Protein Phosphatase Type Research Articles

TAF-Iβ deficiency inhibits proliferation and promotes apoptosis by rescuing PP2A and inhibiting the AKT/GSK-3β pathway in leukemic cells.

Template-activating factor Iβ (TAF-Iβ) has been associated with numerous pathophysiological processes and has been reported as an oncogene responsible for the regulation of important signaling pathways in various types of solid tumor; however, few studies have investigated the role of TAF-Iβ in leukemia. The present study reported the upregulated expression of TAF-Iβ in 36 patients with acute leukemia and six leukemic cell lines. In addition, TAF-Iβ-knockdown (KD) cells were generated via RNA interference. TAF-Iβ KD not only inhibited the proliferation of leukemia cells but also induced apoptosis. Furthermore, it was revealed that the mechanism underlying these effects may be associated with the upregulation of protein phosphatase type 2A and inhibition of the protein kinase B/glycogen synthase kinase-3β signaling pathway. Collectively, the findings demonstrated that TAF-Iβ serves an important role in various types of leukemia and may be considered as a potential therapeutic target for the treatment of leukemia.

Essential role of GEXP15, a specific Protein Phosphatase type 1 partner, in Plasmodium berghei in asexual erythrocytic proliferation and transmission.

The essential and distinct functions of Protein Phosphatase type 1 (PP1) catalytic subunit in eukaryotes are exclusively achieved through its interaction with a myriad of regulatory partners. In this work, we report the molecular and functional characterization of Gametocyte EXported Protein 15 (GEXP15), a Plasmodium specific protein, as a regulator of PP1. In vitro interaction studies demonstrated that GEXP15 physically interacts with PP1 through the RVxF binding motif in P. berghei. Functional assays showed that GEXP15 was able to increase PP1 activity and the mutation of the RVxF motif completely abolished this regulation. Immunoprecipitation assays of tagged GEXP15 or PP1 in P. berghei followed by immunoblot or mass spectrometry analyses confirmed their interaction and showed that they are present both in schizont and gametocyte stages in shared protein complexes involved in the spliceosome and proteasome pathways and known to play essential role in parasite development. Phenotypic analysis of viable GEXP15 deficient P. berghei blood parasites showed that they were unable to develop lethal infection in BALB/c mice or to establish experimental cerebral malaria in C57BL/6 mice. Further, although deficient parasites produced gametocytes they did not produce any oocysts/sporozoites indicating a high fitness cost in the mosquito. Global proteomic and phosphoproteomic analyses of GEXP15 deficient schizonts revealed a profound defect with a significant decrease in the abundance and an impact on phosphorylation status of proteins involved in regulation of gene expression or invasion. Moreover, depletion of GEXP15 seemed to impact mainly the abundance of some specific proteins of female gametocytes. Our study provides the first insight into the contribution of a PP1 regulator to Plasmodium virulence and suggests that GEXP15 affects both the asexual and sexual life cycle.

PP2A: A Promising Biomarker and Therapeutic Target in Endometrial Cancer.

Over the last decade, the use of targeted therapies has immensely increased in the treatment of cancer. However, treatment for endometrial carcinomas (ECs) has lagged behind, although potential molecular markers have been identified. This is particularly problematic for the type II ECs, since these aggressive tumors are usually not responsive toward the current standard therapies. Therefore, type II ECs are responsible for most EC-related deaths, indicating the need for new treatment options. Interestingly, molecular analyses of type II ECs have uncovered frequent genetic alterations (up to 40%) in PPP2R1A, encoding the Aα subunit of the tumor suppressive heterotrimeric protein phosphatase type 2A (PP2A). PPP2R1A mutations were also reported in type I ECs and other common gynecologic cancers, albeit at much lower frequencies (0–7%). Nevertheless, PP2A inactivation in the latter cancer types is common via other mechanisms, in particular by increased expression of Cancerous Inhibitor of PP2A (CIP2A) and PP2A Methylesterase-1 (PME-1) proteins. In this review, we discuss the therapeutic potential of direct and indirect PP2A targeting compounds, possibly in combination with other anti-cancer drugs, in EC. Furthermore, we investigate the potential of the PP2A status as a predictive and/or prognostic marker for type I and II ECs.

Plasmodium pseudo-Tyrosine Kinase-like binds PP1 and SERA5 and is exported to host erythrocytes

Pseudokinases play key roles in many biological processes but they are poorly understood compared to active kinases. Eight putative pseudokinases have been predicted in Plasmodium species. We selected the unique pseudokinase belonging to tyrosine kinase like (TKL) family for detailed structural and functional analysis in P. falciparum and P. berghei. The primary structure of PfpTKL lacks residues critical for kinase activity, supporting its annotation as a pseudokinase. The recombinant pTKL pseudokinase domain was able to bind ATP, but lacked catalytic activity as predicted. The sterile alpha motif (SAM) and RVxF motifs of PfpTKL were found to interact with the P. falciparum proteins serine repeat antigen 5 (SERA5) and protein phosphatase type 1 (PP1) respectively, suggesting that pTKL has a scaffolding role. Furthermore, we found that PP1c activity in a heterologous model was modulated in an RVxF-dependent manner. During the trophozoite stages, PbpTKL was exported to infected erythrocytes where it formed complexes with proteins involved in cytoskeletal organization or host cell maturation and homeostasis. Finally, genetic analysis demonstrated that viable strains obtained by genomic deletion or knocking down PbpTKL did not affect the course of parasite intra-erythrocytic development or gametocyte emergence, indicating functional redundancy during these parasite stages.

Spatial Organization of a cAMP/Ca2+‐Regulated Signaling Complex: A Structural Study of an AKAP79‐Scaffolded Complex Containing Type IIβ PKA and Calcineurin

cAMP‐dependent protein kinase (PKA) regulates many cell processes including development, differentiation, and memory. In its resting state, PKA is a tetrameric holoenzyme consisting of a regulatory (R) subunit dimer and two catalytic (C) subunits. Four isoforms of the R‐subunit exist (RIα, RIβ, RIIα, and RIIβ). Although each isoform varies in sequence and quaternary structure, all share a common domain organization. The strength, duration, and specificity of PKA activity within the cell are mostly dependent upon isoform‐specific subcellular localization. The D/D domain interface of the R‐ subunits serves as a docking site for A Kinase Anchoring Proteins (AKAPs) that target PKA to specific cellular locations. In cardiac myocytes and neurons, PKA is localized to Cav1.2 L‐type calcium channels by AKAP79 and up‐regulates calcium influx by phosphorylating the channel's cytosolic C‐terminal tail. AKAP79 also binds the Ca2+/calmodulin‐dependent protein phosphatase type 2B (PP2B, or calcineurin) at a site very close to that of PKA binding. Given the close proximity of the calcineurin and PKA binding sites on AKAP79, calcineurin has been suggested to play roles in the regulation of calcium channels, along with regulation of PKA activity itself. Using a variety of biochemical and biophysical techniques, we have isolated and characterized a stable complex of the C‐terminal tail of AKAP79, RIIβ holoenzyme, and calciuneurin (“ARC” for short). We show a stoichiometry of 1:1:1 (AKAP79ct: RIIβ2‐C2 :CNt) using native mass spectroscopy, and SAXS/SANS techniques confirm that this is a compact complex, suggesting a close interaction between PKA and calcineurin when bound to AKAP79. Altogether, we suggest beyond its function of localizing PKA and CN in close spatial proximity to the Cav1.2 L‐type calcium channel, the ARC signalosome fosters extended protein‐protein interactions and regulations that suggest tight spatiotemporal control. Support for this work was provided through NIH GM034921.Support or Funding InformationJames Hall is an IRACDA fellow and was supported by NIGMS/NIH award K12GM068524. This work was also supported by a grant from the National Institutes of Health GM034921 to S.S. Taylor. This Research used resources at the High Flux Isotope Reactor and Spallation Neutron Source, which are DOE Office of Science User Facilities operated by Oak Ridge National Laboratory. This work utilized SasView software, originally developed by the DANSE project under NSF award DMR‐0520547.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Biological activities of Chromolena odorata (L.) King and Robinson (Asteraceae) collected from Sabah, Malaysia as protein phosphatase type-1 inhibitor

Chromolena Odorata has been traditionally used as wound healer in local community. The present study investigated the anti-kinase and anti-phosphatases activities on methanolic C. odorata extract. Mutant yeast strains used are MKK1P386, MKK1P386_MSG5, PAY704-1 and PAY700-4. Bioassay guided fractionation of C. odorata revealed positive activities of hexane, ethyl acetate and chloroform partitions. Column chromatography of all partitionates later confirmed fraction F2 from chloroform extract had most favorable activity with inhibitory zone ranged between 7±0.0 mm until 15±0.0 mm. Kinetic analysis including maximum enzyme velocity (Vm) and Michealis-Menten constant (Km) were evaluated and compared for both normal and inhibited reactions. Enzyme activity with DiFMUP as substrate showed fraction F2 act as PP1 enzyme inhibitor with the Km value 0.60 mM and Vm value 200 mM/min as compared to the normal enzymatic reaction. Results provided unveil the potential of C. odorata as an effective therapeutic agent.

Biological activities of Chromolena odorata (L.) King and Robinson (Asteraceae) collected from Sabah, Malaysia as protein phosphatase type-1 inhibitor

Biological activities of Chromolena odorata (L.) King and Robinson (Asteraceae) collected from Sabah, Malaysia as protein phosphatase type-1 inhibitor

γ-Tocotrienol-Inhibited Cell Proliferation of Human Gastric Cancer by Regulation of Nuclear Factor-κB Activity.

γ-Tocotrienol (γ-T3) exhibits the activity of anticancer via regulating cell signaling pathways. Nuclear factor-κB (NF-κB), one of the crucial pro-inflammatory factors, is involved in the regulation of cell proliferation, apoptosis, invasion, and migration of tumor. In the present study, NF-κB activity inhibited by γ-T3 was investigated in gastric cancer cells. Cell proliferation, NF-κB activity, active protein phosphatase type 2A (PP2A), and ataxia-telangiectasia mutated (ATM) protein were explored using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), methylene blue, enzyme-linked immunosorbent assay (ELISA), malachite green, luciferase, and Western blotting assays. The effects of γ-T3 on tumor growth and the expression of NF-κB and PP2A proteins were also further examined by implanting human gastric cancer cells in a BALB/c nude mouse model. The results showed that γ-T3 significantly inhibited the cell proliferation and attenuated the NF-κB activity in vitro and in vivo. γ-T3 dramatically increased PP2A activity and protein expression, which suppressed ATM phosphorylation and its translocation to the cytoplasm in gastric cancer cells. Thus, our findings may provide mechanistic insight into effects of γ-T3 on the regulation of NF-κB activity by a PP2A-dependent mechanism and suggest that PP2A may serve as a molecular target for a potential chemopreventive agent.

Characterization of a Protein Phosphatase Type-1 and a Kinase Anchoring Protein in Plasmodium falciparum.

With its multiple regulatory partners, the conserved Protein Phosphatase type-1 (PP1) plays a central role in many functions of the biology of eukaryotic cells, including Plasmodium falciparum. Here, we characterized a protein named PfRCC-PIP, as a major partner of PfPP1. We established its direct interaction in vitro and its presence in complex with PfPP1 in the parasite. The use of Xenopus oocyte model revealed that RCC-PIP can interact with the endogenous PP1 and act in synergy with suboptimal doses of progesterone to trigger oocyte maturation, suggesting a regulatory effect on PP1. Reverse genetic studies suggested an essential role for RCC-PIP since no viable knock-out parasites could be obtained. Further, we demonstrated the capacity of protein region containing RCC1 motifs to interact with the parasite kinase CDPK7. These data suggest that this protein is both a kinase and a phosphatase anchoring protein that could provide a platform to regulate phosphorylation/dephosphorylation processes.

Interactions of cantharidin-like inhibitors with human protein phosphatase-5 in a Mg2+ system: molecular dynamics and quantum calculations.

The serine/threonine protein phosphatase type 5 (PP5) is a promising target for designing new antitumor drugs. This enzyme is a member of the PPP phosphatases gene family, which catalyzes a dephosphorylation reaction: a regulatory process in the signal transduction pathway that controls various biological processes. The aim of this work is to study and compare the inhibition of PP5 by ten cantharidin-like inhibitors in order to bring about contributions relevant to the better comprehension of their inhibitory activity. In this theoretical investigation, we used molecular dynamics techniques to understand the role of key interactions that occur in the protein active site; QM calculations were employed to study the interaction mode of these inhibitors in the enzyme. In addition, atoms in molecules (AIM) calculations were carried out to characterize the chemical bonds among the atoms involved and investigate the orbital interactions with their respective energy values. The obtained results suggest that the Arg275, Asn303, His304, His352, Arg400, His427, Glu428, Val429, Tyr451, and Phe446 residues favorably contribute to the interactions between inhibitors and PP5. However, the Asp271 and Asp244 amino acid residues do not favor such interactions for some inhibitors. Through the QM calculations, we can suggest that the reactional energy of the coordination mechanism of these inhibitors in the PP5 active site is quite important and is responsible for the inhibitory activity. The AIM technique employed in this work was essential to get a better comprehension of the transition states acquired from the mechanism simulation. This work offers insights of how cantharidin-like inhibitors interact with human PP5, potentially allowing the design of more specific and even less cytotoxic drugs for cancer treatments. Graphical Abstract Interactions of cantharidin-like inhibitors with human protein phosphatase-5 in a Mg2+ system.

Virtual screening and bioassay of specific potential inhibitors for Arabidopsis group A protein phosphatases type 2C

Virtual screening and bioassay of specific potential inhibitors for Arabidopsis group A protein phosphatases type 2C

Sensor system based on flexible screen-printed electrodes for electrochemical detection of okadaic acid in seawater

The monitoring of marine dinophysistoxin okadaic acid in seawater can serve as an early alert system for preventing the potential negative effects this toxin can have on the food industry and human health in general. A disposable sensor system for electrochemical detection of this toxin has been developed using screen-printed electrodes. The method described is based on the inhibition of protein phosphatase type 2A by okadaic acid, evaluating the enzyme activity. p-Nitrophenyl phosphate, 4-methylumbelliferyl phosphate and phenyl phosphate have been tested as substrates achieving good limits of detection of 2.7·10–12 M of okadaic acid. The proposed method has been successfully applied to okadaic acid determination in seawater samples. A study of divalent cations present in seawater that can interfere with the measurement has been carried out. The electrode systems were printed on both rigid and textile backing materials to observe the influence of those materials on the final performance of the sensor.

Glc8, a regulator of protein phosphatase type 1, mediates oxidation tolerance, asexual development and virulence in Beauveria bassiana, a filamentous entomopathogenic fungus.

Protein phosphatase type 1 (PP1) plays an important role in cellular metabolism and development in yeast. In PP1 enzyme complex, Glc8 protein is a global regulatory subunit and regulates many physiological processes. However, its biological roles are unexplored in filamentous fungi. In this study, we characterized a yeast ortholog of Glc8 in Beauveria bassiana, a filamentous entomopathogenic fungus. Gene disruption of BbGlc8 had no significant effect on vegetative growth, but resulted in a significant reduction in conidiation (51%) and blastospore yield (55%) in the mutant. The ΔBbGlc8 mutant displayed an enhanced sensitivity to oxidative stress and a weakened virulence as indicated by cuticle infection and intrahemocoel injection assays. Transcriptomic analysis indicated that the genes regulated by BbGlc8 during conidiation were primarily associated with metabolism, cell rescue and cell wall formation. Notably, as a down-regulated gene in ΔBbGlc8 mutant, BbOsmC2 (a member of OsmC protein family) contributes to fungal resistance to salt stress, spore differentiation and virulence. Thus, BbOsmC2 functions as a down-stream target of BbGlc8 during spore differentiation, but not in stress response. Our findings indicate that BbGlc8 contributes to the biocontrol potential of B. bassiana by mediating comprehensive genetic pathways.

Targeted Therapies in Type II Endometrial Cancers: Too Little, but Not Too Late.

Type II endometrial carcinomas (ECs) are responsible for most endometrial cancer-related deaths due to their aggressive nature, late stage detection and high tolerance for standard therapies. However, there are no targeted therapies for type II ECs, and they are still treated the same way as the clinically indolent and easily treatable type I ECs. Therefore, type II ECs are in need of new treatment options. More recently, molecular analysis of endometrial cancer revealed phosphorylation-dependent oncogenic signalling in the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways to be most frequently altered in type II ECs. Consequently, clinical trials tested pharmacologic kinase inhibitors targeting these pathways, although mostly with rather disappointing results. In this review, we highlight the most common genetic alterations in type II ECs. Additionally, we reason why most clinical trials for ECs using targeted kinase inhibitors had unsatisfying results and what should be changed in future clinical trial setups. Furthermore, we argue that, besides kinases, phosphatases should no longer be ignored in clinical trials, particularly in type II ECs, where the tumour suppressive phosphatase protein phosphatase type 2A (PP2A) is frequently mutated. Lastly, we discuss the therapeutic potential of targeting PP2A for (re)activation, possibly in combination with pharmacologic kinase inhibitors.

Abstract 3713: Targeting β-catenin pathway by nanoparticle-mediated delivery of norcantharidin impairs the stemness of triple negative breast cancer cells

Abstract Background Norcantharidin (NCTD) is known as an inhibitor of serine/threonine protein phosphatase types 1 and 2A (PP1/PP2A) and exhibits potent anticancer efficacies. Our preliminary study identified NCTD interfering with the interaction between phospho-β-catenin and PP1/PP2A could downregulate the levels of non-phospho β-catenin in the cytoplasm in Triple Negative Breast Cancer (TNBC) cells. As β-catenin pathway plays an important role in cancer stemness, we hypothesize that NCTD could prevent β-catenin nuclear translocation, impair the stemness and reduce the metastasis of TNBC. However, the inherent features of short half-life and nephrotoxicity hinder NCTD's further in vivo application. The goal of this study was to test the therapeutic effect of targeting β-catenin pathway by nanoparticle-mediated delivery of NCTD against TNBC. Experimental Design NCTD was loaded into an integrin α5 targeting nanoparticle (NP) (RGD-LPH) via complexing with polyethyleneimine. The targeting efficiency of RGD-LPH was tested by the in vivo imaging of RGD-LPH labeled by cyanine 7.5 (cy7.5). The effects of NP-mediated delivery NCTD on TNBC cell malignant behaviors and tumor metastasis were tested in various in vitro and in vivo models. Results We found that RGD-LPH NP-mediated delivery of NCTD significantly reduced the active and total β-catenin levels as evidenced by Western Blot. The self-renewal capacity was remarkably blocked by RGD-LPH, as examined by sphere formation. RGD-LPH also suppressed features of cancer cells related to proliferation, as examined by MTT, clonogenic assay and soft agar colony formation. In particular, compared to free NCTD, RGD-LPH demonstrated extended-release profile and long-last effect in the long-term experiments such as clonogenic assay and soft agar colony formation assay. In vivo, systemic delivery of RGD-LPH(cy7.5) indicated a preferential uptake in primary tumor and lung of the tumor-bearing mice. Efficacy studies indicated reduced metastasis of mice from RGD-LPH treatment. Conclusion: Collectively, our results strongly suggest that NCTD being encapsulated in RGD-LPH, which warrants further development for preclinical and clinical studies as a novel TNBC therapeutic approach, is a robust modulator of Wnt signaling with anti-metastasis activity. Citation Format: Yunfei Li, Zhishan Wang, Yajuan Xiao, Chengfeng Yang. Targeting β-catenin pathway by nanoparticle-mediated delivery of norcantharidin impairs the stemness of triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3713.

Molecular identification of BrHAB2a, one of the two AtHAB2-like proteins in Brassica rapa, is an important component of ABA signaling

Abscisic acid (ABA) signaling is a vital physiological step that is used by many land plants to fight against environmental stress. As components of the linear ABA signaling pathway, clade A protein phosphatases type 2C (PP2C-As) are mainly inhibited by PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYLs)-type receptors upon their binding to ABA. Here, we show that the genome of Brassica rapa encodes 14 putative clade A PP2C-like proteins (BrPP2C-As). Two of these BrPP2C-As, Bra025964 and Bra016595, show high similarity to the HAB2 (Homology to ABI2) protein in Arabidopsis. RNAseq data reveal that nearly all BrPP2C-As, like BrHAB2a (Bra025964) and BrHAB2b (Bra016595), were highly expressed in at least one tissue. Overexpression of BrHAB2a conferred ABA insensitivity to Arabidopsis thaliana seedlings. Furthermore, the phosphatase activity of BrHAB2a could be inhibited by AtPYL1 or BrPYL1 in the presence of ABA. Overall, these results suggest that BrHAB2a is a functional PP2C-A like protein phosphotase and a key component of ABA signaling in Brassica rapa.

Successful overexpression of wild-type inhibitor-2 of PP1 in cardiovascular cells.

About half of the cardiac serine/threonine phosphatase activity is due to the activity of protein phosphatase type 1 (PP1). The activity of PP1 can be inhibited by an endogenous protein for which the expression inhibitor-2 (I-2) has been coined. We have previously described a transgenic mouse overexpressing a truncated form of I-2. Here, we have described and initially characterized several founders that overexpress the non-truncated (i.e., full length) I-2 in the mouse heart (TG) and compared them with non-transgenic littermates (WT). The founder with the highest overexpression of I-2 displayed under basal conditions no difference in contractile parameters (heart rate, developed tension, and its first derivate) compared to WT. The relative level of PP1 inhibition was similar in mice overexpressing the non-truncated as well as the truncated form of I-2. For comparison, we overexpressed I-2 by an adenoviral system in several cell lines (myocytes from a tumor-derived cell line (H9C2), neonatal rat cardiomyocytes, smooth muscle cells from rat aorta (A7R5)). We noted gene dosage-dependent staining for I-2 protein in infected cells together with reduced PP1 activity. Finally, I-2 expression in neonatal rat cardiomyocytes led to an increase of Ca2+ transients by about 60%. In summary, we achieved immunologically confirmed overexpression of wild-type I-2 in cardiovascular cells which was biochemically able to inhibit PP1 in the whole heart (using I-2 transgenic mice) as well as in isolated cells including cardiomyocytes (using I-2 coding virus) indirectly underscoring the importance of PP1 for cardiovascular function.

Forskolin increases the effect of everolimus on aromatase inhibitor-resistant breast cancer cells.

Aromatase inhibitor (AI) resistance is a major obstacle in the treatment of estrogen receptor-positive breast cancer. Everolimus (EVE) ameliorates AI-resistant breast cancer and is therefore used in cancer treatment. However, some patients show resistance to EVE. Here, we used 30 clones of long-term estrogen-deprived (LTED) MCF-7 cells as a model of AI-resistant breast cancer. We examined changes in protein phosphatase type 2A (PP2A) and cancerous inhibitor of PP2A (CIP2A), a negative regulator of PP2A, in LTED cells treated with EVE. In LTED cells with high sensitivity to EVE, CIP2A expression decreased at low EVE concentrations; however, in LTED cells poorly sensitive to EVE, CIP2A and PP2A did not change upon exposure to EVE. Therefore, we hypothesized that there is a relation between expression of CIP2A and sensitivity to EVE. Knockdown of CIP2A increased the sensitivity to EVE in three clones poorly sensitive to EVE. Additionally, we found that treatment with FSK, which activates PP2A, increased the sensitivity of the cells to EVE. Our data point to CIP2A and PP2A as novel therapeutic targets for AI-resistant breast cancer.

Marine Longilenes, Oxasqualenoids with Ser-Thr Protein Phosphatase 2A Inhibition Activity.

The red seaweed Laurencia viridis is a rich source of oxygenated secondary metabolites that were derived from squalene. We report here the structures of three novel compounds, (+)-longilene peroxide (1), longilene (2), and (+)-prelongilene (3) that were isolated from this alga, in addition to other substances, 4 and 5, resulting from their acid-mediated degradation. The effect of compounds 1 and 3 against Ser-Thr protein phosphatase type 2A (PP2A) was evaluated, showing that (+)-longilene peroxide (1) inhibited PP2A (IC50 11.3 μM). In order to explain the interaction between PP2A and compounds 1 and 3, molecular docking simulations onto the PP2A enzyme-binding region were used.

Spatial Organization of a cAMP/Ca2+‐Regulated Signaling Complex: A Solution Structural Small‐Angle X‐ray and Neutron Scattering Study of an AKAP79‐Scaffolded Complex Containing Type IIβ PKA and Calcineurin

cAMP‐dependent protein kinase (PKA) regulates many cell processes including development, differentiation, and memory. In its resting state, PKA is a tetrameric holoenzyme consisting of a regulatory (R) subunit dimer and two catalytic (C) subunits. Four isoforms of the R‐subunit exist (RIα, RIβ, RIIα, and RIIβ). Although each isoform varies in sequence and quaternary structure, all share a common domain organization. The strength, duration, and specificity of PKA activity within the cell are mostly dependent upon isoform specific subcellular localization. The D/D domain interface of the R‐subunits serves as a docking site for A Kinase Anchoring Proteins (AKAPs) that target PKA to specific cellular locations. In cardiac myocytes and neurons, PKA is localized to Cav1.2 L‐type calcium channels by AKAP79 and up‐regulates calcium influx by phosphorylating the channel's cytosolic C‐terminal tail. AKAP79 also binds the Ca2+/calmodulin‐dependent protein phosphatase type 2B (PP2B, or calcineurin) at a site very close to that of PKA binding. Given the close proximity of the calcineurin and PKA binding sites on AKAP79, calcineurin has been suggested to play roles in regulation of calcium channels, along with regulation of PKA activity itself. Using a variety of biochemical and biophysical techniques, we have isolated and characterized a stable complex of the C‐terminal tail of AKAP79, RIIβ holoenzyme, and calcuneurin (“ARC” for short). We show a stoichiometry of 1:1:1 (AKAP79: RIIβ2C2:CN) using native mass spectroscopy, and SAXS/SANS techniques confirm that this is a compact complex, suggesting a close interaction between PKA and calcineurin when bound to AKAP79. Altogether, we suggest beyond its function of localizing PKA and CN in close spatial proximity to the Cav1.2 L‐type calcium channel, the ARC signalosome fosters extended protein‐protein interactions and regulations that suggest tight spatiotemporal control.Support or Funding InformationJames Hall is a San Diego IRACDA Fellow supported by National Institutes of Health (NIH)/NIGMS K12 GM06852 Award. Support for this work was provided through NIH GM034921.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.