR parameters that were measured. These data show that inhibition of the regulation of glycolysis by PFKFB2 does not diminish the anti-fibrotic effect of metformin in a model of renal fibrosis.

Quantitative proteomic analysis reveals apoE4‐dependent phosphorylation of the actin regulating protein VASP

AbstractBackgroundApolipoprotein E4 (APOE4) is the major genetic risk factor for Alzheimer’s disease (AD). Current evidence suggests that neuronal expression of apoE4 is sufficient to promote neuropathology via a gain‐of‐function mechanism; however, the precise molecular mechanisms mediating this process are still unclear. To elucidate specific protein targets and cellular pathways dysregulated due to apoE4 expression, we performed global proteomic profiling of ubiquitylation and phosphorylation signaling in Neuro‐2a cells stably expressing either apoE3 or apoE4.MethodNeuro‐2a cells (apoE3 or apoE4) were lysed and proteins digested into peptides. Peptides were enriched for either phosphorylation or ubiquitylation post‐translational modifications, and the resulting peptides were detected and quantified using a label‐free quantitative proteomics workflow. Modification sites were detected by MaxQuant, and statistical analysis was performed using MSstats to identify significantly regulated modification sites (p<0.05) between apoE3 and apoE4‐expressing cells. Immunoblotting was performed to validate protein phosphorylation sites of interest.ResultThe expression of apoE3 versus apoE4 in Neuro‐2a cells results in the differential regulation of thousands of ubiquitylation and phosphorylation sites. Gene ontology and pathway analysis of these regulated sites reveal an enrichment for a variety of cellular processes, including proteasomal biology, redox homeostasis, spindle organization, as well as cytoskeletal regulation. Accordingly, among the most differentially regulated phosphorylation sites is the vasodilator‐stimulated phosphoprotein, VASP, in which there is a 14‐fold increase in phosphorylation at position S235 in Neuro2a‐apoE4‐expressing cells (p=5.5e‐10) as compared to Neuro2a‐apoE3. This phosphorylation was reduced upon siRNA knockdown of APOE4, as well as by pharmacological inhibition of PKA, indicating that VASP S235 is phosphorylated by PKA in an apoE4‐dependent manner. VASP S235 phosphorylation results in reduced filopodia and cytoskeletal filament formation. Thus, we hypothesize that phosphorylation of VASP at S235 represents a mechanism regulating the well‐established defects in neurite outgrowth and dendritic spine formation in apoE4‐expressing neurons.ConclusionOur data demonstrate that VASP S235 is highly phosphorylated by PKA in apoE4‐expressing neurons compared to apoE3. Future experiments will evaluate the effect of VASP S235 phosphorylation on actin cytoskeleton remodeling and neurite outgrowth and will be extended to the evaluation of PKA inhibitors as a therapeutic opportunity in apoE4‐associated AD.

Phosphorylation of S11 in PHR1 negatively controls its transcriptional activity.

Plant responses to phosphate starvation (-Pi) are very well characterized at the biochemical and molecular levels. The expression of thousands of genes is modified under this stress condition, depending on the action of Phosphate starvation response 1 (PHR1). Existing data indicate that neither the PHR1 transcript nor the quantity or localization of its protein increase during nutrient stress, raising the question of how its activity is regulated. Here, we present data showing that SnRK1 kinase is able to phosphorylate some phosphate starvation response proteins (PSRs), including PHR1. Based on a model of the three-dimensional structure of the catalytic subunit SnRK1α1, docking simulations predicted the binding modes of peptides from PHT1;8, PHO1 and PHR1 with SnRK1. PHR1 recombinant protein interacted in vitro with the catalytic subunits SnRK1α1 and SnRK1α2. A BiFC assay corroborated the in vivo interaction between PHR1 and SnRK1α1 in the cytoplasm and nucleus. Analysis of phosphorylated residues suggested the presence of one phosphorylated site containing the SnRK1 motif at S11, and mutation in this residue disrupted the incorporation of 32 P, suggesting that it is a major phosphorylation site. Electrophoretic mobility shift assay results indicated that the binding of PHR1 to P1BS motifs was not influenced by phosphorylation. Importantly, transient expression assays in Arabidopsis protoplasts showed a decrease in PHR1 activity in contrast with the S11A mutant, suggesting a role for Ser11 as a negative regulatory phosphorylation site. Taken together, these findings suggest that phosphorylation of PHR1 at Ser11 is a mechanism to control the PHR1-mediated adaptive response to -Pi.

Phosphorylation of Ser82 on IRF3 acts as negative-feedback regulation of IRF3-dependent innate immunity.

Interferon Regulatory Factor 3 (IRF3) is essential for the production of type I interferon (IFN) during virus infection; however, the mechanism underlying its regulation remains to be elucidated. Here we have identified a novel negative regulatory phosphorylation site on IRF3. In this study, we discovered that Ser82 phosphorylation on IRF3 abrogates virus-induced IFN-β activation. Furthermore, our results clarified the mechanism in which Ser82 phosphorylation on IRF3 retains the function of dimerization and nuclear import, but abolishes the promoter binding ability of IRF3. In addition, Ser82 phosphorylation on IRF3 serves as a negative feedback mechanism for the type I IFN response. These findings elucidate a previously unknown mechanism for negatively regulating IRF3 to finely tune type I IFN response.

Bayesian analysis of dynamic phosphoproteomic data identifies protein kinases mediating GPCR responses

BackgroundA major goal in the discovery of cellular signaling networks is to identify regulated phosphorylation sites (“phosphosites”) and map them to the responsible protein kinases. The V2 vasopressin receptor is a G-protein coupled receptor (GPCR) that is responsible for regulation of renal water excretion through control of aquaporin-2-mediated osmotic water transport in kidney collecting duct cells. Genome editing experiments have demonstrated that virtually all vasopressin-triggered phosphorylation changes are dependent on protein kinase A (PKA), but events downstream from PKA are still obscure.MethodsHere, we used: 1) Tandem mass tag-based quantitative phosphoproteomics to experimentally track phosphorylation changes over time in native collecting ducts isolated from rat kidneys; 2) a clustering algorithm to classify time course data based on abundance changes and the amino acid sequences surrounding the phosphosites; and 3) Bayes’ Theorem to integrate the dynamic phosphorylation data with multiple prior “omic” data sets covering expression, subcellular location, known kinase activity, and characteristic surrounding sequences to identify a set of protein kinases that are regulated secondary to PKA activation.ResultsPhosphoproteomic studies revealed 185 phosphosites regulated by vasopressin over 15 min. The resulting groups from the cluster algorithm were integrated with Bayes’ Theorem to produce corresponding ranked lists of kinases likely responsible for each group. The top kinases establish three PKA-dependent protein kinase modules whose regulation mediate the physiological effects of vasopressin at a cellular level. The three modules are 1) a pathway involving several Rho/Rac/Cdc42-dependent protein kinases that control actin cytoskeleton dynamics; 2) mitogen-activated protein kinase and cyclin-dependent kinase pathways that control cell proliferation; and 3) calcium/calmodulin-dependent signaling.ConclusionsOur findings identify a novel set of downstream small GTPase effectors and calcium/calmodulin-dependent kinases with potential roles in the regulation of water permeability through actin cytoskeleton rearrangement and aquaporin-2 trafficking. The proposed signaling network provides a stronger hypothesis for the kinases mediating V2 vasopressin receptor responses, encouraging future targeted examination via reductionist approaches. Furthermore, the Bayesian analysis described here provides a template for investigating signaling via other biological systems and GPCRs.7VJHPzA8Cu6VMRYsgYqSsgVideo abstract

Effect of a C‐terminal Electrostatic Interaction Domain on KCC3 Function

BackgroundThe K:Cl cotransporters (KCCs) are key players to maintain cellular volume, mediate epithelial transport and intracellular chloride concentration, and regulate GABA neurotransmission. Under isotonic conditions, KCCs are normally inhibited due to the phosphorylation of residues located within the cytoplasmic domains. On the contrary, dephosphorylation of those residues in response to hypotonic cell swelling stimulates transport activity. Dysfunction of these proteins and the processes involved in their regulation have been associated with various human diseases by alteration of ionic homeostasis mainly in the kidney and the brain. Based on the cryo‐EM structures recently reported of human KCCs, revealing structural determinants for regulation in both N‐ and C‐terminal domains, in the present study we analyzed the effect of a structural domain located at the C‐ terminal involved in electrostatic interactions. It has been stated that the side chains of residues as Gln (Q), Arg (R) and Lys (K) tend to form hydrogen bonds promoting the stabilization of tertiary and quaternary protein structures. However, whether the phosphorylation status is modified after the electrostatic disruption is unknown.MethodsXenopus laevis oocytes and HEK 293 cells were either microinjected or transfected with KCC3a wild type (WT) or C‐terminal mutants, Q905A, Q905/K934A (2M), Q905/K934/R937A (3M) and Q905/K934/R937/K938A (4M), key residues involved in electrostatic interactions and located within the C‐terminal domain of the cotransporter. The phosphorylation status of KCC3a in response to isotonic and hypotonic conditions was evaluated by immunoblot analysis of three key regulatory phosphorylation sites (pSer96, pThr991 and pThr1048). In parallel, phosphorylation status of SPAK/OSR1 activating regulatory site pSer373/pSer325 was analyzed under the same conditions.ResultsCell swelling‐induced dephosphorylation is not observed on C‐terminal electrostatic interaction domain mutants compared to WT KCC3 in X. laevis oocytes and HEK cells. All three regulatory‐phosphorylation sites in KCC3a mutants remained phosphorylated under hypotonic conditions where dephosphorylation is required for the protein to become active. These results suggest that the disruption of the hydrogen bonds could disrupt the proper folding of the protein and thus interfere with the regulatory dephosphorylation/phosphorylation signaling pathway which is known to activate the transporter after cell swelling.ConclusionsOur results show that the four residues in the electrostatic interaction domain of the C‐termini are involved in KCC3a response to hypotonic conditions affecting the phosphorylation status of the cotransporter. The side chains of these residues seem to be important in maintaining tertiary conformation given their ability to form hydrogen bonds and thus, the disruption of their interactions affects the cotransporter ability to respond to osmotic and cell volume changes.

Novel Regulation of Nitric Oxide Synthase 3 (NOS3) Isoform by Acetylation at Lysine 610

NOS3‐mediated NO regulates endothelial and kidney epithelial function. Lysine acetylation is a dynamic post‐translational modification influencing cell signaling. Our lab and others showed NOS3 is regulated by multiple deacetylases. We therefore sought to uncover novel lysine acetylation sites of NOS3 and investigated lysines in the NOS3 reductase domain, K607, K610, K727, and K731. We hypothesized that deacetylation of one or more of these lysines on NOS3 would impact NO production. In order to test this hypothesis, we created NOS3 constructs containing lysine to arginine mutations at these sites mimicking deacetylation. These plasmids were transfected into COS7 cells and expression was confirmed with Western blot. NO production was assessed by measuring nitrite accumulation by HPLC in the media over 48 hours. Mutation of all four lysines or lysines K607 and K610 dramatically increased NO production whereas K727 and K731 did not change NO production compared to wildtype (pmol NO2/mg protein, WT: 2271 ± 729.3; K607/610/727/731R: 15176 ± 2759, p=0.046 vs WT; K607/610R: 9431 ± 1772, p=0.0019 vs WT; K727/731R: 3523 ± 805.8, p=0.918). We further investigated single mutation of K607 and K610. We found K610R (10564 ± 1883 pmol NO2/mg protein, p=0.002 vs WT), but not K607R (1589 ± 412.0 pmol NO2/mg protein, p>0.99) to have increased NO production compared to WT (1547 ± 180.8 pmol NO2/mg protein). We then hypothesized that acetylation of K610 on NOS3 would decrease NO production compared to deacetylation of K610. We mutated K610 to glutamine mimicking acetylation of this lysine. Contrary to what we hypothesized, we found K610Q to produce greater NO than both WT and K610R (pmol NO2/mg protein, WT: 2196 ± 606.1, K610Q: 30212 ± 880.2, p<0.0001 WT vs K610Q; K610R:13389 ± 1086, p<0.0001 K610R vs K610Q). Previous reports show that amino acids 605‐612 coordinate regulatory phosphorylation sites of NOS3. We therefore investigated NOS3 K610R and K610Q relative to WT at key phosphorylation regulatory sites by western blot. We found K610R and K610Q to have reduced phosphorylation at T495 (RDU; WT: 1.00 ± 0.252; K610R: 0.820 ± 0.251, p=0.0257; K610Q: 0.711 ± 0.205, p=0.0046), increased phosphorylation at S1177 (WT: 1.00 ± 0.125; K610R: 1.58 ± 0.2921, p=0.038; K610Q: 1.93 ± 0.358, p=0.0082), and no change in phosphorylation at S633 (WT: 1.00 ± 0.054; K610R: 0.885 ± 0.0536, p=0.126; K610Q: 0.955 ± 0.0779, p=0.319). Additionally, K610R but not K610Q was found to have increased S615 phosphorylation (WT: 1.00 ± 0.112; K610R: 2.01 ± 0.259, p=0.0003; K610Q: 1.07 ± 0.112, p=0.682). These data suggest acetylation of K610 would significantly increase NO production and regulate NOS3 phosphorylation sites in endothelial cells and/or kidney epithelial cells. We propose that acetylation/deacetylation of K610 would have dramatic consequences for blood pressure regulation as well as water and electrolyte homeostasis.

Proteome-wide quantitative RNA-interactome capture identifies phosphorylation sites with regulatory potential in RBM20

Cellular mRNA-binding proteins (mRBPs) are major posttranscriptional regulators of gene expression. Although many posttranslational modification sites in mRBPs have been identified, little is known about how these modifications regulate mRBP function. Here, we developed quantitative RNA-interactome capture (qRIC) to quantify the fraction of mRBPs pulled down with polyadenylated mRNAs. Combining qRIC with phosphoproteomics allowed us to systematically compare pull-down efficiencies of phosphorylated and nonphosphorylated forms of mRBPs. Almost 200 phosphorylation events affected pull-down efficiency compared with the unmodified mRBPs and thus have regulatory potential. Our data capture known regulatory phosphorylation sites in ELAVL1, SF3B1, and UPF1 and identify potential regulatory sites. Follow-up experiments on the splicing regulator RBM20 revealed multiple phosphorylation sites in the C-terminal disordered region affecting nucleocytoplasmic localization, association with cytoplasmic ribonucleoprotein granules, and alternative splicing. Together, we show that qRIC in conjunction with phosphoproteomics is a scalable method to identify functional posttranslational modification sites in mRBPs.

Modulation of myosin by cardiac myosin binding protein-C peptides improves cardiac contractility in ex-vivo experimental heart failure models

Cardiac myosin binding protein-C (cMyBP-C) is an important regulator of sarcomeric function. Reduced phosphorylation of cMyBP-C has been linked to compromised contractility in heart failure patients. Here, we used previously published cMyBP-C peptides 302A and 302S, surrogates of the regulatory phosphorylation site serine 302, as a tool to determine the effects of modulating the dephosphorylation state of cMyBP-C on cardiac contraction and relaxation in experimental heart failure (HF) models in vitro. Both peptides increased the contractility of papillary muscle fibers isolated from a mouse model expressing cMyBP-C phospho-ablation (cMyBP-CAAA) constitutively. Peptide 302A, in particular, could also improve the force redevelopment rate (ktr) in papillary muscle fibers from cMyBP-CAAA (nonphosphorylated alanines) mice. Consistent with the above findings, both peptides increased ATPase rates in myofibrils isolated from rats with myocardial infarction (MI), but not from sham rats. Furthermore, in the cMyBP-CAAA mouse model, both peptides improved ATPase hydrolysis rates. These changes were not observed in non-transgenic (NTG) mice or sham rats, indicating the specific effects of these peptides in regulating the dephosphorylation state of cMyBP-C under the pathological conditions of HF. Taken together, these studies demonstrate that modulation of cMyBP-C dephosphorylation state can be a therapeutic approach to improve myosin function, sarcomere contractility and relaxation after an adverse cardiac event. Therefore, targeting cMyBP-C could potentially improve overall cardiac performance as a complement to standard-care drugs in HF patients.

FGF1 and insulin control lipolysis by convergent pathways

Inexorable increases in insulin resistance, lipolysis, and hepatic glucose production (HGP) are hallmarks of type 2 diabetes. Previously, we showed that peripheral delivery of exogenous fibroblast growth factor 1 (FGF1) has robust anti-diabetic effects mediated by the adipose FGF receptor (FGFR) 1. However, its mechanism of action is not known. Here, we report that FGF1 acutely lowers HGP by suppressing adipose lipolysis. On a molecular level, FGF1 inhibits the cAMP-protein kinase A axis by activating phosphodiesterase 4D (PDE4D), which separates it mechanistically from the inhibitory actions of insulin via PDE3B. We identify Ser44 as an FGF1-induced regulatory phosphorylation site in PDE4D that is modulated by the feed-fast cycle. These findings establish the FGF1/PDE4 pathway as an alternate regulator of the adipose-HGP axis and identify FGF1 as an unrecognized regulator of fatty acid homeostasis.

Mutation of Regulatory Phosphorylation Sites in PFKFB2 Does Not Affect Metformin's Protective Effects Against Renal Fibrosis

Mutation of Regulatory Phosphorylation Sites in PFKFB2 Does Not Affect Metformin's Protective Effects Against Renal Fibrosis