The metal-dependent protein phosphatase (PPM/PP2C) family regulates innate immune and cell death pathways through reversible phosphorylation. Although these enzymes contain a conserved third Mg2+/Mn2+ ion (M3) that is essential for activity, its chemical role in phosphate hydrolysis has remained unclear. Here, we report studies that reveal PPM1B promotes cell death during Pseudomonas aeruginosa infection and utilizes a trinuclear metal center in which M3 directly coordinates the substrate phosphate, positioning it for in-line SN2 hydrolysis. In addition to substrate orientation, M3 positions a water molecule to protonate the departing alkoxide, stabilizing the leaving-group. Functionally, M3 substitutes for the arginine clamp in phosphoprotein phosphatases (PPP), revealing that these evolutionarily distinct phosphatase families have converged on the same chemical strategy through fundamentally different catalytic architectures. Together, these findings define a three-metal mechanism in PPM phosphatases and identify the M3 site as a rare and potentially druggable feature for immune and infectious diseases.

Rationale: Fungi and plants sense environmental CO2 with a special group of PP2C phosphatases. These phosphatases contain a unique C-terminal intrinsically disordered region (IDR) enriched with Ser/Thr residues which have relatively high affinities for CO2. Hypercapnia, but not pH changes, triggers PP2C phase separation in a manner that is dependent on the Ser/Thr residues of the IDR. CO2 is proposed to act as a ‘molecular glue’ that bridges PP2Cs together at the IDR, activating compartmentalized cell signals in response to hypercapnia. In mammals, hypercapnia regulates inflammation, mitochondrial metabolism, alveolar fluid clearance, dyspnea, and vasomotor tone, however, it is unknown whether humans sense CO2 through a similar mechanism. PPM1B is a human homolog of PP2C, therefore, we hypothesize that PPM1B senses CO2 through phase separation. Methods: Full length PPM1B and PPM1B with a deletion of the C-terminal IDR (PPM1BΔIDR) were transfected into Rosetta 2 (DE3) cells and expression was induced with 0.5 mM of IPTG for 5 hours. Proteins were then purified using immobilized metal affinity, anion exchange, and size exclusion chromatography and labeled with the Alexa Fluor 647 far-red fluorescence dye. Buffered solution with either 15 µM of far-red labeled PPM1B or PPM1BΔIDR were imaged before and after the addition of hypercapnia (10% CO2) using fluorescence microscopy. Results: Far-red labelling of PPM1B and PPM1BΔIDR was confirmed with fluorescent western blotting and spectral scanning, and labelled proteins retained phosphatase activity. At baseline, recombinant proteins showed little to no biomolecule condensates at 0% CO2. After the addition of 10% CO2, PPM1B formed biomolecular condensates, however, no protein droplets were detected in PPM1BΔIDR. Conclusion: Preliminary data suggest hypercapnia triggers PPM1B phase separation in vitro, and that the CO2 responsive region of the protein is encoded within the C-terminal IDR. Future studies are warranted to test whether CO2 activates PPM1B phase separation in cells and identify the signaling pathways that are regulated by PPM1B during hypercapnia.

Liprin-α1 is a widely expressed scaffolding protein responsible for regulating cellular processes such as focal adhesion, cell motility, and synaptic transmission. Liprin-α1 interacts with many proteins including ELKS, GIT1, liprin-β, and LAR-family receptor tyrosine protein phosphatase. Through these protein-protein interactions, liprin-α1 assembles large higher-order molecular complexes; however, the regulation of this complex assembly/disassembly is unknown. Liquid-liquid phase separation (LLPS) is a process that concentrates proteins within cellular nano-domains to facilitate efficient spatiotemporal signaling in response to signaling cascades. While there is no report that liprin-α1 spontaneously undergoes LLPS, we found that GFP-liprin-α1 expressed in HEK293 cells occasionally forms droplet-like condensates. MS-based interactomics identified Protein Phosphatase 2A (PP2A)/B56δ (PPP2R5D) trimers as specific interaction partners of liprin-α1 through a canonical Short Linear Interaction Motif (SLiM) in its N-terminal dimerization domain. Mutation of this SLiM nearly abolished PP2A interaction, and resulted in significantly increased LLPS. GFP-liprin-α1 showed significantly increased droplet formation in HEK293 cells devoid of B56δ (PPP2R5D knockout), suggesting that PPP2R5D/PP2A holoenzyme inhibits liprin-α1 LLPS. Guided by reported liprin-α1 Ser/Thr phosphorylation sites, we found liprin-α1 phospho-mimetic mutant at serine 763 (S763E) is sufficient to drive its LLPS. Domain mapping studies of liprin-α1 indicated that the intrinsically disordered region, the N-terminal dimerization domain, and the SAM domains are all necessary for liprin-α1 LLPS. Finally, expression of p.E420K, a human PPP2R5D variant causing Houge-Janssens Syndrome type 1 (also known as Jordan's Syndrome), significantly compromised suppression of liprin-α1 LLPS. Our work identified B56δ-PP2A holoenzyme as an inhibitor of liprin-α1 LLPS via regulation at multiple phosphorylation sites.

Intrinsically disordered regions (IDR) and short linear motifs (SLiMs) play pivotal roles in the intricate signaling networks governed by phosphatases and kinases. B56δ (encoded by PPP2R5D) is a regulatory subunit of protein phosphatase 2A (PP2A) with long IDRs that harbor a substrate-mimicking SLiM and multiple phosphorylation sites. De novo missense mutations in PPP2R5D cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Our single-particle cryo-EM structures of the PP2A-B56δ holoenzyme reveal that the long, disordered arms at the B56δ termini fold against each other and the holoenzyme core. This architecture suppresses both the phosphatase active site and the substrate-binding protein groove, thereby stabilizing the enzyme in a closed latent form with dual autoinhibition. The resulting interface spans over 190 Å and harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is coupled to an allosteric network responsive to phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations increase the holoenzyme activity and perturb the phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the normal variant.

Genetic germline variants of PPP2R5D (encoding: phosphoprotein phosphatase 2 regulatory protein 5D) result in PPP2R5D-related disorder (Jordan's Syndrome), which is characterized by intellectual disability, hypotonia, seizures, macrocephaly, autism spectrum disorder, and delayed motor skill development. The disorder originates from de novo single nucleotide mutations, generating missense variants that act in a dominant manner. Pathogenic mutations altering 13 different amino acids have been identified, with the E198K variant accounting for similar to 40% of reported cases. However, the generation of a heterozygous E198K variant cell line to study the molecular effects of the pathogenic mutation has been challenging. Here, we use CRISPR-PRIME genomic editing to introduce a transition (c.592G>A) in a single PPP2R5D allele in HEK293 cells, generating E198K-heterozygous lines to complement existing E420K variant lines. We generate global protein and phosphorylation profiles of WT, E198K, and E420K cell lines and find unique and shared changes between variants and WT cells in kinase- and phosphatase-controlled signaling cascades. We observed ribosomal protein S6 (RPS6) hyperphosphorylation as a shared signaling alteration, indicative of increased ribosomal protein S6-kinase activity. Treatment with rapamycin or an RPS6-kinase inhibitor (LY2584702) suppressed RPS6 phosphorylation in both, suggesting upstream activation of mTORC1/p70S6K. Intriguingly, our data suggests ERK-dependent activation of mTORC1 in both E198K and E420K variant cells, with additional AKT-mediated mTORC1 activation in the E420K variant. Thus, although upstream activation of mTORC1 differs between PPP2R5D-related disorder genotypes, inhibition of mTORC1 or RPS6 kinases warrants further investigation as potential therapeutic strategies for patients.

PP2A-serine/threonine protein phosphatases function as heterotrimeric holoenzymes, composed of a common scaffold (A-subunit encoded by PPP2R1A/PPP2R1B), a common catalytic (C-subunit encoded by PPP2CA/PPP2CB), and one of many variable regulatory (B) subunits. The site of phosphoprotein phosphatase (PPP) hydrolysis features a bimetal system (M1/M2), an associated bridge hydroxide [W1(OH−)], and a highly-conserved core sequence. In the presumptive common mechanism, the phosphoprotein’s seryl/threonyl phosphate coordinates the M1/M2 system, W1(OH−) attacks the central P atom, rupturing the antipodal bond, and simultaneously, a histidine/aspartate tandem protonates the exiting seryl/threonyl alkoxide. Based on studies of PPP5C, a conserved arginine proximal to M1 is also expected to bind the substrate’s phosphate group in a bidentate fashion. However, in PP2A isozymes, the role of the arginine (Arg89) in hydrolysis is not clear because two independent structures for PP2A(PPP2R5C) and PP2A(PPP2R5D) show that Arg89 engages in a weak salt bridge at the B:C interface. These observations raise the question of whether hydrolysis proceeds with or without direct involvement of Arg89. The interaction of Arg89 with B:Glu198 in PP2A(PPP2R5D) is significant because the pathogenic E198K variant of B56δ is associated with irregular protein phosphorylation levels and consequent developmental disorders (Jordan’s Syndrome; OMIM #616355). In this study, we perform quantum-based hybrid [ONIOM(UB3LYP/6-31G(d):UPM7)] calculations on 39-residue models of the PP2A(PPP2R5D)/pSer (phosphoserine) system to estimate activation barriers for hydrolysis in the presence of bidentate Arg89-substrate binding and when Arg89 is otherwise engaged in the salt-bridge interaction. Our solvation-corrected results yield ΔH‡ ≈ ΔE‡ = +15.5 kcal/mol for the former case, versus +18.8 kcal/mol for the latter, indicating that bidentate Arg89-substrate binding is critical for optimal catalytic function of the enzyme. We speculate that PP2A(PPP2R5D) activity is suppressed by B:Glu198 sequestration of C:Arg89 under native conditions, whereas the PP2A(PPP2R5D)-holoenzyme containing the E198K variant has a positively-charged lysine in this position that alters normal function.

enrichment associated with known signaling networks, aberrant activation of mTORC1 signaling emerged from the data collected comparing wild type and variant cells, suggesting inhibitors of mTORC1 (e.g., rapamycin) may be useful to partially restore normal function to PPP2R5D E420K variant cells.

PDF file - 2199K, Figure 1S. Inhibition of PP4C mRNA and protein expression by treatment with antisense oligodeoxynucleotides. Figure 2S. Images selected from time-lapse live cell imaging of H2B-GFP expressing HeLa cells representing typical behavior of cell populations treated with antisense oligonucleotides targeting PP2ACα. Figure 3S. Comparison of the effects produced by fostriecin, mismatch controls, and antisense oligodeoxynucleotides targeting PP2ACα or PP4C. Figure 4S. Representative confocal microscopic images non-mitotic cells treated with antisense targeting PP4C.

Table S1. Comparison of the PPP5C-LB-100 model with known structures of PPP5C-inhibitor complexes; Supplementary Figure S1 shows. Isomorphous difference Fourier (Fo-Fo) map of PPP5C-LB-100 - PPP5C-4TE, created in PHENIX, contoured at 3σ. Red and green coloring indicate negative and positive density, respectively. The red arrow highlights the negative density for the C5 methyl in 4TE that is not present in LB-100. From the map, there is no apparent positive density for the methylpiperazine ring of LB-100; Supplementary Figure S2 shows the mass spectrometry analysis of LB-100 revealing a mass of 269.15, which corresponds to the mass of protonated LB-100; theoretical mass of 268.31.

An increasing number of mutations associated with devastating human diseases are diagnosed by whole-genome/exon sequencing. Recurrent de novo missense mutations have been discovered in B56δ (encoded by PPP2R5D), a regulatory subunit of protein phosphatase 2A (PP2A), that cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Single-particle cryo-EM structures show that the PP2A-B56δ holoenzyme possesses closed latent and open active forms. In the closed form, the long, disordered arms of B56δ termini fold against each other and the holoenzyme core, establishing dual autoinhibition of the phosphatase active site and the substrate-binding protein groove. The resulting interface spans over 190 Å and harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is close to an allosteric network responsive to activation phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations perturb the activation phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the wild variant.

Activating mutations in fibroblast growth factor receptor 3 (FGFR3) and inactivating mutations in the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase both result in decreased production of cyclic GMP in chondrocytes and severe short stature, causing achondroplasia (ACH) and acromesomelic dysplasia, type Maroteaux, respectively. Previously, we showed that an NPR2 agonist BMN-111 (vosoritide) increases bone growth in mice mimicking ACH (Fgfr3(Y367C/+)). Here, because FGFR3 signaling decreases NPR2 activity by dephosphorylating the NPR2 protein, we tested whether a phosphatase inhibitor (LB-100) could enhance BMN-111-stimulated bone growth in ACH. Measurements of cGMP production in chondrocytes of living tibias, and of NPR2 phosphorylation in primary chondrocytes, showed that LB-100 counteracted FGF-induced dephosphorylation and inactivation of NPR2. In ex vivo experiments with Fgfr3(Y367C/+) mice, the combination of BMN-111 and LB-100 increased bone length and cartilage area, restored chondrocyte terminal differentiation, and increased the proliferative growth plate area, more than BMN-111 alone. The combination treatment also reduced the abnormal elevation of MAP kinase activity in the growth plate of Fgfr3(Y367C/+) mice and improved the skull base anomalies. Our results provide a proof of concept that a phosphatase inhibitor could be used together with an NPR2 agonist to enhance cGMP production as a therapy for ACH.

Functional genomic approaches have facilitated the discovery of rare genetic disorders and improved efforts to decipher their underlying etiology. PPP2R5D-related disorder is an early childhood onset condition characterized by intellectual disability, hypotonia, autism-spectrum disorder, macrocephaly, and dysmorphic features. The disorder is caused by de novo single nucleotide changes in PPP2R5D, which generate heterozygous dominant missense variants. PPP2R5D is known to encode a B'-type (B'56δ) regulatory subunit of a PP2A-serine/threonine phosphatase. To help elucidate the molecular mechanisms altered in PPP2R5D-related disorder, we used a CRISPR-single-base editor to generate HEK-293 cells in which a single transition (c.1258G>A) was introduced into one allele, precisely recapitulating a clinically relevant E420K variant. Unbiased quantitative proteomic and phosphoproteomic analyses of endogenously expressed proteins revealed heterozygous-dominant changes in kinase/phosphatase signaling. These data combined with orthogonal validation studies revealed a previously unrecognized interaction of PPP2R5D with AKT in human cells, leading to constitutively active AKT-mTOR signaling, increased cell size, and uncoordinated cellular growth in E420K-variant cells. Rapamycin reduced cell size and dose-dependently reduced RPS6 phosphorylation in E420K-variant cells, suggesting that inhibition of mTOR1 can suppress both the observed RPS6 hyperphosphorylation and increased cell size. Together, our findings provide a deeper understanding of PPP2R5D and insight into how the E420K-variant alters signaling networks influenced by PPP2R5D. Our comprehensive approach, which combines precise genome editing, isobaric tandem mass tag labeling of peptides generated from endogenously expressed proteins, and concurrent liquid chromatography-mass spectrometry (LC-MS3), also provides a roadmap that can be used to rapidly explore the etiologies of additional genetic disorders.

RATIONALE:Vascular smooth muscle cells (SMCs) exhibit remarkable plasticity and can undergo dedifferentiation upon pathological stimuli associated with disease and interventions.OBJECTIVE:Although epigenetic changes are critical in SMC phenotype switching, a fundamental regulator that governs the epigenetic machineries regulating the fate of SMC phenotype has not been elucidated.METHODS AND RESULTS:Using SMCs, mouse models, and human atherosclerosis specimens, we found that FAK (focal adhesion kinase) activation elicits SMC dedifferentiation by stabilizing DNMT3A (DNA methyltransferase 3A). FAK in SMCs is activated in the cytoplasm upon serum stimulation in vitro or vessel injury and active FAK prevents DNMT3A from nuclear FAK-mediated degradation. However, pharmacological or genetic FAK catalytic inhibition forced FAK nuclear localization, which reduced DNMT3A protein via enhanced ubiquitination and proteasomal degradation. Reduced DNMT3A protein led to DNA hypomethylation in contractile gene promoters, which increased SMC contractile protein expression. RNA-sequencing identified SMC contractile genes as a foremost upregulated group by FAK inhibition from injured femoral artery samples compared with vehicle group. DNMT3A knockdown in injured arteries reduced DNA methylation and enhanced contractile gene expression supports the notion that nuclear FAK-mediated DNMT3A degradation via E3 ligase TRAF6 (TNF [tumor necrosis factor] receptor-associated factor 6) drives differentiation of SMCs. Furthermore, we observed that SMCs of human atherosclerotic lesions exhibited decreased nuclear FAK, which was associated with increased DNMT3A levels and decreased contractile gene expression.CONCLUSIONS:This study reveals that nuclear FAK induced by FAK catalytic inhibition specifically suppresses DNMT3A expression in injured vessels resulting in maintaining SMC differentiation by promoting the contractile gene expression. Thus, FAK inhibitors may provide a new treatment option to block SMC phenotypic switching during vascular remodeling and atherosclerosis.

Serine/threonine protein phosphatase-5 (PP5; PPP5C) is a member of the phosphoprotein phosphatase (PPP) gene family. The PPP catalytic domains feature a bimetal system (M1/M2), an associated bridge hydroxide (W1(OH−)), an M1-bound water/hydroxide (W2), and a highly conserved core sequence. The PPPs are presumed to share a common mechanism: The seryl/threonyl phosphoryl group of the phosphoprotein coordinates the metal ions, W1(OH−) attacks the central phosphorous atom, rupturing the antipodal phosphoester bond and releasing the phosphate-free protein. Also, a histidine/aspartate tandem is responsible for protonating the exiting seryl/threonyl alkoxide. Here, we employed quantum-based computations on a large section of the PP5 catalytic site. A 33-residue, ONIOM(UB3LYP/6-31G(d):UPM7) model was built to perform computations using methylphosphate dianion as a stand-in substrate for phosphoserine/phosphothreonine. We present a concerted transition state (TS) in which W1(OH−) attacks the phosphate center at the same time that the exiting seryl/threonyl alkoxide is protonated directly by the His304/Asp274 tandem, with W2 assigned as a water molecule: W2(H2O). Arg275, proximal to M1, stabilizes the substrate and TS by binding both the ester oxygen (Oγ) and a phosphoryl oxygen (O1) in a bidentate fashion; in the product state, Tyr451 aids in decoupling Arg275 from O1 of the product phosphate ion. The reaction is exothermic (ΔH = −2.0 kcal/mol), occurs in a single step, and has a low activation barrier (ΔH‡ = +10.0 kcal/mol). Our work is an improvement over an earlier computational study that also found bond rupture and alkoxide protonation to be concerted, but concluded that Arg275 is deprotonated during the reactant and TS stages of the pathway. In that earlier study, the critical electron-withdrawal role that Arg275 plays during the hydroxide attack was not correctly accounted for.

ABSTRACTActivating mutations in fibroblast growth factor receptor 3 (FGFR3) and inactivating mutations in the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase both result in decreased production of cyclic GMP (cGMP) in chondrocytes and severe short stature, causing achondroplasia (ACH) and acrosomelic dysplasia type Maroteaux, respectively. Previously we showed that an NPR2 agonist BMN-111 (vosoritide) increases bone growth in mice mimicking ACH (Fgfr3Y367C/+), and that in control growth plate chondrocytes, FGFR3 signaling decreases NPR2 activity by dephosphorylating the NPR2 protein. Here we tested whether a phosphatase inhibitor (LB-100) could enhance bone growth in ACH. Inex vivoimaging experiments using a FRET sensor to measure cGMP production in chondrocytes of living tibias from newborn mice, LB-100 counteracts the FGF-induced dephosphorylation and inactivation of NPR2. Inex vivoexperiments withFgfr3Y367C/+mice, LB-100 in combination with BMN-111 increases the rate of femur growth by ∼25% vs BMN-111 alone, restores chondrocyte terminal differentiation, increases the proliferative growth plate area of the femur, and reduces the activity of the MAP kinase pathway. Our results provide a proof of concept that a phosphatase inhibitor could be used together with an NPR2 agonist to enhance cGMP production as a therapy for ACH.GRAPHICAL ABSTRACT

During inflammation, mediators increase calcium influx into endothelial cells through store‐operated calcium entry (SOCE) channels. Increasing calcium influx through the SOCE current ISOC promotes endothelial barrier disruption through the formation of inter‐endothelial cell gaps. Inter‐endothelial cell gap formation leads to increased permeability. Identifying mechanisms of ISOC inhibition may aid in the development of therapeutic strategies against endothelial permeability‐associated vascular inflammation. Our studies indicate FK506‐binding protein 51 (FKBP51), in conjunction with protein phosphatase 5 (PPP5C), inhibit ISOC and calcium entry‐induced inter‐endothelial cell gap formation. Further, we determined that the calcium‐binding protein S100A6 is necessary for the PPP5C‐FKBP51‐mediated inhibition of ISOC in pulmonary microvascular endothelial cells (PMVECs). In PMVECs, S100A6 interacts with the ISOC channel in a calcium‐dependent manner, i.e., following an increase in cytosolic calcium. However, we do not know whether calcium entry specifically through the ISOC channel or through other SOCE channels that provides the calcium source to promote S100A6 interaction with the ISOC channel.MethodsPMVECs were used for co‐precipitation and immunocytochemistry‐based studies. When cells are treated with rolipram (R) plus thapsigargin (Tg), ISOC and other SOCE channels are activated. However, when cells are treated with Tg alone, ISOC is not activated, but other SOCE channels are activated. The TRPC4 subunit of the ISOC channel was immunoprecipitated and probed for co‐precipitation of S100A6.ResultsFollowing R/Tg treatment, robust co‐precipitation of S100A6 with TRPC4 was observed, whereas following Tg alone only minor co‐precipitation was seen. Immunocytochemistry revealed that in untreated cells S100A6 localization is principally cytosolic. Following R/Tg treatment, but not Tg alone, a distinct membrane‐delimited pool of S100A6 was observed, particularly at sites of cell‐cell contact. Membrane localization was confirmed by co‐localization with wheat germ agglutinin (WGA).ConclusionOverall, our data reveal that calcium entry specifically through the ISOC channel is an important determinant of S100A6 translocation to the plasma membrane promoting interaction of S100A6 with the ISOC channel.Support or Funding InformationNIH HL R56HL107778

ISOC is a cation current permeating the ISOC channel. In pulmonary endothelial cells, ISOC activation leads to formation of inter-endothelial cell gaps and barrier disruption. The immunophilin FK506-binding protein 51 (FKBP51), in conjunction with the serine/threonine protein phosphatase 5C (PPP5C), inhibits ISOC . Free PPP5C assumes an autoinhibitory state, which has low "basal" catalytic activity. Several S100 protein family members bind PPP5C increasing PPP5C catalytic activity in vitro. One of these family members, S100A6, exhibits a calcium-dependent translocation to the plasma membrane. The goal of this study was to determine whether S100A6 activates PPP5C in pulmonary endothelial cells and contributes to ISOC inhibition by the PPP5C-FKBP51 axis. We observed that S100A6 activates PPP5C to dephosphorylate tau T231. Following ISOC activation, cytosolic S100A6 translocates to the plasma membrane and interacts with the TRPC4 subunit of the ISOC channel. Global calcium entry and ISOC are decreased by S100A6 in a PPP5C-dependent manner and by FKBP51 in a S100A6-dependent manner. Further, calcium entry-induced endothelial barrier disruption is decreased by S100A6 dependent upon PPP5C, and by FKBP51 dependent upon S100A6. Overall, these data reveal that S100A6 plays a key role in the PPP5C-FKBP51 axis to inhibit ISOC and protect the endothelial barrier against calcium entry-induced disruption.