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Abstract Background Fas-associated factor 1 (FAF1) is a multidomain protein that interacts with diverse partners to affect numerous cellular processes. Previously, we discovered two Small Ubiquitin-like Modifier (SUMO)-interacting motifs (SIMs) within FAF1 that are crucial for transcriptional modulation of mineralocorticoid receptor. Recently, we identified Sin3A-associated protein 130 (SAP130), a putative sumoylated protein, as a candidate FAF1 interaction partner by yeast two-hybrid screening. However, it remained unclear whether SAP130 sumoylation might occur and functionally interact with FAF1. Results In this study, we first show that SAP130 can be modified by SUMO1 at Lys residues 794, 878 and 932 both in vitro and in vivo. Mutation of these three SUMO-accepting Lys residues to Ala had no impact on SAP130 association with Sin3A or its nuclear localization, but the mutations abrogated the association of SAP130 with the FAF1. The mutations also potentiated SAP130 trans-repression activity and attenuated SAP130-mediated promotion of cell growth. Additionally, SUMO1-modified SAP130 was less stable than unmodified SAP130. Transient transfection experiments further revealed that FAF1 mitigated the trans-repression and cell proliferation-promoting functions of SAP130, and promoted SAP130 degradation by enhancing its polyubiquitination in a sumoylation-dependent manner. Conclusions Together, these results demonstrate that sumoylation of SAP130 regulates its biological functions and that FAF1 plays a crucial role in controlling the SUMO-dependent regulation of transcriptional activity and protein stability of SAP130.

Abstract Background Protein phosphatase 2A (PP2A) is one of the major protein phosphatases in eukaryotic cells and is essential for cellular homeostasis. PP2A is a heterotrimer comprising the dimeric AC core enzyme and a highly variable regulatory B subunit. Distinct B subunits help the core enzyme gain full activity toward specific substrates and contribute to diverse cellular roles of PP2A. PP2A has been thought to play a tumor suppressor and the B56γ3 regulatory subunit was shown to play a key tumor suppressor regulatory subunit of PP2A. Nevertheless, we uncovered a molecular mechanism of how B56γ3 may act as an oncogene in colorectal cancer (CRC). Methods Polyclonal pools of CRC cells with stable B56γ3 overexpression or knockdown were generated by retroviral or lentiviral infection and subsequent drug selection. Co-immunoprecipitation(co-IP) and in vitro pull-down analysis were applied to analyze the protein–protein interaction. Transwell migration and invasion assays were applied to investigate the role of B56γ3 in affecting motility and invasive capability of CRC cells. The sensitivity of CRC cells to 5-fluorouracil (5-FU) was analyzed using the PrestoBlue reagent assay for cell viability. Immunohistochemistry (IHC) was applied to investigate the expression levels of phospho-AKT and B56γ3 in paired tumor and normal tissue specimens of CRC. DataSets of TCGA and GEO were analyzed to investigate the correlation of B56γ3 expression with overall survival rates of CRC patients. Results We showed that B56γ3 promoted epithelial-mesenchymal transition (EMT) and reduced the sensitivity of CRC cells to 5-FU through upregulating AKT activity. Mechanistically, B56γ3 upregulates AKT activity by targeting PP2A to attenuate the p70S6K-mediated negative feedback loop regulation on PI3K/AKT activation. B56γ3 was highly expressed and positively correlated with the level of phospho-AKT in tumor tissues of CRC. Moreover, high B56γ3 expression is associated with poor prognosis of a subset of patients with CRC. Conclusions Our finding reveals that the B56γ3 regulatory subunit-containing PP2A plays an oncogenic role in CRC cells by sustaining AKT activation through suppressing p70S6K activity and suggests that the interaction between B56γ3 and p70S6K may serve as a therapeutic target for CRC. Video Abstract

Abstract The prognosis of patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer has considerably improved. However, no reliable treatment besides anti-HER2 strategies has been available. FTY720, a small-molecule compound used for treating refractory multiple sclerosis, has been reported to have beneficial effects against cancers. We therefore evaluated the efficacy of FTY720 in trastuzumab-resistant breast cancer cells and investigated the possible mechanism involved. This study evaluated morphological changes after FTY720 treatment. Antiproliferative WST-1 assays and LDH Cytotoxicity Assay Kits were used to determine the treatment effects of drugs, whereas Western blot analysis was used to evaluate protein expression. Apoptotic events were investigated through annexin V staining and TUNEL assays using flow cytometry. FTY720 was effective in trastuzumab-resistant breast cancer cell lines despite the presence of PIK3CA mutation. Studied on a xenograft mouse model, FTY720-treated groups had statistically significantly poorer HCC1954 xenograft growth in vivo compared with the control group. Our findings suggest that FTY720 can overcome resistance to trastuzumab therapy in patients with HER2-positive breast cancer, with FTY720 plus trastuzumab might offer even better efficacy in vitro and in vivo.

Abstract Hypoxic-ischemic (HI) encephalopathy is the major cause of mortality and disability in newborns. The neurovascular unit is a major target of acute and chronic brain injury, and therapies that protect simultaneously both neurons and vascular endothelial cells from neonatal HI injury are in demand. Insulin receptors and its key downstream molecule-insulin receptor substrate −1 (IRS-1) are potential neuroprotective targets and expressed both in neuron and endothelial cells. To investigate whether IRS-1 can act similarly in neurons and vascular endothelial cells in protecting neurovascular units and brain form HI injury, we found that neuron-specific IRS-1 transgenic rats showed reduced neurovascular injury and infarct volumes, whereas endothelial-specific IRS-1 transgenic rats showed increased blood-brain barrier (BBB) disruption and exaggerated neurovascular injury after neonatal HI brain injury. Endothelial-specific IRS-1 overexpression increased vascular permeability and disassembled the tight junction protein (zonula occludens-1) complex. Inhibition of mammalian target of rapamycin (mTOR) by rapamycin preserved tight junction proteins and attenuated BBB leakage and neuronal apoptosis after HI in the endothelial-specific IRS-1 transgenic pups. Together, our findings suggested that neuronal and endothelial IRS-1 had opposite effects on the neurovascular integrity and damage after neonatal HI brain injury and that endothelial IRS-1 worsens neurovascular integrity after HI via mTOR-mediated tight junction protein disassembly.

The effects of lacosamide (LCS, Vimpat®), an anti-convulsant and analgesic, on voltage-gated Na+ current (INa) were investigated. LCS suppressed both the peak (transient, INa(T)) and sustained (late, INa(L)) components of INa with the IC50 values of 78 and 34 μM found in GH3 cells and of 112 and 26 μM in Neuro-2a cells, respectively. In GH3 cells, the voltage-dependent hysteresis of persistent INa (INa(P)) during the triangular ramp pulse was strikingly attenuated, and the decaying time constant (τ) of INa(T) or INa(L) during a train of depolarizing pulses was further shortened by LCS. The recovery time course from the INa block elicited by the preceding conditioning train can be fitted by two exponential processes, while the single exponential increase in current recovery without a conditioning train was adequately fitted. The fast and slow τ’s of recovery from the INa block by the same conditioning protocol arose in the presence of LCS. In Neuro-2a cells, the strength of the instantaneous window INa (INa(W)) during the rapid ramp pulse was reduced by LCS. This reduction could be reversed by tefluthrin. Moreover, LCS accelerated the inactivation time course of INa activated by pulse train stimulation, and veratridine reversed its decrease in the decaying τ value in current inactivation. The docking results predicted the capability of LCS binding to some amino-acid residues in sodium channels owing to the occurrence of hydrophobic contact. Overall, our findings unveiled that LCS can interact with the sodium channels to alter the magnitude, gating, voltage-dependent hysteresis behavior, and use dependence of INa in excitable cells.

The non-linear voltage-dependent hysteresis (Hys(V)) of voltage-gated ionic currents can be robustly activated by the isosceles-triangular ramp voltage (Vramp) through digital-to-analog conversion. Perturbations on this Hys(V) behavior play a role in regulating membrane excitability in different excitable cells. A variety of small molecules may influence the strength of Hys(V) in different types of ionic currents elicited by long-lasting triangular Vramp. Pirfenidone, an anti-fibrotic drug, decreased the magnitude of Ih’s Hys(V) activated by triangular Vramp, while dexmedetomidine, an agonist of α2-adrenoceptors, effectively suppressed Ih as well as diminished the Hys(V) strength of Ih. Oxaliplatin, a platinum-based anti-neoplastic drug, was noted to enhance the Ih’s Hys(V) strength, which is thought to be linked to the occurrence of neuropathic pain, while honokiol, a hydroxylated biphenyl compound, decreased Ih’s Hys(V). Cell exposure to lutein, a xanthophyll carotenoid, resulted in a reduction of Ih’s Hys(V) magnitude. Moreover, with cell exposure to UCL-2077, SM-102, isoplumbagin, or plumbagin, the Hys(V) strength of erg-mediated K+ current activated by triangular Vramp was effectively diminished, whereas the presence of either remdesivir or QO-58 respectively decreased or increased Hys(V) magnitude of M-type K+ current. Zingerone, a methoxyphenol, was found to attenuate Hys(V) (with low- and high-threshold loops) of L-type Ca2+ current induced by long-lasting triangular Vramp. The Hys(V) properties of persistent Na+ current (INa(P)) evoked by triangular Vramp were characterized by a figure-of-eight (i.e., ∞) configuration with two distinct loops (i.e., low- and high-threshold loops). The presence of either tefluthrin, a pyrethroid insecticide, or t-butyl hydroperoxide, an oxidant, enhanced the Hys(V) strength of INa(P). However, further addition of dapagliflozin can reverse their augmenting effects in the Hys(V) magnitude of the current. Furthermore, the addition of esaxerenone, mirogabalin, or dapagliflozin was effective in inhibiting the strength of INa(P). Taken together, the observed perturbations by these small-molecule modulators on Hys(V) strength in different types of ionic currents evoked during triangular Vramp are expected to influence the functional activities (e.g., electrical behaviors) of different excitable cells in vitro or in vivo.

Carbamazepine (CBZ, Tegretol®) is an anticonvulsant used in the treatment of epilepsy and neuropathic pain; however, several unwanted effects of this drug have been noticed. Therefore, the regulatory actions of CBZ on ionic currents in electrically excitable cells need to be reappraised, although its efficacy in suppressing voltage-gated Na+ current (INa) has been disclosed. This study was undertaken to explore the modifications produced by CBZ on ionic currents (e.g., INa and erg-mediated K+ current [IK(erg)]) measured from Neuro-2a (N2a) cells. In these cells, we found that this drug differentially suppressed the peak (transient, INa(T)) and sustained (late, INa(L)) components of INa in a concentration-dependent manner with effective IC50 of 56 and 18 μM, respectively. The overall current–voltage relationship of INa(T) with or without the addition of CBZ remained unchanged; however, the strength (i.e., ∆area) in the window component of INa (INa(W)) evoked by the short ascending ramp pulse (Vramp) was overly lessened in the CBZ presence. Tefluthrin (Tef), a synthetic pyrethroid, known to stimulate INa, augmented the strength of the voltage-dependent hysteresis (Hys(V)) of persistent INa (INa(P)) in response to the isosceles-triangular Vramp; moreover, further application of CBZ attenuated Tef-mediated accentuation of INa(P)’s Hys(V). With a two-step voltage protocol, the recovery of INa(T) inactivation seen in Neuro-2a cells became progressively slowed by adding CBZ; however, the cumulative inhibition of INa(T) evoked by pulse train stimulation was enhanced during exposure to this drug. Neuro-2a-cell exposure to CBZ (100 μM), the magnitude of erg-mediated K+ current measured throughout the entire voltage-clamp steps applied was mildly inhibited. The docking results regarding the interaction of CBZ and voltage-gate Na+ (NaV) channel predicted the ability of CBZ to bind to some amino-acid residues in NaV due to the existence of a hydrogen bond or hydrophobic contact. It is conceivable from the current investigations that the INa (INa(T), INa(L), INa(W), and INa(P)) residing in Neuro-2a cells are susceptible to being suppressed by CBZ, and that its block on INa(L) is larger than that on INa(T). Collectively, the magnitude and gating of NaV channels produced by the CBZ presence might have an impact on its anticonvulsant and analgesic effects occurring in vivo.

Protein phosphatase 2A (PP2A) is a ubiquitous phospho-serine/threonine phosphatase that controls many diverse cellular functions. The predominant form of PP2A is a heterotrimeric holoenzyme consisting of a scaffolding A subunit, a variable regulatory B subunit, and a catalytic C subunit. The C subunit also associates with other interacting partners, such as α4, to form non-canonical PP2A complexes. We report visualization of PP2A complexes in mammalian cells. Bimolecular fluorescence complementation (BiFC) analysis of PP2A subunit interactions demonstrates that the B subunit plays a key role in directing the subcellular localization of PP2A, and confirms that the A subunit functions as a scaffold in recruiting the B and C subunits to form a heterotrimeric holoenzyme. BiFC analysis also reveals that α4 promotes formation of the AC core dimer. Furthermore, we demonstrate visualization of specific ABC holoenzymes in cells by combining BiFC and fluorescence resonance energy transfer (BiFC-FRET). Our studies not only provide direct imaging data to support previous biochemical observations on PP2A complexes, but also offer a promising approach for studying the spatiotemporal distribution of individual PP2A complexes in cells.

Hepatocellular carcinoma (HCC), a major cause of cancer-related death in Southeast Asia, is frequently associated with hepatitis B virus (HBV) infection. HBV X protein (HBx), encoded by a viral non-structural gene, is a multifunctional regulator in HBV-associated tumor development. We investigated novel signaling pathways underlying HBx-induced liver tumorigenesis and found that the signaling pathway involving IκB kinase β (IKKβ), tuberous sclerosis complex 1 (TSC1), and mammalian target of rapamycin (mTOR) downstream effector S6 kinase (S6K1), was upregulated when HBx was overexpressed in hepatoma cells. HBx-induced S6K1 activation was reversed by IKKβ inhibitor Bay 11-7082 or silencing IKKβ expression using siRNA. HBx upregulated cell proliferation and vascular endothelial growth factor (VEGF) production, and these HBx-upregulated phenotypes were abolished by treatment with IKKβ inhibitor Bay 11-7082 or mTOR inhibitor rapamycin. The association of HBx-modulated IKKβ/mTOR/S6K1 signaling with liver tumorigenesis was verified in a HBx transgenic mouse model in which pIKKβ, pS6K1, and VEGF expression was found to be higher in cancerous than non-cancerous liver tissues. Furthermore, we also found that pIKKβ levels were strongly correlated with pTSC1 and pS6K1 levels in HBV-associated hepatoma tissue specimens taken from 95 patients, and that higher pIKKβ, pTSC1, and pS6K1 levels were correlated with a poor prognosis in these patients. Taken together, our findings demonstrate that HBx deregulates TSC1/mTOR signaling through IKKβ, which is crucially linked to HBV-associated HCC development.

Background: MSP58 is a nucleolar protein associated with rRNA transcription and cell proliferation. Its mechanism of translocation into the nucleus or the nucleolus, however, is not entirely known. In order to address this lack, the present study aims to determine a crucial part of this mechanism: the nuclear localization signal (NLS) and the nucleolar localization signal (NoLS) associated with the MSP58 protein. Results: We have identified and characterized two NLSs in MSP58. The first is located between residues 32 and 56 (NLS1) and constitutes three clusters of basic amino acids (KRASSQALGTIPKRRSSSRFIKRKK); the second is situated between residues 113 and 123 (NLS2) and harbors a monopartite signal (PGLTKRVKKSK). Both NLS1 and NLS2 are highly conserved among different vertebrate species. Notably, one bipartite motif within the NLS1 (residues 44-56) appears to be absolutely necessary for MSP58 nucleolar localization. By yeast two-hybrid, pull-down, and coimmunoprecipitation analysis, we show that MSP58 binds to importin a1 and a6, suggesting that nuclear targeting of MSP58 utilizes a receptor-mediated and energy-dependent import mechanism. Functionally, our data show that both nuclear and nucleolar localization of MSP58 are crucial for transcriptional regulation on p21 and ribosomal RNA genes, and context-dependent effects on cell proliferation. Conclusions: Results suggest that MSP58 subnuclear localization is regulated by two nuclear import signals, and that proper subcellular localization of MSP58 is critical for its role in transcriptional regulation. Our study reveals a molecular mechanism that controls nuclear and nucleolar localization of MSP58, a finding that might help future researchers understand the MSP58 biological signaling pathway. [ABSTRACT FROM AUTHOR]

The transcription factor NF-κB (nuclear factor κB) co-ordinates various gene expressions in response to diverse signals and is a critical regulator of inflammation and innate immunity. Several negative regulators of NF-κB have been identified as downstream targets of NF-κB and function as a feedback control of NF-κB activation. A few protein phosphatases have also been shown to inactivate NF-κB activation. However, little is known about how protein phosphatases detect and respond to NF-κB activation. In the present study, we report a regulatory subunit of PP5 (protein phosphatase 5), G4-1, that physically interacts with IKKβ [IκB (inhibitor of NF-κB) kinase β] and negatively regulates NF-κB activation. The association of G4-1 with IKKβ depends on the kinase activity of IKKβ. Mapping of the G4-1-binding domain of IKKβ reveals that the serine-rich domain in the C-terminus of IKKβ is required for G4-1 binding. When seven autophosphorylated serine residues in this domain were mutated to alanine, the mutant form of IKKβ lost its ability to bind G4-1 and was more potent than the wild-type kinase to activate NF-κB. Knockdown of G4-1 enhanced TNFα (tumour necrosis factor α)-induced NF-κB activity, and knockdown of PP5 totally abolished the inhibitory activity of G4-1 on NF-κB activation. The results of the present study suggest that G4-1 functions as an adaptor to recruit PP5 to the phosphorylated C-terminus of activated IKKβ and to down-regulate the activation of IKKβ. [ABSTRACT FROM AUTHOR]

BAD, a proapoptotic molecule of the BCL2 family, is regulated by reversible phosphorylation. During survival, BAD is sequestered by 14-3-3 through serine 136 phosphorylation and is dissociated from BCL-X[sub L] through serine 155 phosphorylation. We report that phosphoserine 112 (pSer112) dephosphorylation functions as a gatekeeper for BAD-mediated apoptosis. During apoptosis, dephosphorylation of pSer112 preceded pSer136 dephosphorylation. Dephosphorylation of pSer112 accelerated dephosphorylation of pSer136, and inhibition of pSer112 dephosphorylation prevented pSer136 dephosphorylation, indicating that dephosphorylation of pSer112 is required for dephosphorylation of pSer136. Protein phosphatase 2A (PP2A) is the major pSer112 phosphatase. PP2A competed with 14-3-3 for BAD binding, and survival factor withdrawal enhanced PP2A association with BAD. Dephosphorylation of the critical residue, pSer136, could only be blocked by inhibition of all known subfamilies of serine/threonine phosphatases, suggesting that multiple phosphatases are involved in pSer136 dephosphorylation. Inhibition of PP2A rescued FL5.12 cells from apoptosis, demonstrating a physiologic role for PP2A-mediated pSer112 dephosphorylation. Thus, PP2A dephosphorylation of pSer112 is the key initiating event regulating the activation of BAD during interleukin-3 withdrawal-induced apoptosis. [ABSTRACT FROM AUTHOR]

The signaling of glycogen synthase kinase-3β (GSK-3β) has been implicated in stress-induced apoptosis. However, the pro-apoptotic role of GSK-3β is still unclear. Here, we show the involvement of GSK-3β in ceramide-induced mitochondrial apoptosis. Ceramide induced GSK-3β activation via protein dephosphorylation at serine 9. We previously reported that ceramide induced caspase-2 and caspase-8 activation, Bid cleavage, mitochondrial damage, and apoptosis. In this study, we found that caspase-2 activation and the subsequent apoptotic events were abolished by the GSK-3β inhibitors lithium chloride and SB216763, and by GSK-3β knockdown using short interfering RNA. We also found that ceramide-activated protein phosphatase 2A (PP2A) indirectly caused GSK-3β activation, and that the PP2A-regulated PI 3-kinase-Akt pathway was involved in GSK-3β activation. These results indicate a role for GSK-3β in ceramide-induced apoptosis, in which GSK-3β acts downstream of PP2A and the PI 3-kinase-Akt pathway, and upstream of caspase-2 and caspase-8. [ABSTRACT FROM AUTHOR]

Akt is a protein serine/threonine kinase that is involved in the regulation of diverse cellular processes. Phosphorylation of Akt at regulatory residues Thr-308 and Ser-473 leads to its full activation. The protein phosphatase 2A (PP2A) has long been known to negatively regulate Akt activity. The PP2A holoenzyme consists of the structural subunit (A), catalytic subunit (C), and a variable regulatory subunit (B). Here we report the identification of the specific B regulatory subunit that targets the PP2A holoenzyme to Akt. We found endogenous association of PP2A AB55C holoenzymes with Akt by co-immunoprecipitation analyses in pro-lymphoid FL5.12 cells. Akt was shown to associate with ectopically expressed B55α subunit in NIH3T3 cells. The direct interaction between B55α subunit and Akt was confirmed using in vitro pulldown analyses. Intriguingly, we found that overexpression of B55α subunit significantly impaired phosphorylation at Thr-308, but to a lesser extent at Ser-473 of Akt in both FL5.12 and NIH3T3 cells. Concomitantly, phosphorylation of a subset of Akt substrates, including FoxO3a, was substantially decreased by B55α overexpression in these cells. Silencing of B55α expression markedly increased phosphorylation at Thr-308 but not at Ser-473 in both FL5.12 cells and NIH3T3 cells. Consistently, PP2A AB55C holoenzymes preferentially dephosphorylated phospho-Thr-308 rather than phospho-Ser-473 in in vitro dephosphorylation assays. Furthermore, B55α overexpression retarded proliferation of NIH3T3 cells, and knockdown of B55α expression increased survival of FL5.12 cells upon interleukin-3 deprivation. Together, our data demonstrate that B55α-dependent targeting of the PP2A holoenzyme to Akt selectively regulates Akt phosphorylation at Thr-308 to regulate cell proliferation and survival. [ABSTRACT FROM AUTHOR]