Phosphorylation Of Endogenous Proteins Research Articles

Design of a covalent protein-protein interaction inhibitor of SRPKs to suppress angiogenesis and invasion of cancer cells

Serine–arginine (SR) proteins are splicing factors that play essential roles in both constitutive and alternative pre-mRNA splicing. Phosphorylation of their C-terminal RS domains by SR protein kinases (SRPKs) regulates their localization and diverse cellular activities. Dysregulation of phosphorylation has been implicated in many human diseases, including cancers. Here, we report the development of a covalent protein–protein interaction inhibitor, C-DBS, that targets a lysine residue within the SRPK-specific docking groove to block the interaction and phosphorylation of the prototypic SR protein SRSF1. C-DBS exhibits high specificity and conjugation efficiency both in vitro and in cellulo. This self-cell-penetrating inhibitor attenuates the phosphorylation of endogenous SR proteins and subsequently inhibits the angiogenesis, migration, and invasion of cancer cells. These findings provide a new foundation for the development of covalent SRPK inhibitors for combatting diseases such as cancer and viral infections and overcoming the resistance encountered by ATP-competitive inhibitors.

The Enzymatic Function of Kvβ2 Controls Vascular Potassium Channel Activity

Voltage-gated potassium (Kv) channels expressed in vascular smooth muscle control membrane potential and thereby regulate vascular tone and organ perfusion. Native Kv1 channels in the resistance vasculature consist of an alpha pore domain in complex with intracellular beta (Kvβ) proteins, which are active aldo-keto reductases (AKR6A). Recent work suggests that the AKR function of Kvβ2 is required for vascular Kv1 channel NAD(P)(H) redox sensing and O2 sensitivity of resistance arterial tone. Nonetheless, whether acute changes in Kvβ2 AKR function impact vascular Kv1 activity is unknown. We propose that Kv1 gating is sensitive to changes in the levels of endogenous substrates and post-translational modification of Kvβ2. Here, we tested the hypothesis that the availability of the glycolytic byproduct and purported Kvβ substrate, methylglyoxal, and protein kinase C-dependent phosphorylation differentially regulate vascular Kvβ2 function. Using the inside-out configuration of the patch clamp technique, we measured the open probability (nPo) of Kv channels present in patches excised from human coronary smooth muscle cells. Perfusion of methylglyoxal (5 μM) resulted in significantly increased Kv channel nPo (3.3x10−5 ± 9.5x10−6 to 3.2x10−4 ± 2.1x10−4; n=5) and this effect was enhanced (~8-fold) in the presence of 100 μM NADPH, suggesting that Kv sensitivity to methylglyoxal is cofactor-dependent. Based on this, we next tested whether Kvβ phosphorylation regulates AKR catalysis and influences vascular Kv1 redox sensitivity. Using in situ proximity ligation, we found that serine phosphorylation of Kvβ2 in smooth muscle was increased upon treatment with the PKC activator phorbol 12-myristate 13-acetate (PMA; 100 nM), indicating that smooth muscle Kvβ2 is a phosphorylation target under these conditions. In vitro phosphorylation of purified Kvβ2 with PKCα significantly slowed catalysis (11.3 ± 0.3 vs. 6.5 ± 0.2 μmoles/min/mg protein, control and PKCα-treated rat Kvβ2.1, respectively). Pretreatment of cells with 10 nM PMA before excising inside-out membrane patches eliminated the increase in Kv channel nP(o) evoked by methylglyoxal (±NADPH). Moreover, ex vivo treatment of isolated coronary arteries with PMA (10 nM) abolished vasodilation in response to elevated NADH:NAD+ via application of 2–5 mM external L-lactate. Together, these results suggest that vascular smooth muscle Kv1 channels are functionally regulated by endogenous substrates and phosphorylation of intracellular Kvβ proteins. We propose that targeting vascular Kvβ AKR function may represent a novel strategy to modify K+ channel redox sensitivity and thus strengthen the coupling between metabolic demand and organ perfusion. T32-ES011564 and R01HL163818. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Monitoring phosphorylation and acetylation of CRISPR-mediated HiBiT-tagged endogenous proteins

Intracellular pathways transduce signals through changes in post-translational modifications (PTMs) of effector proteins. Among the approaches used to monitor PTM changes are immunoassays and overexpression of recombinant reporter genes. Genome editing by CRISPR/Cas9 provides a new means to monitor PTM changes by inserting reporters onto target endogenous genes while preserving native biology. Ideally, the reporter should be small in order not to interfere with the processes mediated by the target while sensitive enough to detect tightly expressed proteins. HiBiT is a 1.3 kDa reporter peptide capable of generating bioluminescence through complementation with LgBiT, an 18 kDa subunit derived from NanoLuc. Using HiBiT CRISPR/Cas9-modified cell lines in combination with fluorescent antibodies, we developed a HiBiT-BRET immunoassay (a.k.a. Immuno-BRET). This is a homogeneous immunoassay capable of monitoring post-translational modifications on diverse protein targets. Its usefulness was demonstrated for the detection of phosphorylation of multiple signaling pathway targets (EGFR, STAT3, MAPK8 and c-MET), as well as chromatin containing histone H3 acetylation on lysine 9 and 27. These results demonstrate the ability to efficiently monitor endogenous biological processes modulated by post-translational modifications using a small bioluminescent peptide tag and fluorescent antibodies, providing sensitive quantitation of the response dynamics to multiple stimuli.

The protective pattern of chitosan-based active coating on texture stabilization of refrigerated carp fillets from the perspective of proteolysis

Herein, the protective pattern of chitosan coating enriched with green tea extract on texture stabilization of refrigerated grass carp fillets was explored. In general, higher shear force and lower endogenous enzyme activities were observed in coated fillets, with the max. level of cathepsins and calpain decreasing by 30.2 ∼ 39.6 % when compared to the control during storage. Meanwhile, the coating reduced protein composition changes and accumulation of protein degradation products. According to label-free proteomic analysis, the proteome closer to fresh sample was observed in coated fillets than that of the control, as supported by PCA and hierarchical cluster analysis. Meanwhile, 33 differential proteins involved in tissue structure, protein phosphorylation and protein turnover were further screened out, and most DAPs showed up-regulation in coated fillets compared to the control on day 12. Presumably, the coating modulated endogenous enzyme-induced myofibrillar protein degradation and protein phosphorylation level, thereby stabilizing the texture properties of refrigerated fillets.

Interrogating signaling pathways activation and deactivation with homogeneous NanoBiT Cell based kinase immunoassays

Cellular responses such as gene expression, enzyme activities, protein synthesis and translocation are orchestrated through activation of diverse cell signaling pathways. In these pathways, phosphorylation of specific proteins by specific kinases constitute important nodes by which the signal is transduced from an upstream activation event to downstream cellular responses. As a corollary, alteration of these kinase activities can have a detrimental effect on these cellular processes, leading to serious pathologies such as cancer, inflammation and diabetes. Therefore, monitoring these signaling events through the analysis of specific node phosphorylation states can help better understand cell behavior in health and disease states. We developed the NanoBiT cell based kinase immunoassay platform which is a suite of simple homogeneous assays to detect phosphorylation of endogenous proteins of interest in lysed cells. NanoLuc Binary Technology (NanoBiT) is a two‐subunit system based on NanoLuc luciferase that was successfully demonstrated for protein‐protein interaction (PPI) detection. We generated secondary antibody pairs linked to the two Nanoluc components, NanoBiT small and large fragments. A cell lysate containing the protein target is incubated with two primary antibodies that recognize separate epitopes on the protein. These bring NanoBiT secondary antibodies into proximity to form an active NanoLuc luciferase that makes light in proportion to the amount of target protein. When the primary antibody pair includes a phosphospecific antibody, the luminescence reflects the level of the target protein phosphorylation. Unlike current assays such as Western blot or ELISA, that are tedious, require multiple washing steps, and not easily adaptable to HTS, the NanoBiT immunoassay method presented here takes less than two hours to complete in a homogeneous “Add and Read” format. Other advantages include no cell engineering as the phosphorylation of endogenous substrate is detected in any cell type, and only a simple luminometer is required for detection. We tested this system by monitoring the activation of multiple signaling pathways including NF‐κB, mTOR/AKT and JAK/STAT, through specific nodes of phosphorylation such as pIκB, pAKT, and pSTAT3. We also tested different small or large molecule inhibitors of these pathways or their corresponding node kinase (IKK, mTOR, or JAKs) and obtained the expected pharmacology. Our results demonstrate that this bioluminescent technology can be adapted to any signaling pathway node, allowing scientists to streamline the analysis of signaling pathways of interest, study the kinase cellular activity and regulation or identify specific kinase or pathway inhibitors.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 3446: Deciphering key cancer and inflammation signaling pathways with homogeneous bioluminescent cell-based kinase activity assays

Abstract Signaling pathways activation leads to a multitude of cellular responses including modulation of enzyme activity, altered gene expression, and protein translocation or degradation. Specific phosphorylation events by specific kinases in the cell constitute important nodes in signaling pathways. Altered normal kinase activity leads to several undesirable consequences, causing diseases such as cancer and inflammation. Thus, monitoring these signaling events is essential to better understand normal cell behavior and disease states. Current assays to analyze cellular kinase activities can be tedious, nonhomogeneous, and not easily adaptable to HTS or suffer from generation of false hits due to fluorescence interference, signal quenching, or require special instruments for detection. To overcome these shortcomings, we developed a simple homogeneous assay platform to detect phosphorylation of specific endogenous proteins in cells. We took advantage of the NanoLuc Binary Technology (NanoBiT), a two-subunit system based on NanoLuc luciferase that was successfully demonstrated for protein-protein interaction (PPI) detection. We generated a detection system containing secondary antibody pairs linked to the two Nanoluc components, NanoBiT small and large fragments (11 aa peptide and 18 kDa). Cell lysate containing the phosphorylated protein is incubated with two primary antibodies that recognize separate epitopes on a single protein. These will bring NanoBiT secondary antibodies into proximity to form an active NanoLuc luciferase that makes light in proportion to the amount of target protein. When the primary antibody pair includes a phosphospecific antibody, the luminescence reflects the level of target protein phosphorylation. We tested this system by monitoring the activation of signaling pathways involved in cancer, immune and inflammatory responses (NF-κB, BTK and JAK/STAT). In very few steps we detected the expected biologic response of these pathways either by monitoring BTK autophosphorylation, assessing phosphorylation and degradation of IκB or phosphorylation of STAT3 upon activation of the cells with TNFα, or IL6, respectively. We also tested different small- or large-molecule inhibitors of these pathways or their corresponding node kinase (BTK, IKK, or JAKs) and obtained the expected pharmacology. The bioluminescent system presented here has many advantages including “Add and Read” format, no cell engineering required as the phosphorylation of endogenous substrate is detected in any cell type, and only a simple luminometer is required for detection. Our results demonstrate that this technology can be adapted to any signaling pathway analysis, allowing scientists to analyze signaling pathways of interest, study the kinase cellular activity and regulation or identify specific kinase or pathway inhibitors. Citation Format: Hicham Zegzouti, Brian Hwang, Nidhi Nath, Said Goueli. Deciphering key cancer and inflammation signaling pathways with homogeneous bioluminescent cell-based kinase activity assays [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 3446.

Deciphering Key Cancer and Inflammation Signaling Pathways with a Novel Homogeneous Bioluminescent Cell Based Kinase Activity Assays

Signaling pathway activation leads to a multitude of cellular responses including modulation of enzyme activity, altered gene expression, and protein translocation or degradation. Specific phosphorylation events by specific kinases in the cell constitute important nodes in signaling pathways. Altered normal kinase activity leads to several undesirable consequences causing diseases such as cancer and inflammation. Thus, monitoring these signaling events is essential for better understanding of normal cell behavior and disease states. Current assays to analyze cellular kinase activities can be tedious, non‐homogeneous, and not easily adaptable to HTS or suffer from generation of false hits due to fluorescence interference, signal quenching, or require special instruments for detection. To overcome these shortcomings, we developed a simple homogeneous assay platform to detect phosphorylation of specific endogenous proteins in cells. We took advantage of the NanoLuc Binary Technology (NanoBiT), a two‐subunit system based on NanoLuc luciferase that was successfully demonstrated for protein‐protein interaction (PPI) detection. We generated a detection system containing secondary antibody pairs linked to the two Nanoluc components, NanoBiT small and large fragments (11 aa peptide and 18 kDa). Cell lysate containing the phosphorylated protein is incubated with two primary antibodies that recognize separate epitopes on a single protein. These will bring NanoBiT secondary antibodies into proximity to form an active NanoLuc luciferase that makes light in proportion to the amount of target protein. When the primary antibody pair includes a phosphospecific antibody, the luminescence reflects the level of target protein phosphorylation. We tested this system by monitoring the activation of signaling pathways involved in cancer, immune and inflammatory responses (NF‐kB, JAK/STAT, and BTK). With very few steps, we detected the expected biological response of these pathways by monitoring phosphorylation/degradation of IkB, phosphorylation of STAT3, and BTK autophosphorylation. We also tested different small or large molecule inhibitors of these pathways or their corresponding node kinase (IKK, JAKs, and BTK) and obtained the expected pharmacology. The bioluminescent system presented here have many advantages including “Add and Read” format, no cell engineering required as the phosphorylation of endogenous substrate is detected in any cell type including primary cells, and only a simple luminometer is required for detection. Our results demonstrate that this technology can be adapted to any signaling pathway analysis allowing scientists to analyze signaling pathways of interest, study the kinase cellular activity and regulation or identify specific kinase or pathway inhibitors.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CYCLOPS, A DNA-Binding Transcriptional Activator, Orchestrates Symbiotic Root Nodule Development

Nuclear calcium oscillations are a hallmark of symbiotically stimulated plant root cells. Activation of the central nuclear decoder, calcium- and calmodulin-dependent kinase (CCaMK), triggers the entire symbiotic program including root nodule organogenesis, but the mechanism of signal transduction byCCaMK was unknown. We show that CYCLOPS, a direct phosphorylation substrate of CCaMK, isaDNA-binding transcriptional activator. Two phosphorylated serine residues within the N-terminal negative regulatory domain of CYCLOPS are necessary for its activity. CYCLOPS binds DNA in a sequence-specific and phosphorylation-dependent manner and transactivates the NODULE INCEPTION (NIN) gene. A phosphomimetic version of CYCLOPS was sufficient to trigger root nodule organogenesis in the absence of rhizobia and CCaMK. CYCLOPS thus induces a transcriptional activation cascade, in which NIN and a heterotrimeric NF-Y complex act in hierarchical succession to initiate symbiotic root nodule development.

The centrosomal kinase NEK2 is a novel splicing factor kinase involved in cell survival.

NEK2 is a serine/threonine kinase that promotes centrosome splitting and ensures correct chromosome segregation during the G2/M phase of the cell cycle, through phosphorylation of specific substrates. Aberrant expression and activity of NEK2 in cancer cells lead to dysregulation of the centrosome cycle and aneuploidy. Thus, a tight regulation of NEK2 function is needed during cell cycle progression. In this study, we found that NEK2 localizes in the nucleus of cancer cells derived from several tissues. In particular, NEK2 co-localizes in splicing speckles with SRSF1 and SRSF2. Moreover, NEK2 interacts with several splicing factors and phosphorylates some of them, including the oncogenic SRSF1 protein. Overexpression of NEK2 induces phosphorylation of endogenous SR proteins and affects the splicing activity of SRSF1 toward reporter minigenes and endogenous targets, independently of SRPK1. Conversely, knockdown of NEK2, like that of SRSF1, induces expression of pro-apoptotic variants from SRSF1-target genes and sensitizes cells to apoptosis. Our results identify NEK2 as a novel splicing factor kinase and suggest that part of its oncogenic activity may be ascribed to its ability to modulate alternative splicing, a key step in gene expression regulation that is frequently altered in cancer cells.

Globular body production, their anatomy, DNase gel analysis and NDP kinase activity in root tips of Poncirus trifoliata L.

Green globular bodies were developed from Poncirus trifoliata L. root tip explants as a response to addition in the substrate of different growth regulators. From the globular bodies, shoots initiated and grew. Median section of the globular bodies reveals that they are composed of parenchyma cells and originate from the pericycle. The activity of DNases during shoot formation from globular bodies was influenced by the type and concentration of plant growth regulators that were added in the nutrient substrate. Peptide bands formation was also influenced by the increase of BA concentration. Consequently, BA, NAA and IAA combination influenced 5'-triphosphonucleosides (NTPs) appearance and activity in the presence of metal. Peptide bands resulted from the electrophoretic analysis of endogenous protein phosphorylation, proved to be catalytic subunits of NDP kinases, as they all phosphorylate diphosphonucleosides. The enzymes DNases and NDP kinases could be used as a scientific tool for the study of shoot formation from P.trifoliata L. green globular bodies.

Calyculin A from Discodermia Calyx Is a Dual Action Toxin that Blocks Calcium Influx and Inhibits Protein Ser/Thr Phosphatases

Calyculin A (Caly A) is cell permeable toxin widely used in cell biology research as an inhibitor of type 1 and type 2A protein Ser/Thr phosphatases of the PPP family. Here we tested effects of low concentrations of Caly A on proliferation of human cancer and non-cancer cell lines. We found that long-term 0.3 nM Caly A prevented G1 to S phase cell cycle progression in human Hs-68 fibroblasts and ARPE19 epithelial cells, but not human breast cancer MDA-MB-468, MDA-MB-231 and MCF7 cells. These conditions produced no change in cyclin D1 levels or in the phosphorylation of endogenous proteins. However, acute application of 0.3 nM Caly A blocked serum-induced increase in intracellular calcium levels in Hs-68 fibroblasts, but not in MDA-MB-468 breast cancer cells. We propose that subnanomolar Caly A prevents cell cycle progression because it blocks calcium uptake by fibroblasts. This probably involves non-selective cation channels and cancer cell proliferation was not affected because calcium enters these cells by other channels. Our results suggest that calyculin A has dual actions and acts as a channel blocker, in addition to its well-established effects as a phosphatase inhibitor.

Development and Validation of a Single-Well Cell-Based Assay for the Detection of Endogenous Phosphoproteins

We describe a cellular assay for detection of phosphorylation of endogenous proteins, whereby cells are seeded, treated, and assayed for modulation of phosphorylation in a single microplate well. The procedure is coupled to a rapid, one-wash sandwich enzyme-linked immuno-sorbent assay, enabling results to be obtained within 3-4 h from cell seeding. The assay was tested in two separate cellular systems, namely, HeLa and MCF-7 cells. When using the one-well protocol with Akt phosphorylation as a model, the response to a number of agonists was the same as the response obtained using cells treated in a separate microplate, using a conventional lysate transfer approach. The assay procedure was automated, and quantitative pharmacological data on three known inhibitors of the PI3-kinase signaling pathway was obtained within 4 h from seeding cells, with six dispense steps, and a single wash cycle. Thus, the protocol affords a reliable means of assaying for cellular signaling events in different cell types, and is amenable to automation.

Site-specific quantitative analysis of cardiac mitochondrial protein phosphorylation

We report the development of a multiple-reaction monitoring (MRM) strategy specifically tailored to the detection and quantification of mitochondrial protein phosphorylation. We recently derived 68 MRM transitions specific to protein modifications in the respiratory chain, voltage-dependent anion channel, and adenine nucleotide translocase. Here, we have now expanded the total number of MRM transitions to 176 to cover proteins from the tricarboxylic acid cycle, pyruvate dehydrogenase complex, and branched-chain alpha-keto acid dehydrogenase complex. We utilized the transition set to analyze endogenous protein phosphorylation in human heart, mouse heart, and mouse liver. The data demonstrate the potential utility of the MRM workflow for studying the functional details of mitochondrial phosphorylation signaling. This article is part of a Special Issue entitled: From protein structures to clinical applications.

Dysregulation of epithelial Na+ absorption induced by inhibition of the kinases TORC1 and TORC2

Although the serum and glucocorticoid-inducible protein kinase 1 (SGK1) appears to be involved in controlling epithelial Na(+) absorption, its role in this physiologically important ion transport process is undefined. As SGK1 activity is dependent upon target of rapamycin complex 2 (TORC2)-catalysed phosphorylation of SGK1-Ser(422) , we have explored the effects of inhibiting TORC2 and/or TORC1 upon the hormonal control of Na(+) absorption. Na(+) absorption was quantified electrometrically in mouse cortical collecting duct cells (mpkCCD) grown to confluence on permeable membranes. Kinase activities were assessed by monitoring endogenous protein phosphorylation, with or without TORC1/2 inhibitors (TORIN1 and PP242) and the TORC1 inhibitor: rapamycin. Inhibition of TORC1/2 (TORIN1, PP242) suppressed basal SGK1 activity, prevented insulin- and dexamethasone-induced SGK1 activation, and caused modest (10-20%) inhibition of basal Na(+) absorption and substantial (∼80%) inhibition of insulin/dexamethasone-induced Na(+) transport. Inhibition of TORC1 did not impair SGK1 activation or insulin-induced Na(+) transport, but did inhibit (∼80%) dexamethasone-induced Na(+) absorption. Arginine vasopressin stimulated Na(+) absorption via a TORC1/2-independent mechanism. Target of rapamycin complex 2, but not TORC1, is important to SGK1 activation. Signalling via phosphoinositide-3-kinase/TORC2/SGK1 can explain insulin-induced Na(+) absorption. TORC2, but not TORC1, is also involved in glucocorticoid-induced SGK1 activation but its role is permissive. Glucocorticoid-induced Na(+) transport displayed a requirement for TORC1 activity. Therefore, TORC1 and TORC2 contribute to the regulation of Na(+) absorption. Pharmacological manipulation of TORC1/2 signalling may provide novel therapies for Na(+)-sensitive hypertension.

Effects of nominally selective inhibitors of the kinases PI3K, SGK1 and PKB on the insulin‐dependent control of epithelial Na+ absorption

Insulin-induced Na(+) retention in the distal nephron may contribute to the development of oedema/hypertension in patients with type 2 diabetes. This response to insulin is usually attributed to phosphatidylinositol-3-kinase (PI3K)/serum and glucocorticoid-inducible kinase 1 (SGK1) but a role for protein kinase B (PKB) has been proposed. The present study therefore aimed to clarify the way in which insulin can evoke Na(+) retention. We examined the effects of nominally selective inhibitors of PI3K (wortmannin, PI103, GDC-0941), SGK1 (GSK650394A) and PKB (Akti-1/2) on Na(+) transport in hormone-deprived and insulin-stimulated cortical collecting duct (mpkCCD) cells, while PI3K, SGK1 and PKB activities were assayed by monitoring the phosphorylation of endogenous proteins. Wortmannin substantially inhibited basal Na(+) transport whereas PI103 and GDC-0941 had only very small effects. However, these PI3K inhibitors all abolished insulin-induced Na(+) absorption and inactivated PI3K, SGK1 and PKB fully. GSK650394A and Akti-1/2 also inhibited insulin-evoked Na(+) absorption and while GSK650394A inhibited SGK1 without affecting PKB, Akti-1/2 inactivated both kinases. While studies undertaken using PI103 and GDC-0941 show that hormone-deprived cells can absorb Na(+) independently of PI3K, PI3K seems to be essential for insulin induced Na(+) transport. Akti-1/2 does not act as a selective inhibitor of PKB and data obtained using this compound must therefore be treated with caution. GSK650394A, on the other hand, selectively inhibits SGK1 and the finding that GSK650394A suppressed insulin-induced Na(+) absorption suggests that this response is dependent upon signalling via PI3K/SGK1.

Changes in cAMP-dependent phosphorylation of specific cytosol protein in the peri- and postmenopausal ovary.

Changes in cAMP-dependent phosphorylation of 53 kDa protein in a human ovary with respect to the aging process were studied. The phosphorylation of endogenous proteins in ovarian cytosol decreased with aging. However, ovarian aging was accompanied by an increase in the cAMP-dependent phosphorylation of 53 kDa specific protein while the amount of this protein decreased with aging. The plasma gonadotropin levels of women with regular menstrual periods were significantly different from those of women whose menstrual periods had in the recent past become irregular (FSH: p less than 0.01; LH: p less than 0.05); however, there was no significant difference in the plasma estradiol levels between these two groups. Our data show the possible correlations between the non-steroidal ovarian factor (53 kDa protein) and/or its phosphorylation by cAMP-dependent protein kinase and the ovarian aging process.

Development of cell-based assays for cytokine receptor signaling, using an AlphaScreen SureFire assay format

The signal transducers and activators of transcription (STAT) proteins are a small family of signaling proteins that are crucial for cytokine and growth factor receptor-mediated signaling in various blood cell types. Despite their central role in immune and hematopoietic cellular regulation, there are relatively few options for monitoring receptor-mediated JAK/STAT signaling events in a cell-based format, without the need for cellular transfections or labor intensive methodology. Indeed, traditional methods such as the Western blot or ELISA remain a standard method for determining the phosphorylation status of endogenous STAT proteins. Here we present data for the rapid detection of endogenous receptor-mediated phosphorylation of multiple STAT proteins using the bead-based AlphaScreen SureFire technology. With three different cell lines (human acute monocytic leukemia THP-1 cells, human erythroleukemic TF-1 cells, and human T lymphocytic Jurkat cells), we have optimized a rapid and homogeneous methodology for monitoring endogenous, receptor-mediated signaling via STAT 1, STAT 3, or STAT 5 phosphorylation, in response to several agonists. These assays, which can be tailored for both standard research applications or high-throughput drug screening applications, afford quantitative data for receptor-mediated signaling mechanisms in an endogenous, cellular environment.

Phosphorylation of TRPC6 Channels at Thr69 Is Required for Anti-hypertrophic Effects of Phosphodiesterase 5 Inhibition

Activation of Ca(2+) signaling induced by receptor stimulation and mechanical stress plays a critical role in the development of cardiac hypertrophy. A canonical transient receptor potential protein subfamily member, TRPC6, which is activated by diacylglycerol and mechanical stretch, works as an upstream regulator of the Ca(2+) signaling pathway. Although activation of protein kinase G (PKG) inhibits TRPC6 channel activity and cardiac hypertrophy, respectively, it is unclear whether PKG suppresses cardiac hypertrophy through inhibition of TRPC6. Here, we show that inhibition of cGMP-selective PDE5 (phosphodiesterase 5) suppresses endothelin-1-, diacylglycerol analog-, and mechanical stretch-induced hypertrophy through inhibition of Ca(2+) influx in rat neonatal cardiomyocytes. Inhibition of PDE5 suppressed the increase in frequency of Ca(2+) spikes induced by agonists or mechanical stretch. However, PDE5 inhibition did not suppress the hypertrophic responses induced by high KCl or the activation of protein kinase C, suggesting that PDE5 inhibition suppresses Ca(2+) influx itself or molecule(s) upstream of Ca(2+) influx. PKG activated by PDE5 inhibition phosphorylated TRPC6 proteins at Thr(69) and prevented TRPC6-mediated Ca(2+) influx. Substitution of Ala for Thr(69) in TRPC6 abolished the anti-hypertrophic effects of PDE5 inhibition. In addition, chronic PDE5 inhibition by oral sildenafil treatment actually induced TRPC6 phosphorylation in mouse hearts. Knockdown of RGS2 (regulator of G protein signaling 2) and RGS4, both of which are activated by PKG to reduce G alpha(q)-mediated signaling, did not affect the suppression of receptor-activated Ca(2+) influx by PDE5 inhibition. These results suggest that phosphorylation and functional suppression of TRPC6 underlie prevention of pathological hypertrophy by PDE5 inhibition.

Insulin Induces REDD1 Expression through Hypoxia-inducible Factor 1 Activation in Adipocytes

REDD1 (regulated in development and DNA damage responses) is essential for the inhibition of mTORC1 (mammalian target of rapamycin complex) signaling pathway in response to hypoxia. REDD1 expression is regulated by many stresses such as hypoxia, oxidative stress, and energy depletion. However, the regulation of REDD1 expression in response to insulin remains unknown. In the present study, we demonstrate that in murine and in human adipocytes, insulin stimulates REDD1 expression. Insulin-induced REDD1 expression occurs through phosphoinositide 3-kinase/mTOR-dependent pathways. Moreover, using echinomycin, a hypoxia-inducible factor 1 (HIF-1) inhibitor, and HIF-1alpha small interfering RNA, we demonstrate that insulin stimulates REDD1 expression only through the transcription factor HIF-1. In conclusion, our study shows that insulin stimulates REDD1 expression in adipocytes.

Bacterial tyrosine kinases: novel targets for antibacterial therapy?

The resistance of pathogenic bacteria to current antibiotics has become a crucial public health problem. To combat this resistance, there is a constant need for antibacterial drugs with new modes of action on therapeutic targets. Recent data have shown that a variety of cellular processes essential for bacterial survival and virulence are regulated by the phosphorylation of certain endogenous proteins catalyzed by specific tyrosine kinases. In this article, I highlight a selection of recent findings that confirm the central role of protein tyrosine phosphorylation in the control of bacterial physiology. Based on this knowledge, potential applications in the discovery of novel antibiotics are proposed.