Biphasic Phosphorylation Research Articles

Key roles for phosphorylation and the Coiled-coil domain in TRIM56-mediated positive regulation of TLR3-TRIF–dependent innate immunity

Tripartite-motif protein-56 (TRIM56) positively regulates the induction of type I interferon (IFN) response via the TLR3 pathway by enhancing IRF3 activation and depends on its C-terminal residues 621-750 for interacting with the adaptor TRIF. However, the precise underlying mechanism and detailed TRIM56 determinants remain unclear. Herein, we show ectopic expression of murine TRIM56 also enhances TLR3-dependent IFN-β promoter activation, suggesting functional conservation. We found that endogenous TRIM56 and TRIF formed a complex early (0.5-2 h) after poly-I:C stimulation and that TRIM56 overexpression also promoted activation of NF-κB by poly-I:C but not that by TNF-α or IL-1β, consistent with a specific effect on TRIF prior to the bifurcation of NF-κB and IRF3. Using transient transfection and Tet-regulated cell lines expressing various TRIM56 mutants, we demonstrated the Coiled-coil domain and a segment spanning residues ∼434-610, but not the B-box or residues 355-433, were required for TRIM56 augmentation of TLR3 signaling. Moreover, alanine substitution at each putative phosphorylation site, Ser471, Ser475 and Ser710, abrogated TRIM56 function. Concordantly, mutants bearing Ser471Ala, Ser475Ala, or Ser710Ala, or lacking the Coiled-coil domain, all lost the capacity to enhance poly-I:C-induced establishment of an antiviral state. Furthermore, the Ser710Ala mutation disrupted the TRIM56-TRIF association. Using phospho-specific antibodies, we detected biphasic phosphorylation of TRIM56 at Ser471 and Ser475 following TLR3 stimulation, with the early phase occurring at ∼0.5-1 h, prior to IRF3 phosphorylation. Together, these data reveal novel molecular details critical for the TRIM56 augmentation of TLR3-dependent antiviral response and highlight important roles for TRIM56 scaffolding and phosphorylation.

KOPI: Kinase inhibitOr Proteome Impact analysis

Kinase inhibitors often exert on/off-target effects, and efficient data analysis is essential for assessing these effects on the proteome. We developed a workflow for rapidly performing such a proteomic assessment, termed as kinase inhibitor proteome impact analysis (KOPI). We demonstrate KOPI’s utility with staurosporine (STS) on the leukemic K562 cell proteome. We identified systematically staurosporine’s non-kinome interactors, and showed for the first time that it caused paradoxical hyper- and biphasic phosphorylation.

Abstract 15736: The Sphk1/s1p Signaling Axis Regulates Pulmonary Vascular Inflammation by Activating Nf-kb and Nlrp3 Inflammasome

Rationale: Pulmonary vascular inflammation is activated during the development of pulmonary arterial hypertension (PAH). Sphingosine kinase 1 (Sphk1) catalyzes the formation of sphingosine-1-phosphate (S1P), which has been shown to promote inflammation by activating nuclear factor kappa B (NF-κB) and the nucleotide oligomerization domain-like receptor 3 (NLRP3) inflammasome. However, the impact of S1P on NLRP3 inflammasome activation in PAH is unknown. Here, we tested whether Sphk1/S1P regulates pulmonary vascular inflammation via activating NF-κB and the NLRP3 inflammasome. Methods: Human pulmonary arterial smooth muscle cells (PASMCs) and endothelial cells (PAECs) were treated with S1P or were exposed to hypoxia. Sphk1 plasmid or siRNA was used to increase and decrease Sphk1/S1P levels, respectively. Activation of NF-κB signaling and the NLRP3 inflammasome (increased NLRP3, cleaved caspase-1, IL-1β and IL-18) were detected by western blots or real-time PCR. ELISA was used to measure secretion of IL-1β. Results: S1P treatment led to a biphasic phosphorylation and degradation of IκBα accompanied by increased phosphorylation and expression of NF-κB in PASMCs. S1P treatment also resulted in increased expression of inflammasome mediators, NLRP3 and cleaved caspase-1, enhanced intracellular expression of IL-1b and IL-18, and higher levels of secreted IL-1β compared to controls. Notably, over expression of Sphk1 in PASMCs led to increased NF-κB and NLRP3 expression, whereas suppression of Sphk1 expression reduced NLRP3, cleavage of IL-1β and IL-18 expression and phosphorylation of NF-κB. Similar phenomena with respect to inflammasome activation and IL-1β secretion was observed in PASMCs exposed to hypoxia. Increased NF-κB and NLRP3 activation, cleavage of caspase-1 were also observed in S1P treated PAECs, however no changes in IL-1β secretion levels were detected. Conclusion: These findings suggest that S1P induces pulmonary vascular pro-inflammatory signaling via activation of NF-κB and the NLRP3 inflammasome, and that the S1P/Sphk1 signaling axis may be involved in this context. Activation of the inflammasome may be an important mechanism underlying PAH pathogenesis, targeting of which may be exploited for the treatment of PAH.

Convergent ERK1/2, p38 and JNK mitogen activated protein kinases (MAPKs) signalling mediate catecholoestradiol-induced proliferation of ovine uterine artery endothelial cells.

The catechol metabolites of 17β-oestradiol (E2 β), 2-hydroxyoestradiol (2-OHE2 ) and 4-hydroxyoestradiol (4-OHE2 ), stimulate proliferation of pregnancy-derived ovine uterine artery endothelial cells (P-UAECs) through β-adrenoceptors (β-ARs) and independently of the classic oestrogen receptors (ERs). Herein we show that activation of ERK1/2, p38 and JNK mitogen activated protein kinases (MAPKs) is necessary for 2-OHE2 - and 4-OHE2 -induced P-UAEC proliferation, as well as proliferation induced by the parent hormone E2 β and other β-AR signalling hormones (i.e. catecholamines). Conversely, although 2-OHE2 and 4-OHE2 rapidly activate phosphatidylinositol 3-kinase (PI3K), its activation is not involved in catecholoestradiol-induced P-UAEC proliferation. We also show for the first time the signalling mechanisms involved in catecholoestradiol-induced P-UAEC proliferation; which converge at the level of MAPKs with the signalling mechanisms mediating E2 β- and catecholamine-induced proliferation. The present study advances our understanding of the complex signalling mechanisms involved in regulating uterine endothelial cell proliferation during pregnancy. Previously we demonstrated that the biologically active metabolites of 17β-oestradiol, 2-hydroxyoestradiol (2-OHE2 ) and 4-hydroxyoestradiol (4-OHE2 ), stimulate pregnancy-specific proliferation of uterine artery endothelial cells derived from pregnant (P-UAECs), but not non-pregnant ewes. However, unlike 17β-oestradiol, which induces proliferation via oestrogen receptor-β (ER-β), the catecholoestradiols mediate P-UAEC proliferation via β-adrenoceptors (β-AR) and independently of classic oestrogen receptors. Herein, we aim to further elucidate the signalling mechanisms involved in proliferation induced by catecholoestradiols in P-UAECs. P-UAECs were treated with 2-OHE2 and 4-OHE2 for 0, 0.25, 0.5, 1, 2, 4, 12 and 24h, to analyse activation of mitogen activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K)-AKT. Specific inhibitors for ERK1/2 MAPK (PD98059), p38 MAPK (SB203580), JNK MAPK (SP600125), or PI3K (LY294002) were used to determine the involvement of individual kinases in agonist-induced P-UAEC proliferation. 2-OHE2 and 4-OHE2 stimulated biphasic phosphorylation of ERK1/2, slow p38 and JNK phosphorylation over time, and rapid monophasic AKT phosphorylation. Furthermore, ERK1/2, p38 and JNK MAPKs, but not PI3K, were individually necessary for catecholoestradiol-induced proliferation. In addition, when comparing the signalling mechanisms of the catecholoestradiols, to 17β-oestradiol and catecholamines, we observed that convergent MAPKs signalling pathways facilitate P-UAEC proliferation induced by all of these hormones. Thus, all three members of the MAPK family mediate the mitogenic effects of catecholoestradiols in the endothelium during pregnancy. Furthermore, the convergent signalling of MAPKs involved in catecholoestradiol-, 17β-oestradiol- and catecholamine-induced endothelial cell proliferation may be indicative of unappreciated evolutionary functional redundancy to facilitate angiogenesis and ensure maintenance of uterine blood flow during pregnancy.

Prostaglandin E2-stimulated prostanoid EP4 receptors induce prolonged de novo prostaglandin E2 synthesis through biphasic phosphorylation of extracellular signal-regulated kinases mediated by activation of protein kinase A in HCA-7 human colon cancer cells

Approximately two decades have passed since E-type prostanoid 4 (EP4) receptors were cloned, and the signaling pathways mediated by these receptors have since been implicated in cancer development through the alliance of Gαi-protein/phosphatidylinositol 3-kinase (PI3K)/extracellular signal-regulated kinases (ERKs) activation. Although prostanoid EP4 receptors were initially identified as Gαs-coupled receptors, the specific/distinctive role(s) of prostanoid EP4 receptor-induced cAMP/protein kinase A (PKA) pathways in cancer development have not yet been elucidated in detail. We previously reported using HCA-7 human colon cancer cells that prostaglandin E2 (PGE2)-stimulated prostanoid EP4 receptors induced cyclooxygenase-2 (COX-2) as an initiating event in development of colon cancer. Moreover, this induction of COX-2 was mediated by transactivation of epidermal growth factor (EGF) receptors. However, direct activation of EGF receptors by EGF also induced similar amounts of COX-2 in this cell line. Thus, the emergence of unique role(s) for prostanoid EP4 receptors is expected by clarifying the different signaling mechanisms between PGE2-stimulated prostanoid EP4 receptors and EGF-stimulated EGF receptors to induce COX-2 and produce PGE2. We here demonstrated that prostanoid EP4 receptor activation by PGE2 in HCA-7 cells led to PKA-dependent re-activation of ERKs, which resulted in prolonged de novo synthesis of PGE2. Although EGF-stimulated EGF receptors in cells also induced COX-2 and the de novo synthesis of PGE2, the activation of this pathway was transient and not mediated by PKA. Therefore, the novel mechanism underlying prolonged de novo synthesis of PGE2 has provided an insight into the importance of prostanoid EP4 receptor-mediated Gαs-protein/cAMP/PKA pathway in development of colon cancer.

The Ang-(1–7)/Mas-1 axis attenuates the expression and signalling of TGF-β1 induced by AngII in mouse skeletal muscle

AngII (angiotensin II) induces pathological conditions such as fibrosis in skeletal muscle. In this process, AngII increases ROS (reactive oxygen species) and induces a biphasic phosphorylation of p38 MAPK (mitogen-activated protein kinase). In addition, AngII stimulates the expression and production of TGF (transforming growth factor)-β1 via a mechanism dependent on ROS production mediated by NADPH oxidase (NOX) and p38 MAPK activation. In the present study, we investigated whether Ang-(1-7) [angiotensin-(1-7)], through the Mas-1 receptor, can counteract the signalling induced by AngII in mouse skeletal muscle and cause a decrease in the expression and further activity of TGF-β1in skeletal muscle cells. Our results show that Ang-(1-7) decreased the expression of TGF-β1 induced by AngII in a dose-dependent manner. In addition, we observed that Ang-(1-7) prevented the increase in TGF-β1 expression induced by AngII, ROS production dependent on NOX and the early phase of p38 MAPK phosphorylation. Interestingly, Ang-(1-7) also prevented the late phase of p38 MAPK phosphorylation, Smad-2 phosphorylation and Smad-4 nuclear translocation, an increase in transcriptional activity, as determined using the p3TP-lux reporter, and fibronectin levels, all of which are dependent on the TGF-β1 levels induced by AngII. We also demonstrated that Ang-(1-7) prevented the increase in TGF-β1, fibronectin and collagen content in the diaphragm of mice infused with AngII. All of these effects were reversed by the administration of A779, indicating the participation of Mas-1. In conclusion, our findings support the hypothesis that Ang-(1-7) decreases the expression and further biological activity of TGF-β1 induced by AngII invitro and invivo.

Ras Protein Activation Is a Key Event in Activity-dependent Survival of Cerebellar Granule Neurons

Neuronal activity promotes the survival of cerebellar granule neurons (CGNs) during the postnatal development of cerebellum. CGNs that fail to receive excitatory inputs will die by apoptosis. This process could be mimicked in culture by exposing CGNs to either a physiological concentration of KCl (5 mm or K5) plus N-methyl-d-aspartate (NMDA) or to 25 mm KCl (K25). We have previously described that a 24-h exposure to NMDA (100 μm) or K25 at 2 days in vitro induced long term survival of CGNs in K5 conditions. Here we have studied the molecular mechanisms activated at 2 days in vitro in these conditions. First we showed that NMDA or K25 addition promoted a rapid stimulation of PI3K and a biphasic phosphorylation on Ser-473 of Akt, a PI3K substrate. Interestingly, we demonstrated that only the first wave of Akt phosphorylation is necessary for the NMDA- and K25-mediated survival. Additionally, we detected that both NMDA and K25 increased ERK activity with a similar time-course. Moreover, our results showed that NMDA-mediated activation of the small G-protein Ras is necessary for PI3K/Akt pathway activation, whereas Rap1 was involved in NMDA phosphorylation of ERK. On the other hand, Ras, but not Rap1, mediates K25 activation of PI3K/Akt and MEK/ERK pathways. Because neuroprotection by NMDA or K25 is mediated by Ras (and not by Rap1) activation, we propose that Ras stimulation is a crucial event in NMDA- and K25-mediated survival of CGNs through the activation of PI3K/Akt and MEK/ERK pathways.

Role of pro renin receptor in Ang II-mediated EGF receptor transactivation

Prorenin-induced intracellular signaling pathway is not fully elucidated. We investigated whether the (pro)renin receptor mediates epidermal growth factor (EGF) receptor transactivation through angiotensin (Ang) II-dependent and -independent pathways in human embryo kidney 293 cells. Prorenin (2 nmol/L) caused biphasic phosphorylation of EGF receptor (Tyr992) and extracellular signal-regulated kinase (ERK) 1/2, peaking at 5 minutes followed by a decrease and a second peak at 60-120 minutes, whereas EGF receptor (Tyr1068) and Src were phosphorylated at only 120 minutes. These prorenin-induced phosphorylation processes were inhibited by (pro)renin receptor siRNA. Similarly, Ang II type 1 (AT1) receptor blocker (ARB) or AT1receptor siRNA completely inhibited prorenin-induced phosphorylation of EGF receptor (Tyr1068) and Src, as well as the late peaks of EGF receptor (Tyr992) and ERK 1/2. However, early peaks of EGF receptor (Tyr992) and ERK 1/2 at 5 minutes were not effectively blocked by ARB or AT1receptor siRNA. Incubation with prorenin significantly increased Ang II levels of cell lysate. These data indicate that the (pro)renin receptor mediates EGF receptor transactivation in both Ang II-dependent and -independent pathways.

In Vitro Stretch Injury Induces Time- and Severity-Dependent Alterations of STEP Phosphorylation and Proteolysis in Neurons

Striatal-enriched tyrosine phosphatase (STEP) has been identified as a component of physiological and pathophysiological signaling pathways mediated by N-methyl-d-aspartate (NMDA) receptor/calcineurin/calpain activation. Activation of these pathways produces a subsequent change in STEP isoform expression or activation via dephosphorylation. In this study, we evaluated changes in STEP phosphorylation and proteolysis in dissociated cortical neurons after sublethal and lethal mechanical injury using an in vitro stretch injury device. Sublethal stretch injury produces minimal changes in STEP phosphorylation at early time points, and increased STEP phosphorylation at 24 h that is blocked by the NMDA-receptor antagonist APV, the calcineurin-inhibitor FK506, and the sodium channel blocker tetrodotoxin. Lethal stretch injury produces rapid STEP dephosphorylation via NR2B-containing NMDA receptors, but not calcineurin, and a subsequent biphasic phosphorylation pattern. STEP(61) expression progressively increases after sublethal stretch with no change in calpain-mediated STEP(33) formation, while lethal stretch injury results in STEP(33) formation via a NR2B-containing NMDA receptor pathway within 1 h of injury. Blocking calpain activation in the initial 30 min after stretch injury increases the ratio of active STEP in cells and blocks STEP(33) formation, suggesting that STEP is an early substrate of calpain after mechanical injury. There is a strong correlation between the amount of STEP(33) formed and the degree of cell death observed after lethal stretch injury. In summary, these data demonstrate that previously characterized pathways of STEP regulation via the NMDA receptor are generally conserved in mechanical injury, and suggest that calpain-mediated cleavage of STEP(33) should be further examined as an early marker of neuronal fate after stretch injury.

Metalloestrogenic Effects of Quantum Dots

To investigate the metalloestrogenic effects of cadmium telluride quantum dots (QDs) in both human breast cancer cells and in vivo in mice. Human breast cancer cells (MCF-7 cells) were utilized to study QDs, cadmium and 17β-estradiol induced estrogen-related genomic and nongenomic signaling. Female prepubescent and ovariectomized adult mice were treated with CdTe QDs to assess whether QD-induced estrogenicity would lead to uterine changes. Our findings demonstrate that in vitro cadmium-containing QDs induce cellular proliferation, estrogen receptor α activation, and biphasic phosphorylation of AKT and ERK1/2, comparable with 17β-estradiol. Green QDs elicited a more robust estrogenic response than orange QDs. Addition of the selective estrogen receptor antagonist, ICI 182780, completely abolished all QD-induced estrogenic effects, suggesting that QD-induced estrogenic signaling is mediated via the estrogen receptor. In vivo, chronic treatment of mice with QDs led to a two- to three-fold increase in uterine weight, comparable or greater than 17β-estradiol. These findings suggest that certain cadmium-containing nanocrystals are endocrine disruptors, whose effects can exceed those induced by ionic cadmium or 17β-estradiol.

Differential effects of Akt pathway inhibitors on IL-1β-induced protein phosphorylation in human fibroblast-like synoviocytes

Context: Interleukin (IL)-1β activates various signal transduction pathways including p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and Akt in human fibroblast-like synoviocytes (HFLS).Objective: We investigated the effects of an Akt inhibitor, a phosphatidylinositol 3-kinase (PI3K) inhibitor, and Akt RNAi knockdown on IL-1β-induced protein phosphorylation in HFLS to clarify the role of the PI3K/Akt signaling pathway in the phosphorylation of the inhibitor of κB (IκB)α and heat shock protein 27 (HSP27).Materials and methods: A multiplex suspension array system was used for the detection of phosphorylated proteins.Results: IL-1β induced biphasic phosphorylation of IκBα, with the first phase occurring 10 min after IL-1β stimulation, and this was augmented by treatment with Akt inhibitor IV. However, this phenomenon was not observed after treatment with LY-294002, a PI3K inhibitor. Furthermore, Akt inhibitor IV suppressed ERK2 phosphorylation, whereas LY-294002 and Akt RNAi had no effect. In contrast, Akt inhibitor IV, LY-294002, and Akt RNAi augmented HSP27 phosphorylation.Discussion and conclusions: Modulation of different stages of the PI3K/Akt pathway may differentially affect the phosphorylation of IκBα and HSP27 in HFLS.

P2Y6 Receptors and ADAM17 Mediate Low-Dose Gamma-Ray-Induced Focus Formation (Activation) of EGF Receptor

The EGF receptor (EGFR) is frequently expressed in tumors of epithelial origin. Although it is well known that ionizing radiation induces activation of EGFR, the mechanism remains unknown. Recently, we reported that activation of P2Y receptors is involved in γ-radiation-induced activation of extracellular signal-regulated kinase1/2 (ERK1/2), which is dependent on activation of EGFR. Here we focused on the mechanism of activation of EGFR in response to low-dose γ radiation, mainly in terms of the activation-associated formation of EGFR foci in A549 cells. Irradiation of cells with 0.1 Gy γ rays induced biphasic phosphorylation of EGFR and ERK1/2 as well as biphasic formation of EGFR foci. The radiation-induced focus formation of EGFR was abolished by ecto-nucleotidase, P2Y receptor antagonists and knockdown of P2Y6 receptor, suggesting the involvement of extracellular nucleotides and activation of P2Y6 receptors in this process. Further, a disintegrin and metalloprotease 17 (ADAM17) is expressed in A549 cells and an ADAM17 inhibitor significantly blocked the radiation-induced focus formation of EGFR. We conclude that activation of both P2Y6 receptors and ADAM17 mediates the low-dose γ-radiation-induced activation of EGFR, as evaluated in terms of focus formation, in A549 cells.

ERK signaling leads to mitochondrial dysfunction in extracellular zinc‐induced neurotoxicity

A zinc-induced signaling pathway leading to extracellular signal-regulated kinase 1/2 (ERK1/2) activation and subsequent neuronal death has been investigated. We find that an extracellular zinc application stimulates biphasic phosphorylation of ERK1/2 and p38 MAPK in rat cultured neurons. The activation of ERK1/2, but not p38, is responsible for zinc neurotoxicity as only U0126, a MEK inhibitor that blocks ERK1/2 phosphorylation, significantly protects cortical neurons from zinc exposure. Over-expression of a dominant negative Ras mutant blocks zinc-induced Elk1-dependent gene expression in neurons, indicating the involvement of Ras activation in the zinc pathway leading to ERK phosphorylation and Elk1 signaling. We also find that zinc treatment results in neuronal mitochondrial hyperpolarization. Importantly, both U0126 and bongkrekic acid, an inhibitor of the mitochondrial adenine nucleotide translocase, effectively reduce zinc-triggered mitochondrial changes. As bongkrekic acid also prevents zinc-triggered neuronal death but not ERK1/2 phosphorylation, activation of MAPK signaling precedes and is required for mitochondrial dysfunction and cell death. These results provide new insight on the mechanism of extracellular zinc-induced toxicity in which the regulation of mitochondrial function by the Ras/MEK/ERK pathway is closely associated with neuronal viability.

Abstract A31: mTOR/S6K1 and MAPK/RSK signaling pathways coordinately regulate estrogen receptor α serine 167 phosphorylation

Abstract Purpose of the Study: Phosphorylation of estrogen receptor α (ERα) Ser167 is associated with development of endocrine resistance, and is a prognostic marker for the disease. Hereby we dissect the signaling pathways responsible for Ser167 phosphorylation. Experimental Procedures: MCF7 cells were grown in standard media. For experiments, cells were deprived of serum for 24h, and were pre-treated with 20ng/mL rapamycin for 30 min and/or 10 µM U0126 for 2h, and then stimulated with 100nM insulin or 100ng/mL PMA. Cell extracts were prepared, and proteins were resolved on SDS-PAGE, followed by immunoblots. For the siRNA studies, double-stranded RNAs for RSK1 and RSK2 were transfected using Lipofectamine2000 (Invitrogen). At 72h post-transfection, cells were lysed using 1X Passive Lysis Buffer (Promega). For luciferase reporter assays, cells were transfected with plasmids encoding for firefly luciferase under control of three ERE, and control renilla luciferase. At 48h post-transfection, cells were harvested and relative luciferase activity was measured using the Dual Luciferase Reporter Assay System and Glomax 20/20 luminometer (Promega). Data: mTOR/S6K1 and MAPK/RSK contribute non-overlapping inputs into ERα activation via Ser167 phosphorylation. The degree of input of each pathway depends on the nature of the activating signal. In unsynchronized cells that are growing in the presence of serum or in cells acutely stimulated with insulin, which preferentially activated the mTOR/S6K1 pathway, we observed that the MAPK/RSK pathway has a minor contribution to Ser167 phosphorylation. However, the MAPK/RSK contribution is more evident following acute stimulation with PMA, which activates both the mTOR/S6K1 and MAPK/RSK pathways. In response to PMA we observed significant rapamycin-insensitive Ser167 phosphorylation, which was U0126-sensitive. Using RNAi against RSK1/2 isoforms we demonstrated that this rapamycin-insensitive phosphorylation is mediated by RSK. In addition to being sensitive to different stimuli, the mTOR and MAPK pathways are also activated with different kinetics. S6K1 and RSK perform a non-redundant role with regard to ERα Ser167 phosphorylation, and are responsible for a biphasic phosphorylation pattern. The early-activated RSK acts in a priming role, while the late-responding S6K1 is responsible for the maintenance of high-level phosphorylation. Estrogen receptor is an important clinical target in treatment of breast cancer. However, resistance to anti-estrogen therapy often develops. One of the mechanisms of resistance is through mitogenic estrogen-independent activation of ERα. Thus, a question arises whether dual inhibition of the mTOR and MAPK pathways may result in a better clinical response. We investigated the effect of a combination of rapamycin and U0126 on transcriptional activity mediated by ERα. We demonstrated that the combination produces a greater inhibition of ERα activity than each agent alone. This strategy may be effective for combating resistance to anti-estrogen or endocrine therapy. Conclusions: The data presented here reveal that the effect of rapamycin therapy in breast cancer could be augmented by combination with MEK inhibitors, which would target two major pathways leading to ERα activation and stimulation of breast cancer cell proliferation. Citation Information: Cancer Res 2009;69(23 Suppl):A31.

Conditional Activation of Bcr-Abl Kinase Delineates Time-Dependent Transforming Events in Hematopoietic Cells.

Abstract 2162Poster Board II-139Cytoplasmic Bcr-Abl kinase evokes a diverse array of transforming signals and is responsible for development of Philadelphia chromosome-positive (Ph+) leukemias. In this fusion protein, disruption or deletion of a N-terminal coiled-coil (CC) domain of Bcr results in substantially decreased or absent tyrosine kinase activity and defective cellular transformation, indicating the crucial role of Abl oligomerization in constitutive activation of its intrinsic kinase activity. Fusion of the estrogen receptor (ER) ligand binding domain (LBD) to the C-terminus of Abl generates a ligand-activatable and transformation-competent tyrosine kinase dimer. However, additional sequences in Bcr may also be required for cellular transformation since Bcr-Abl mutants containing just the extreme CC domain cannot transform fibroblasts.To revisit the mechanism of Bcr-Abl-induced leukemogenesis and especially to delineate early events upon Bcr-Abl activation, we applied this fusion technology to construct DCCp190ER, a p190Bcr-Abl mutant including ER-LBD at the C-terminus but not CC domain at the N-terminus. Cytokine-dependent murine Ba/F3 and human TF-1 cells were retrovirally transduced with DCCp190ER as well as wild-type p190 (wt), DCCp190 and vector control, respectively. Such a series of transformants were subjected to not only biological assays but also biochemical analysis such as anti- phosphotyrosine immunoblot and NF-kB assay.DCCp190ER, but not DCCp190, can support cytokine-independent growth of both Ba/F3 and TF-1 cells in a dose-dependent manner on 4-HT, a synthetic ER-ligand, although excess of 4-HT is rather toxic to these cell lines. Optimal concentration of 4-HT has been determined as 0.5mM. The growth stimulatory effect of 4-HT on DCCp190ER -transduced cells was efficiently canceled by imatinib. Expectedly, upon 4-HT binding, a number of phosphotyrosil proteins could be detected using 4G10 MAb. Protein tyrosine phosphorylation was induced within 10 min after ligand stimulation and a part of proteins revealed a biphasic phosphorylation pattern during the time course. The overall protein profiles of DCCp190ER-induced tyrosine phosphorylation including CrkL and Stat5 resembled p190wt- induced those, but a significant time-interval beyond a day was required for the former to be comparable to the latter in signal intensity (Figure 1). Curiously this holds true for autophosphorylated DCCp190ER. [Display omitted] It is evidenced that Bcr-Abl constitutively activates NF-kB signaling pathway via unknown mechanism. TF-1/DCCp190ER cells were lentivirally transduced with NF-kB/luciferase (kB/Luc) reporter construct and were subjected to monitoring Luc activity upon ligand stimulation. Unstimulated TF-1/DCCp190ER-kB/Luc cells revealed a weak but significant Luc activity over the background. GM-CSF, but not 4-HT, moderately enhanced their Luc activities after 24 hrs of stimulation. On the contrary, 4-fold up-regulation of Luc activity was observed after 6 days culture with 4-HT (Figure 2), suggesting the delayed cellular transformation by DCCp190ER, which is compatible with delayed enhancement of tyrosine phosphorylation. Finally, we investigated gene expression profiling of TF-1 cells induced by DCCp190ER upon 4-HT binding. As a result, expression of a number of genes including TRAIL, GATA2 and endoglin were altered in response to Bcr-Abl activation by treatment with 4-HT for 4 days. The present results suggested that Bcr-Abl kinase may evoke early transforming signals in a unique time-dependent manner, leading to established transformed phenotype. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

A Novel Protein Kinase A-independent, β-Arrestin-1-dependent Signaling Pathway for p38 Mitogen-activated Protein Kinase Activation by β2-Adrenergic Receptors

A growing body of evidence has demonstrated that p38 mitogen-activated protein kinase (MAPK) has a crucial role in various physiological and pathological processes mediated by beta(2)-adrenergic receptors (beta(2)-ARs). However, the detailed mechanism of beta(2)-ARs-induced p38 MAPK activation has not yet been fully defined. The present study demonstrates a novel kinetic model of p38 MAPK activation induced by beta(2)-ARs in human embryonic kidney 293A cells. The beta(2)-AR agonist isoproterenol induced a time-dependent biphasic phosphorylation of p38 MAPK: the early phase peaked at 10 min, and was followed by a delayed phase that appeared at 90 min and was sustained for 6 h. Interestingly, inhibition of the cAMP/protein kinase A (PKA) pathway failed to affect the early phosphorylation but abolished the delayed activation. By contrast, silencing of beta-arrestin-1 expression by small interfering RNA inhibited the early phase activation of p38 MAPK. Furthermore, the NADPH oxidase complex is a downstream target of beta-arrestin-1, as evidenced by the fact that isoproterenol-induced Rac1 activation was also suppressed by beta-arrestin-1 knockdown. In addition, early phase activation of p38 MAPK was prevented by inactivation of Rac1 and NADPH oxidase by pharmacological inhibitors, overexpression of a dominant negative mutant of Rac1, and p47(phox) knockdown by RNA interference. Of note, we demonstrated that only early activation of p38 MAPK is involved in isoproterenol-induced F-actin rearrangement. Collectively, these data suggest that the classic cAMP/PKA pathway is responsible for the delayed activation, whereas a beta-arrestin-1/Rac1/NADPH oxidase-dependent signaling is a heretofore unrecognized mechanism for beta(2)-AR-mediated early activation of p38 MAPK.

Endothelin-1 Couples βPix to p66Shc: Role of βPix in Cell Proliferation through FOXO3a Phosphorylation and p27kip1Down-Regulation Independently of Akt

The phosphorylation of forkhead transcription factor FOXO3a by Akt is critical regulator of cell proliferation induced by serum. We show that endothelin-1 (ET-1) stimulation of primary human mesangial cells (HMCs) induces betaPix and p66Shc up-regulation, resulting in the formation of the betaPix/p66Shc complex. In transformed HMCs, ET-1 induces a biphasic phosphorylation of p66Shc and FOXO3a. The second phase leads to p27(kip1) down-regulation independently of Akt. Depletion of betaPix blocks the second phase of p66Shc and FOXO3a phosphorylation and prevents p27(kip1) down-regulation induced by ET-1. Depletion of either betaPix or p66Shc inhibits ET-1-induced cell proliferation. The expression of beta(1)Pix induces FOXO3a phosphorylation through activation of Rac1, ERK1/2, and p66Shc. Using either p66Shc- or Akt-depleted cells; we show that beta(1)Pix-induced FOXO3a phosphorylation requires p66Shc but not Akt. beta(1)Pix-induced p27(kip1) down-regulation was blocked by U0126 but not by wortmannin. Endogenous betaPix and FOXO3a are constitutively associated with endogenous p66Shc. FOXO3a and p66Shc binding requires beta(1)Pix homodimerization. Expression of beta(1)Pix homodimerization deficient mutant abrogates beta(1)Pix-induced p27(kip1) down-regulation and cell proliferation. Our results identify p66Shc and FOXO3a as novel partners of beta(1)Pix and represent the first direct evidence of beta(1)Pix in cell proliferation via Erk/p66Shc-dependent and Akt-independent mechanisms.

A Critical Role of Protein Kinase Cδ Activation Loop Phosphorylation in Formyl-Methionyl-Leucyl-Phenylalanine-Induced Phosphorylation of p47phox and Rapid Activation of Nicotinamide Adenine Dinucleotide Phosphate Oxidase

Generation of superoxide by professional phagocytes is an important mechanism of host defense against bacterial infection. Several protein kinase C (PKC) isoforms have been found to phosphorylate p47(phox), resulting in its membrane translocation and activation of the NADPH oxidase. However, the mechanism by which specific PKC isoforms regulate NADPH oxidase activation remains to be elucidated. In this study, we report that PKCdelta phosphorylation in its activation loop is rapidly induced by fMLF and is essential for its ability to catalyze p47(phox) phosphorylation. Using transfected COS-7 cells expressing gp91(phox), p22(phox), p67(phox), and p47(phox) (COS-phox cells), we found that a functionally active PKCdelta is required for p47(phox) phosphorylation and reconstitution of NADPH oxidase. PKCbetaII cannot replace PKCdelta for this function. Characterization of PKCdelta/PKCbetaII chimeras has led to the identification of the catalytic domain of PKCdelta as a target of regulation by fMLF, which induces a biphasic (30 and 180 s) phosphorylation of Thr(505) in the activation loop of mouse PKCdelta. Mutation of Thr(505) to alanine abolishes the ability of PKCdelta to catalyze p47(phox) phosphorylation in vitro and to reconstitute NADPH oxidase in the transfected COS-phox cells. A correlation between fMLF-induced activation loop phosphorylation and superoxide production is also established in the differentiated PLB-985 human myelomonoblastic cells. We conclude that agonist-induced PKCdelta phosphorylation is a novel mechanism for NADPH oxidase activation. The ability to induce PKCdelta phosphorylation may distinguish a full agonist from a partial agonist for superoxide production.

Inhibition of histone deacetylases prevents cytokine-induced toxicity in beta cells

The immune-mediated elimination of pancreatic beta cells in type 1 diabetes involves release of cytotoxic cytokines such as IL-1beta and IFNgamma, which induce beta cell death in vitro by mechanisms that are both dependent and independent of nitric oxide (NO). Nuclear factor kappa B (NFkappaB) is a critical signalling molecule in inflammation and is required for expression of the gene encoding inducible NO synthase (iNOS) and of pro-apoptotic genes. NFkappaB has recently been shown to associate with chromatin-modifying enzymes histone acetyltransferases and histone deacetylases (HDAC), and positive effects of HDAC inhibition have been obtained in several inflammatory diseases. Thus, the aim of this study was to investigate whether HDAC inhibition protects beta cells against cytokine-induced toxicity. The beta cell line, INS-1, or intact rat islets were precultured with HDAC inhibitors suberoylanilide hydroxamic acid or trichostatin A in the absence or presence of IL-1beta and IFNgamma. Effects on insulin secretion and NO formation were measured by ELISA and Griess reagent, respectively. iNOS levels and NFkappaB activity were measured by immunoblotting and by immunoblotting combined with electrophoretic mobility shift assay, respectively. Viability was analysed by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and apoptosis by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and histone-DNA complex ELISA. HDAC inhibition reduced cytokine-mediated decrease in insulin secretion and increase in iNOS levels, NO formation and apoptosis. IL-1beta induced a bi-phasic phosphorylation of inhibitor protein kappa Balpha (IkappaBalpha) with the 2nd peak being sensitive to HDAC inhibition. No effect was seen on IkappaBalpha degradation and NFkappaB DNA binding. HDAC inhibition prevents cytokine-induced beta cell apoptosis and impaired beta cell function associated with a downregulation of NFkappaB transactivating activity.

Sustained activation of Src-family tyrosine kinases by ischemia: A potential mechanism mediating extracellular signal-regulated kinase cascades in hippocampal dentate gyrus

In the present report, we investigated the association between the sustained activation of Src family tyrosine kinases (primarily Src kinase) with the biphasic phosphorylation of extracellular signal-regulated kinase (ERK) induced by ischemia in the rat hippocampal CA3/dentate gyrus subfield. Post-ischemia reperfusion resulted in the phosphorylation of ERK in a Ras-dependent manner; down-regulation of NMDA receptors or Src family protein kinases by ketamine or 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2) potently antagonized the activation of ERK, indicating that NMDA receptors and Src family tyrosine kinases are essential for the up-regulation of ERK activity following ischemic stimuli. Additionally, an ischemia-induced association between RKIP and Raf-1 resulted in the inhibition of the ERK signaling cascade through an inhibition of Src-mediated Raf-1 phosphorylation at Tyr340/341 residues. This ischemia-induced inhibition of ERK was not associated with other downstream pathways involving Raf-1 phosphorylation at Ser 259 elicited by protein kinase B (Akt). Dissociation of Raf-1 from RKIP by 24 h reperfusion or (4S)-3-[(E)-but-2-enoyl]-4-benzyl-2-oxazolidinone (locostatin) influenced the second phase of ERK activation elicited by the Src–Raf cassette. We propose that, following ischemia, the Src family tyrosine kinases are critical for modulation of the Ras/Raf/MEK/ERK cascade, in which RKIP is involved in biphasic phosphorylation of ERK via a blockade of Src–Raf cascades.