Protein phosphatase 2A - structure, function and role in neurodevelopmental disorders.

Neurodevelopmental disorders (NDDs), including intellectual disability (ID), autism and schizophrenia, have high socioeconomic impact, yet poorly understood etiologies. A recent surge of large-scale genome or exome sequencing studies has identified a multitude of mostly de novo mutations in subunits of the protein phosphatase 2A (PP2A) holoenzyme that are strongly associated with NDDs. PP2A is responsible for at least 50% of total Ser/Thr dephosphorylation in most cell types and is predominantly found as trimeric holoenzymes composed of catalytic (C), scaffolding (A) and variable regulatory (B) subunits. PP2A can exist in nearly 100 different subunit combinations in mammalian cells, dictating distinct localizations, substrates and regulatory mechanisms. PP2A is well established as a regulator of cell division, growth, and differentiation, and the roles of PP2A in cancer and various neurodegenerative disorders, such as Alzheimer's disease, have been reviewed in detail. This Review summarizes and discusses recent reports on NDDs associated with mutations of PP2A subunits and PP2A-associated proteins. We also discuss the potential impact of these mutations on the structure and function of the PP2A holoenzymes and the etiology of NDDs.

S-acylation-dependent membrane microdomain localization of the regulatory Kvß2.1 subunit.

The voltage-dependent potassium (Kv) channel Kvß family was the first identified group of modulators of Kv channels. Kvß regulation of the α-subunits, in addition to their aldoketoreductase activity, has been under extensive study. However, scarce information about their specific α-subunit-independent biology is available. The expression of Kvßs is ubiquitous and, similar to Kv channels, is tightly regulated in leukocytes. Although Kvß subunits exhibit cytosolic distribution, spatial localization, in close contact with plasma membrane Kv channels, is crucial for a proper immune response. Therefore, Kvß2.1 is located near cell surface Kv1.3 channels within the immunological synapse during lymphocyte activation. The objective of this study was to analyze the structural elements that participate in the cellular distribution of Kvßs. It was demonstrated that Kvß peptides, in addition to the cytoplasmic pattern, targeted the cell surface in the absence of Kv channels. Furthermore, Kvß2.1, but not Kvß1.1, targeted lipid raft microdomains in an S-acylation-dependent manner, which was concomitant with peptide localization within the immunological synapse. A pair of C-terminal cysteines (C301/C311) was mostly responsible for the specific palmitoylation of Kvß2.1. Several insults altered Kvß2.1 membrane localization. Therefore, growth factor-dependent proliferation enhanced surface targeting, whereas PKC activation impaired lipid raft expression. However, PSD95 stabilized Kvß2.1 in these domains. This data shed light on the molecular mechanism by which Kvß2.1 clusters into immunological synapses during leukocyte activation.

γ-2 and GSG1L bind with comparable affinities to the tetrameric GluA1 core.

BACKGROUND: The AMPA-type ionotropic glutamate receptor mediates fast excitatory neurotransmission in the brain. A variety of auxiliary subunits regulate its gating properties, assembly, and trafficking, but it is unknown if the binding of these auxiliary subunits to the receptor core is dynamically regulated. Here we investigate the interplay of the two auxiliary subunits γ-2 and GSG1L when binding to the AMPA receptor composed of four GluA1 subunits. METHODS: We use a three-color single-molecule imaging approach in living cells, which allows the direct observation of the receptors and both auxiliary subunits. Colocalization of different colors can be interpreted as interaction of the respective receptor subunits. RESULTS: Depending on the relative expression levels of γ-2 and GSG1L, the occupancy of binding sites shifts from one auxiliary subunit to the other, supporting the idea that they compete for binding to the receptor. Based on a model where each of the four binding sites at the receptor core can be either occupied by γ-2 or GSG1L, our experiments yield apparent dissociation constants for γ-2 and GSG1L in the range of 2.0-2.5/µm2. CONCLUSIONS: The result that both binding affinities are in the same range is a prerequisite for dynamic changes of receptor composition under native conditions.

Calcium-Activated K+ Channels (KCa) and Therapeutic Implications.

Potassium channels are the most diverse and ubiquitous family of ion channels found in cells. The Ca2+ and voltage gated members form a subfamily that play a variety of roles in both excitable and non-excitable cells and are further classified on the basis of their single channel conductance to form the small conductance (SK), intermediate conductance (IK) and big conductance (BK) K+ channels.In this chapter, we will focus on the mechanisms underlying the gating of BK channels, whose function is modified in different tissues by different splice variants as well as the expanding array of regulatory accessory subunits including ß, γ and LINGO subunits. We will examine how BK channels are modified by these regulatory subunits and describe how the channel gating is altered by voltage and Ca2+ whilst setting this in context with the recently published structures of the BK channel. Finally, we will discuss how BK and other calcium-activated channels are modulated by novel ion channel modulators and describe some of the challenges associated with trying to develop compounds with sufficient efficacy, potency and selectivity to be of therapeutic benefit.

Regulatory γ1 subunits defy symmetry in functional modulation of BK channels.

Structural symmetry is a hallmark of homomeric ion channels. Nonobligatory regulatory proteins can also critically define the precise functional role of such channels. For instance, the pore-forming subunit of the large conductance voltage and calcium-activated potassium (BK, Slo1, or KCa1.1) channels encoded by a single KCa1.1 gene assembles in a fourfold symmetric fashion. Functional diversity arises from two families of regulatory subunits, ß and γ, which help define the range of voltages over which BK channels in a given cell are activated, thereby defining physiological roles. A BK channel can contain zero to four ß subunits per channel, with each ß subunit incrementally influencing channel gating behavior, consistent with symmetry expectations. In contrast, a γ1 subunit (or single type of γ1 subunit complex) produces a functionally all-or-none effect, but the underlying stoichiometry of γ1 assembly and function remains unknown. Here we utilize two distinct and independent methods, a Forster resonance energy transfer-based optical approach and a functional reporter in single-channel recordings, to reveal that a BK channel can contain up to four γ1 subunits, but a single γ1 subunit suffices to induce the full gating shift. This requires that the asymmetric association of a single regulatory protein can act in a highly concerted fashion to allosterically influence conformational equilibria in an otherwise symmetric K+ channel.

The calmodulin-binding tetraleucine motif of KCNE4 is responsible for association with Kv1.3.

The voltage-dependent potassium (Kv) channel Kv1.3 regulates leukocyte proliferation, activation, and apoptosis, and altered expression of this channel is linked to autoimmune diseases. Thus, the fine-tuning of Kv1.3 function is crucial for the immune system response. The Kv1.3 accessory protein, potassium voltage-gated channel subfamily E (KCNE) subunit 4, acts as a dominant negative regulatory subunit to both enhance inactivation and induce intracellular retention of Kv1.3. Mutations in KCNE4 also cause immune system dysfunction. Although the formation of Kv1.3-KCNE4 complexes has profound consequences for leukocyte physiology, the molecular determinants involved in the Kv1.3-KCNE4 association are unknown. We now show that KCNE4 associates with Kv1.3 via a tetraleucine motif situated within the carboxy-terminal domain of this accessory protein. This motif would function as an interaction platform, in which Kv1.3 and Ca2+/calmodulin compete for the KCNE4 interaction. Finally, we propose a structural model of the Kv1.3-KCNE4 complex. Our experimental data and the in silico structure suggest that the KCNE4 interaction hides a forward-trafficking motif within Kv1.3 in addition to adding a strong endoplasmic reticulum retention signature to the Kv1.3-KCNE4 complex. Thus, the oligomeric composition of the Kv1.3 channelosome fine-tunes the precise balance between anterograde and intracellular retention elements that control the cell surface expression of Kv1.3 and immune system physiology.-Solé, L., Roig, S. R., Sastre, D., Vallejo-Gracia, A., Serrano-Albarrás, A., Ferrer-Montiel, A., Fernández-Ballester, G., Tamkun, M. M., Felipe, A. The calmodulin-binding tetraleucine motif of KCNE4 is responsible for association with Kv1.3.

High immunological response against a Trypanosoma equiperdum protein that exhibits homology with the regulatory subunits of mammalian cAMP-dependent protein kinases.

Previously, we have identified a protein in Trypanosoma equiperdum that possesses homology with the regulatory (R) subunits of the mammalian cAMP-dependent protein kinase (PKA). The recombinant T. equiperdum PKA R-like protein was expressed in bacteria and purified to homogeneity. Mice polyclonal antibodies were raised against the recombinant R-like protein to serologically evaluate its humoral immune response. High titers of specific sera antibodies were obtained against the parasite R-like protein by indirect enzyme-linked immunosorbent assay (ELISA), and immunoblots revealed that this protein was specifically recognized by the hyperimmune mice sera. Cellular proliferation assays using splenic B cells from the immunized mice showed higher values when the recombinant T. equiperdum R-like protein was employed than when concanavalin A was utilized as an unspecific mitogen. Two healthy horses that were experimentally infected using either T. equiperdum or Trypanosoma evansi showed a curve response characterized by the appearance of anti-T. equiperdum PKA R-like protein antibody production in sera using indirect ELISA. The recombinant parasite PKA R-like protein was also recognized by sera from naturally trypanosome-infected horses using western blotting. These findings demonstrated that the T. equiperdum PKA R-like protein is an antigen that exhibits cross-reaction with T. equiperdum and T. evansi.

Protein phosphatase 2A regulatory subunit B56ß modulates erythroid differentiation.

Anemia due to attenuated erythroid terminal differentiation is one of the most common hematological disorders occurring at all stages of life. We previously demonstrated that catalytic subunit α of protein phosphatase 2A (PP2Acα) modulates fetal liver erythropoiesis. However the corresponding PP2A regulatory subunit in this process remains unknown. In this study, we report that chemical inhibition of PP2A activity with okadaic acid impairs hemin-induced erythroid differentiation. Interestingly, B56 family member B56ß is the only regulatory subunit whose expression is induced by both erythropoietin in fetal liver cells and hemin in erythroleukemia K562 cells. Finally, knockdown of B56ß attenuates hemin-induced K562 erythroid differentiation. Collectively, our data identify B56ß as the potential functional regulatory subunit of PP2A in erythroid differentiation, shedding light on new target for precise modulation of PP2A activity for treatment of anemia and related diseases.

Immunological identification of a cAMP-dependent protein kinase regulatory subunit-like protein from the Trypanosoma equiperdum TeAp-N/D1 isolate.

Polyclonal immunoglobulin Y (IgY) antibodies were produced in chicken eggs against the purified R(II)-subunit of the cAMP-dependent protein kinase (PKA) from pig heart, which corresponds to the Sus scrofa R(II)α isoform. In order to evaluate whether Trypanosoma equiperdum possessed PKA R-like proteins, parasites from the Venezuelan TeAp-N/D1 strain were examined using the generated anti-R(II) IgY antibodies. Western blot experiments revealed a 57-kDa polypeptide band that was distinctively recognized by these antibodies. Likewise, polyclonal antibodies raised in mice ascites against the recombinant T. equiperdum PKA R-like protein recognized the PKA R(II)-subunit purified from porcine heart and the recombinant human PKA R(I)ß-subunit by immunoblotting. However, a partially purified fraction of the parasite PKA R-like protein was not capable of binding cAMP, implying that this protein is not a direct downstream cAMP effector in T. equiperdum. Although the function of the S. scrofa PKA R(II)α and the T. equiperdum PKA R-like protein appear to be different, their cross-reactivity together with results obtained by bioinformatics techniques corroborated the high level of homology exhibited by both proteins. Moreover, its presence in other trypanosomatids suggests an important cellular role of PKA R-like proteins in parasite physiology.

Regulations of m6A and other RNA modifications and their roles in cancer.

RNA modification is an essential component of the epitranscriptome, regulating RNA metabolism and cellular functions. Several types of RNA modifications have been identified to date; they include N6-methyladenosine (m6A), N1-methyladenosine (m1A), 5-methylcytosine (m5C), N7-methylguanosine (m7G), N6,2'-O-dimethyladenosine (m6Am), N4-acetylcytidine (ac4C), etc. RNA modifications, mediated by regulators including writers, erasers, and readers, are associated with carcinogenesis, tumor microenvironment, metabolic reprogramming, immunosuppression, immunotherapy, chemotherapy, etc. A novel perspective indicates that regulatory subunits and post-translational modifications (PTMs) are involved in the regulation of writer, eraser, and reader functions in mediating RNA modifications, tumorigenesis, and anticancer therapy. In this review, we summarize the advances made in the knowledge of different RNA modifications (especially m6A) and focus on RNA modification regulators with functions modulated by a series of factors in cancer, including regulatory subunits (proteins, noncoding RNA or peptides encoded by long noncoding RNA) and PTMs (acetylation, SUMOylation, lactylation, phosphorylation, etc.). We also delineate the relationship between RNA modification regulator functions and carcinogenesis or cancer progression. Additionally, inhibitors that target RNA modification regulators for anticancer therapy and their synergistic effect combined with immunotherapy or chemotherapy are discussed.

KCNE4-dependent modulation of Kv1.3 pharmacology.

The voltage-dependent potassium channel Kv1.3 is a promising therapeutic target for the treatment of autoimmune and chronic inflammatory disorders. Kv1.3 blockers are effective in treating multiple sclerosis (fampridine) and psoriasis (dalazatide). However, most Kv1.3 pharmacological antagonists are not specific enough, triggering potential side effects and limiting their therapeutic use. Functional Kv are oligomeric complexes in which the presence of ancillary subunits shapes their function and pharmacology. In leukocytes, Kv1.3 associates with KCNE4, which reduces the surface abundance and enhances the inactivation of the channel. This mechanism exerts profound consequences on Kv1.3-related physiological responses. Because KCNE peptides alter the pharmacology of Kv channels, we studied the effects of KCNE4 on Kv1.3 pharmacology to gain insights into pharmacological approaches. To that end, we used margatoxin, which binds the channel pore from the extracellular space, and Psora-4, which blocks the channel from the intracellular side. While KCNE4 apparently did not alter the affinity of either margatoxin or Psora-4, it slowed the inhibition kinetics of the latter in a stoichiometry-dependent manner. The results suggested changes in the Kv1.3 architecture in the presence of KCNE4. The data indicated that while the outer part of the channel mouth remains unaffected, KCNE4 disturbs the intracellular architecture of the complex. Various leukocyte types expressing different Kv1.3/KCNE4 configurations participate in the immune response. Our data provide evidence that the presence of these variable architectures, which affect both the structure of the complex and their pharmacology, should be considered when developing putative therapeutic approaches.

Protein Phosphatase 2A Regulates Phenotypic and Metabolic Alteration of Microglia Cells in HFD-Associated Vascular Dementia Mice via TNF-α/Arg-1 Axis.

Protein phosphatase 2A (PP2A), the activity of which is dictated by the composition of its regulatory subunit, is strongly related to the progression of neurodegenerative disease. The potential role of PP2A on the phenotypic transition of microglial cells under obese conditions is poorly explored. An understanding of the role of PP2A and identification of regulatory subunits contributing to microglial phenotypic transitions in obese condition may serve as a therapeutic target for obesity-associated neurodegeneration. C57BL/6 mice were exposed to obese-associated vascular dementia conditions by performing unilateral common carotid artery occlusion on obese mice of microglial polarization and PP2A activity using flow cytometry, real-time PCR, western blotting, and immunoprecipitation enzymatic assay, followed identifications of PP2A regulatory subunits using LCMS and RT-PCR. Chronic HFD feeding significantly increased the populations of infiltrated macrophages, showing a high percentage of CD86+ in VaD mice, and the expression of pro-inflammatory cytokines, and we observed that PP2A modulates metabolic reprogramming of microglia by regulating OXPHOS/ECAR activity. Using Co-IP and LCMS, we identified the six specific regulatory subunits, namely PPP2R2A, PPP2R2D, PPP2R5B, PPP2R5C, PPP2R5D, and PPP2R5E, that are associated with microglial-activation during obesity-associated-VaD. Interestingly, pharmacological up-regulation of PP2A more significantly suppressed the expression of TNF-alpha than other pro-inflammatory-cytokines and increased the expression of Arginase-1, suggesting that PP2A modulates microglial-phenotypic transitions through TNF-α/Arg-1 axis. Our present findings demonstrate microglial polarization in HFD associated with VaD, and point towards a therapeutic target by providing specific PP2A regulatory-subunits implicated in microglial activation during obesity-related-vascular-dementia.

Oligomerization and Spatial Distribution of Kvß1.1 and Kvß2.1 Regulatory Subunits.

Members of the regulatory Kvß family modulate the kinetics and traffic of voltage-dependent K+ (Kv) channels. The crystal structure of Kv channels associated with Kvß peptides suggests a α4/ß4 composition. Although Kvß2 and Kvß1 form heteromers, evidence supports that only Kvß2.1 forms tetramers in the absence of α subunits. Therefore, the stoichiometry of the Kvß oligomers fine-tunes the activity of hetero-oligomeric Kv channel complexes. We demonstrate that Kvß subtypes form homo- and heterotetramers with similar affinities. The Kvß1.1/Kvß2.1 heteromer showed an altered spatial distribution in lipid rafts, recapitulating the Kvß1.1 pattern. Because Kvß2 is an active partner of the Kv1.3-TCR complex at the immunological synapse (IS), an association with Kvß1 would alter this location, shaping the immune response. Differential regulation of Kvßs influences the traffic and architecture of the Kvß heterotetramer, modulating Kvß-dependent physiological responses.

The Multifaceted Role of Protein Phosphatase 1 in Plasmodium.

Protein phosphatase type 1 (PP1) forms a wide range of Ser/Thr-specific phosphatase holoenzymes which contain one catalytic subunit (PP1c), present in all eukaryotic cells, associated with variable subunits known as regulatory proteins. It has recently been shown that regulators take a leading role in the organization and the control of PP1 functions. Many studies have addressed the role of these regulators in diverse organisms, including humans, and investigated their link to diseases. In this review we summarize recent advances on the role of PP1c in Plasmodium, its interactome and regulators. As a proof of concept, peptides interfering with the regulator binding capacity of PP1c were shown to inhibit the growth of P. falciparum, suggesting their potential as drug precursors.

Protein Kinase A (PRKA) Activity Is Regulated by the Proteasome at the Onset of Human Sperm Capacitation.

The proteasome increases its activity at the onset of sperm capacitation due to the action of the SACY/PRKACA pathway; this increase is required for capacitation to progress. PRKA activity also increases and remains high during capacitation. However, intracellular levels of cAMP decrease in this process. Our goal was to evaluate the role of the proteasome in regulating PRKA activity once capacitation has started. Viable human sperm were incubated in the presence and absence of epoxomicin or with 0.1% DMSO. The activity of PRKA; the phosphorylation pattern of PRKA substrates (pPRKAs); and the expression of PRKAR1, PRKAR2, and AKAP3 were evaluated by Western blot. The localization of pPRKAs, PRKAR1, PRKAR2, and AKAP3 was evaluated by immunofluorescence. Treatment with epoxomicin changed the localization and phosphorylation pattern and decreased the percentage of pPRKAs-positive sperm. PRKA activity significantly increased at 1 min of capacitation and remained high throughout the incubation. However, epoxomicin treatment significantly decreased PRKA activity after 30 min. In addition, PRKAR1 and AKAP3 were degraded by the proteasome but with a different temporal kinetic. Our results suggest that PRKAR1 is the target of PRKA regulation by the proteasome.

Functional Consequences of the Variable Stoichiometry of the Kv1.3-KCNE4 Complex.

The voltage-gated potassium channel Kv1.3 plays a crucial role during the immune response. The channel forms oligomeric complexes by associating with several modulatory subunits. KCNE4, one of the five members of the KCNE family, binds to Kv1.3, altering channel activity and membrane expression. The association of KCNEs with Kv channels is the subject of numerous studies, and the stoichiometry of such associations has led to an ongoing debate. The number of KCNE4 subunits that can interact and modulate Kv1.3 is unknown. KCNE4 transfers important elements to the Kv1.3 channelosome that negatively regulate channel function, thereby fine-tuning leukocyte physiology. The aim of this study was to determine the stoichiometry of the functional Kv1.3-KCNE4 complex. We demonstrate that as many as four KCNE4 subunits can bind to the same Kv1.3 channel, indicating a variable Kv1.3-KCNE4 stoichiometry. While increasing the number of KCNE4 subunits steadily slowed the activation of the channel and decreased the abundance of Kv1.3 at the cell surface, the presence of a single KCNE4 peptide was sufficient for the cooperative enhancement of the inactivating function of the channel. This variable architecture, which depends on KCNE4 availability, differentially affects Kv1.3 function. Therefore, our data indicate that the physiological remodeling of KCNE4 triggers functional consequences for Kv1.3, thus affecting cell physiology.

Controlling Ser/Thr protein phosphatase PP1 activity and function through interaction with regulatory subunits.

Protein phosphatase 1 is a major Ser/Thr protein phosphatase activity in eukaryotic cells. It is composed of a catalytic polypeptide (PP1C), with little substrate specificity, that interacts with a large variety of proteins of diverse structure (regulatory subunits). The diversity of holoenzymes that can be formed explain the multiplicity of cellular functions under the control of this phosphatase. In quite a few cases, regulatory subunits have an inhibitory role, downregulating the activity of the phosphatase. In this chapter we shall introduce PP1C and review the most relevant families of PP1C regulatory subunits, with particular emphasis in describing the structural basis for their interaction.

Structural Features that Distinguish Inactive and Active PI3K Lipid Kinases.

PI3K lipid kinases signal through the PI3K/Akt pathway, regulating cell growth and proliferation. While the structural features that distinguish between the active and inactive states of protein kinases are well established, that has not been the case for lipid kinases, and neither was the structural mechanism controlling the switch between the two states. Class I PI3Ks are obligate heterodimers with catalytic and regulatory subunits. Here, we analyze PI3K crystal structures. Structures with the nSH2 (inactive state) are featured by collapsed activation loop (a-loop) and an IN kinase domain helix 11 (kα11). In the active state, the a-loop is extended and kα11 in the OUT conformation. Our analysis suggests that the nSH2 domain in the regulatory subunit regulates activation, catalysis and autoinhibition through the a-loop. Inhibition, activation and catalytic scenarios are shared by class IA PI3Ks; the activation is mimicked by oncogenic mutations and the inhibition offers an allosteric inhibitor strategy.

Sequence Analysis of the cAMP-Dependent Protein Kinase Regulatory Subunit-Like Protein From Trypanosoma brucei.

PURPOSE: Study the N-terminal, C-terminal, and linker regions of the TbPKAr using homology modeling. METHODS: The amino acid sequences of the N-terminal, C-terminal, and linker regions of the TbPKAr were individually examined by means of BLAST analysis and in silico secondary structure predictions with several programs. RESULTS: The TbPKAr C-terminal region, showed a well-folded α/ß structure, which consists of two concurrent flattened ß-barrel-shaped domains that are separated by an elongated central α-helix similar to its mammalian counterpart, the TbPKAr linker region contains a PKA phosphorylation site and was predicted to be rather disordered. Our analysis also indicated that the TbPKAr N-terminal region lacks a docking/dimerization domain but is enriched in motifs known as leucine-rich repeats (LRR). CONCLUSION: The replacement of the docking/dimerization domain by different structural motifs suggests the inability of TbPKAr to form homodimers; however, the function of the TbPKAr N-terminal LRR-containing domain in Kinetoplastidae parasites is still unknown.

LSD1 Activates PI3K/AKT Signaling Through Regulating p85 Expression in Prostate Cancer Cells.

Lysine specific demethylase 1 (LSD1) functions as a transcriptional repressor through demethylating active histone marks such as mono- or di-methylated histone 3 lysine 4 (H3K4) and interacting with histone deacetylases. However, LSD1 can also act as an activator through demethylating repressive histone marks and possibly non-histone proteins. In prostate cancer (PCa) cells, LSD1 mediates the transcriptional activity of androgen receptor (AR), a ligand dependent nuclear transcription factor that drives PCa initiation and progression to the castration-resistant prostate cancer (CRPC). However, it is unclear whether LSD1 also regulates other growth promoting pathways independent of AR signaling in PCa cells. In this study, we show that LSD1 can activate PI3K/AKT pathways in absence of androgen stimulation, and we further demonstrate that LSD1 transcriptionally regulates the expression of PI3K regulatory subunit, p85, possibly through epigenetic reprogramming of enhancer landscape in PCa cells. Our study suggests that LSD1 has dual functions in promoting PCa development, that it enhances AR signaling through its coactivator function, and that it activates PI3K/AKT signaling through increasing p85 gene expression.