Shc Phosphorylation Research Articles

Protein Kinases in Obesity, and the Kinase-Targeted Therapy.

The action of protein kinases and protein phosphatases is essential for multiple physiological responses. Each protein kinase displays its own unique substrate specificity and a regulatory mechanism that may be modulated by association with other proteins. Protein kinases are classified as dual-specificity kinases and dual-specificity phosphatases. Dual-specificity phosphatases are important signal transduction enzymes that regulate various cellular processes in coordination with protein kinases and play an important role in obesity. Impairment of insulin signaling in obesity is largely mediated by the activation of the inhibitor of kappa B-kinase beta and the c-Jun N-terminal kinase (JNK). Oxidative stress and endoplasmic reticulum (ER) stress activate the JNK pathway which suppresses insulin biosynthesis. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) are important for proper regulation of glucose metabolism in mammals at both the hormonal and cellular levels. Additionally, obesity-activated calcium/calmodulin dependent-protein kinase II/p38 suppresses insulin-induced protein kinase B phosphorylation by activating the ER stress effector, activating transcription factor-4. To alleviate lipotoxicity and insulin resistance, promising targets are pharmacologically inhibited. Nifedipine, calcium channel blocker, stimulates lipogenesis and adipogenesis by downregulating AMPK and upregulating mTOR, which thereby enhances lipid storage. Contrary to the nifedipine, metformin activates AMPK, increases fatty acid oxidation, suppresses fatty acid synthesis and deposition, and thus alleviates lipotoxicity. Obese adults with vascular endothelial dysfunction have greater endothelial cells activation of unfolded protein response stress sensors, RNA-dependent protein kinase-like ER eukaryotic initiation factor-2 alpha kinase (PERK), and activating transcription factor-6. The transcriptional regulation of adipogenesis in obesity is influenced by AGC (protein kinase A (PKA), PKG, PKC) family signaling kinases. Obesity may induce systemic oxidative stress and increase reactive oxygen species in adipocytes. An increase in intracellular oxidative stress can promote PKC-β activation. Activated PKC-β induces growth factor adapter Shc phosphorylation. Shc-generated peroxides reduce mitochondrial oxygen consumption and enhance triglyceride accumulation and lipotoxicity. Liraglutide attenuates mitochondrial dysfunction and reactive oxygen species generation. Co-treatment of antiobesity and antidiabetic herbal compound, berberine with antipsychotic drug olanzapine decreases the accumulation of triglyceride. While low-dose rapamycin, metformin, amlexanox, thiazolidinediones, and saroglitazar protect against insulin resistance, glucagon-like peptide-1 analog liraglutide inhibits palmitate-induced inflammation by suppressing mTOR complex 1 (mTORC1) activity and protects against lipotoxicity.

Abstract 3419: TY-1091, a highly selective and potent second-generation RET inhibitor, demonstrates superior antitumor activity in multiple RET-mutant models

Abstract Proto-oncoprotein RET (rearranged during transfection) is a receptor tyrosine kinase and belongs to the cadherin superfamily. RET fusion proteins and gatekeeper mutations represent strong cancer drivers involved in the development of a variety of cancers. Although selective RET inhibitors selpercatinib (LOXO-292) and pralsetinib (BLU667) were recently marketed for patients with RET-dependent NSCLC and thyroid cancers, RET solvent front mutations G810R/S/C/V and other secondary mutations such as Y806C/N and V728A have been identified as mechanisms counting for acquired resistance to the two drugs and limits the applications of the kinase inhibitors. To provide novel RET inhibitors effective to a broader spectrum of RET fusions and mutations, we identified TY-1091 as a novel next-generation RET inhibitor through the in-house RET program and systemic screening of RET compound candidates. TY-1091 was characterized for its anti-tumor activity through in vitro and in vivo testing of a variety of RET-dependent tumor models including a panel of 17 engineered RET mutant Ba/F3 cell lines and 2 cancer cell models. The results show that TY-1091 inhibits wild type and major RET mutants (IC50, nM): RET G810S (9.5 nM), RET V804M/L/E (2.8-12.6 nM), and double mutants RET V804M/G810S (23.5 nM) and M918T/G810S (47.0 nM) through a biochemical screening against a panel of 17 engineered RET mutant Ba/F3 cell lines. Importantly, the inhibitory activity of TY-1091 is much higher than that of first-generation RET inhibitor Cabozantinib, and comparable to other second-generation compounds LOXO-292 and BLU-667. Consistent with its mode of action, TY-1091 grants extraordinary inhibition effects to tumor cell proliferation compared to its peer compounds LOXO-292 and BLU-667 (including TT (thyroid cancer, RET C634W) and LC2/ad (NSCLC, CCDC6-RET)), and the inhibition of the RET pathway activation, i.e., inhibition of RET phosphorylation (IC50 < 1 nM), SHC phosphorylation (IC50 = 4.6 nM) and ERK phosphorylation (IC50 = 9.8 nM) in Ba/F3-KIF5B-RET cells further validated the above in vitro phenotype. The therapeutic potential of TY-1091 was then further validated through mouse pharmacology in that TY-1091 demonstrated remarkable anti-tumor efficacy in a variety of xenograft models including Ba/F3 KIF5B-RET (wt), Ba/F3 KIF5B-RET (V804L), TT (thyroid cancer, RET C634W), and LC2/ad (NSCLC, CCDC6-RET). Detailed data will be presented. In summary, TY-1091 is a highly potent, orally available, and safe small molecule inhibitor to pan-RET mutations in cancer, and may attenuate SFMs-mediated resistance to existing RET therapy. The IND clearance from the US FDA was received and Phase I clinical investigations of TY-1091 shall be launched in the US soon. *To Whom Correspondence should be addressed to: Jun Li, Chengshan Niu and Yusheng Wu Citation Format: Chengshan Niu, Maolin Zheng, Huan Wang, Kaige Ji, Meihua Li, Guohui Wang, Rongzhen Ni, Apeng Liang, Aishen Gong, Yazhen Zhang, Hui Su, Mingyu Jiang, Shaoqing Chen, Xiugui Chen, Jun Li, Yusheng Wu. TY-1091, a highly selective and potent second-generation RET inhibitor, demonstrates superior antitumor activity in multiple RET-mutant models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3419.

TrkC-T1, the Non-Catalytic Isoform of TrkC, Governs Neocortical Progenitor Fate Specification by Inhibition of MAP Kinase Signaling.

Neocortical projection neurons are generated by neural progenitor cells (NPCs) within the ventricular and subventricular zone. While early NPCs can give rise to both deep and upper layer neurons, late progenitors are restricted to upper layer neurogenesis. The molecular mechanisms controlling the differentiation potential of early versus late NPCs are unknown. Here, we report a novel function for TrkC-T1, the non-catalytic isoform of the neurotrophin receptor TrkC, that is distinct from TrkC-TK+, the full-length isoform. We provide direct evidence that TrkC-T1 regulates the switch in NPC fate from deep to upper layer neuron production. Elevated levels of TrkC-T1 in early NPCs promote the generation of deep layer neurons. Conversely, downregulation of TrkC-T1 in these cells promotes upper layer neuron fate. Furthermore, we show that TrkC-T1 exerts this control by interaction with the signaling adaptor protein ShcA. TrkC-T1 prevents the phosphorylation of Shc and the downstream activation of the MAP kinase (Erk1/2) pathway. In vivo manipulation of the activity of ShcA or Erk1/2, directly affects cortical neuron cell fate. We thus show that the generation of upper layer neurons by late progenitors is dependent on the downregulation of TrkC-T1 in late progenitor cells and the resulting activation of the ShcA/Erk1/2 pathway.

Tetrandrine Protects Hearts Against Ischemia‐Reperfusion Injury Via The Inhibition of p66Shc Phosphorylation And Mitochondrial Translocation

Introduction Increased Mitochondrial (Mito) reactive oxygen species (ROS) generation is a major cause of cardiac ischemia/reperfusion (I/R) injury. p66 Shc is an adaptor protein that regulates multiple pathways governing Mito redox state and ROS generation. Tetrandrine (TET), a bisbenzylisoquinoline alkaloid that is extracted from the root of Stephania tetrandra S. Moore, has been used to treat cardiovascular diseases and hypertension. Here we investigate whether Tet exerts cardioprotection in I/R injury and its underlying mechanism mechanisms. Here we studied the effects of TET on phosphorylation and Mito translocation of p66 Shc, Mito oxidative stress/damage, cardiac function and infarct size in mouse heart model of I/R injury. Methods: Hearts (n = 14 each) isolated from C57BL/6 mice were perfused in the Langendorf mode. After 30-min equilibration, the heart was treated with TET or vehicle for 10 min, then subjected to global, no-flow normothermic ischemia for 30 min followed by reperfusion with the corresponding treatment for 120 min. Cardiac function and myocardial energy metabolism were measured with 31 P-NMR spectroscopy continuously and simultaneously. Effluent was collected to detect the release of lactate dehydrogenase. At the end of the reperfusion, half of the hearts were stained with 2,3,5-triphenyltetrazolium chloride to evaluate infarct size, whereas the other half were freeze-clamped and whole protein lysate, cytosolic and Mito fractions were prepared from the clamped tissues to assess the phosphorylation at ser36 and Mito translocation of p66 SHC. Mito H2O2, malondialdehyde (MDA), and protein carbonyls were measured to assess ROS generation and oxidative damage to lipids and proteins, respectively. Results 3 μM TET significantly reduced the heart infarct size and improved cardiac functional recovery after I/R injury. Consistently, TET reversed I/R-induced increases in H2O2, MDA, and protein carbonyls. Interestingly, 3 μM TET significantly decreased the I/R-induced phosphorylation of p66 Shc at serine 36 and its translocation to mitochondria. Conclusions: Our data indicate that TET protects the heart against myocardial I/R injury by preventing Mito oxidative damage via mechanisms that are likely to suppressing p66 Shc phosphorylation and Mito accumulation. Targeting p66 Shc phosphorylation and translocation may serve as new therapeutic strategies for I/R injury.

Insulin signaling in health and disease.

The molecular mechanisms of cellular insulin action have been the focus of much investigation since the discovery of the hormone 100 years ago. Insulin action is impaired in metabolic syndrome, a condition known as insulin resistance. The actions of the hormone are initiated by binding to its receptor on the surface of target cells. The receptor is an α2β2 heterodimer that binds to insulin with high affinity, resulting in the activation of its tyrosine kinase activity. Once activated, the receptor can phosphorylate a number of intracellular substrates that initiate discrete signaling pathways. The tyrosine phosphorylation of some substrates activates phosphatidylinositol-3-kinase (PI3K), which produces polyphosphoinositides that interact with protein kinases, leading to activation of the kinase Akt. Phosphorylation of Shc leads to activation of the Ras/MAP kinase pathway. Phosphorylation of SH2B2 and of Cbl initiates activation of G proteins such as TC10. Activation of Akt and other protein kinases produces phosphorylation of a variety of substrates, including transcription factors, GTPase-activating proteins, and other kinases that control key metabolic events. Among the cellular processes controlled by insulin are vesicle trafficking, activities of metabolic enzymes, transcriptional factors, and degradation of insulin itself. Together these complex processes are coordinated to ensure glucose homeostasis.

TEL-JAK2 constitutively activates the extracellular signal–regulated kinase (ERK), stress-activated protein/Jun kinase (SAPK/JNK), and p38 signaling pathways

The ets transcription factor, TEL, undergoes chromosomal rearrangements with the tyrosine kinase JAK2. TEL-JAK2 is constitutively active, confers cell line factor independence, and activates signal transducer and activator of transcription-1 (STAT1), STAT3, and STAT5. Data from bone marrow transplantation models suggest that STAT5 activation does not account for the entire disease phenotype induced by TEL-JAK2. This study examined additional signaling pathways that are activated by TEL-JAK2. TEL-JAK2 expression in Ba/F3 cells results in constitutive association and tyrosine phosphorylation of Shc and Ship-1 and, consequently, recruitment of Grb2 to TEL-JAK2. Direct Grb2 recruitment is also possible because a putative Grb2 binding site, Tyr314, is present on TEL-JAK2(5-19) and TEL-JAK2(5-12). Studies with a TEL-JAK2(5-19)Tyr314Phe mutant support a role for Tyr314 in Grb2 recruitment, because Grb2 association with TEL-JAK2(5-19)Tyr314Phe is significantly reduced. Interestingly, TEL-JAK2(5-19)Tyr314Phe shows reduced Ras activation when compared with TEL-JAK2(4-17), TEL-JAK2(5-12), and TEL-JAK2(5-19). Analysis of extracellular signal-regulated kinase-1/2 (ERK1/2), stress-activated protein/Jun kinase (SAPK/JNK), and p38 demonstrates the activation of SAPK/JNK and phosphorylation of p38 by all TEL-JAK2 isoforms. TEL-JAK2(5-12) and TEL-JAK2(5-19) preferentially phosphorylate ERK2, whereas TEL-JAK2(4-17) phosphorylated ERK2 at lower levels. Inhibition studies demonstrated that ERK1/2 activation was necessary for Ba/F3 factor independence mediated by TEL-JAK2(5-19), while inhibition of SAPK/JNK or p38 activity had no effect. Our data reveal the requirement of ERK activation by TEL-JAK2(5-19) in Ba/F3 cells and suggest that TEL-JAK2 leukemogenic potential may be mediated in part through ERK1/2.

1‑Palmitoyl‑2‑linoleoyl‑3‑acetyl‑rac‑glycerol ameliorates EGF‑induced MMP‑9 expression by promoting receptor desensitization in MDA‑MB‑231 cells.

Activated epidermal growth factor receptors (EGFRs) are crucial for inducing metastasis in cancer cells by promoting matrix metalloproteinase (MMP) expression. The present study was designed to investigate the effects of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) on MMP expression in epidermal growth factor (EGF)-stimulated breast cancer cells in vitro. EGF stimulation induced internalization of its cognate receptor, EGFR, for stimulus-desensitization. These internalized receptors, complexed with the ubiquitin ligase c-Cbl and EGFR pathway substrate 15 (EPS15) (for degradation), were evaluated by confocal microscopy at 5–90 min time intervals. During intracellular trafficking of EGFRs, EGF-induced signaling cascades were analyzed by examining EGFR and SHC phosphorylation. Modulation of MMP expression was assessed by evaluating the activity of transcription factor AP-1 using a luciferase assay. PLAG accelerated the assembly of EGFRs with c-Cbl and EPS15 and promoted receptor degradation. This faster intracellular EGFR degradation reduced AP-1-mediated MMP expression. PLAG stimulation upregulated thioredoxin-interacting protein (TXNIP) expression, and this mediated the accelerated receptor internalization. This PLAG-induced increase in EGFR trafficking was blocked in TXNIP-silenced cells. By downregulating MMP expression, PLAG effectively attenuated EGF-induced mobility and invasiveness in these cancer cells. These data suggest that PLAG may be a potential therapeutic agent for blocking metastasis.

Abstract 1794: The FES-related tyrosine kinase associates with and activates the insulin-like growth factor 1 receptor at sites of cell adhesion

Abstract Insulin-like Growth Factor-1 signaling has a well-described function in facilitating tumourigenesis and promoting tumour growth. Attempts to suppress IGF signals at the level of the IGF-1 Receptor have been disappointing, with kinase inhibitors and blocking antibodies generally showing poor efficacy in clinical trials. To address the mechanisms for this lack of efficacy we sought to identify proteins that modulate the cytotoxic response to IGF-1R tyrosine kinase inhibition using a functional siRNA screen. We identified the non-receptor tyrosine FES-related (FER) kinase as a mediator of sensitivity to the IGF-1R tyrosine kinase inhibitor in MCF-7 cells. We found that FER and the IGF-1R co-locate and can be co-immunoprecipitated from different cell types. Ectopic expression of FER strongly enhanced IGF-1R expression and phosphorylation on the atuophosphorylation sites at tyrosines 950 and 1131. FER phosphorylated these sites in an IGF-1R kinase-independent manner and also enhanced IGF-1-mediated phosphorylation of SHC, and activation of either the AKT or MAPK signaling pathways in breast cancer cell lines. The IGF-1R, β1 Integrin, FER and its substrate cortactin were all observed to be co-located in cell adhesion complexes, the disruption of which reduced IGF-1R expression and activity. High FER expression correlates with phosphorylation of SHC in breast cancer cell lines and with a poor prognosis in patient cohorts. FER and SHC phosphorylation and IGF-1R expression could be suppressed with a known ALK inhibitor that shows high specificity for FER kinase. Overall, we conclude that FER-enhances IGF-1R expression, phosphorylation and signaling to promote cooperative growth and adhesion signaling that may facilitate cancer progression and resistance to IGF-1R inhibition. Citation Format: Joanna Stanicka, Leonie Rieger, Orla T. Cox, Sandra O'Shea, Michael Coleman, Ciara O'Flanagan, Barbara Addario, Nuala McCabe, Richard Kennedy, Rosemary O'Connor. The FES-related tyrosine kinase associates with and activates the insulin-like growth factor 1 receptor at sites of cell adhesion [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 1794.

TrkB-mediated activation of the phosphatidylinositol-3-kinase/Akt cascade reduces the damage inflicted by oxygen-glucose deprivation in area CA3 of the rat hippocampus.

The selective vulnerability of hippocampal area CA1 to ischemia-induced injury is a well-known phenomenon. However, the cellular mechanisms that confer resistance to area CA3 against ischemic damage remain elusive. Here, we show that oxygen-glucose deprivation-reperfusion (OGD-RP), an invitro model that mimic the pathological conditions of the ischemic stroke, increases the phosphorylation level of tropomyosin receptor kinase B (TrkB) in area CA3. Slices preincubated with brain-derived neurotrophic factor (BDNF) or 7,8-dihydroxyflavone (7,8-DHF) exhibited reduced depression of the electrical activity triggered by OGD-RP. Consistently, blockade of TrkB suppressed the resistance of area CA3 to OGD-RP. The protective effect of TrkB activation was limited to area CA3, as OGD-RP caused permanent suppression of CA1 responses. At the cellular level, TrkB activation leads to phosphorylation of the accessory proteins SHC and Gab as well as the serine/threonine kinase Akt, members of the phosphoinositide 3-kinase/Akt (PI-3-K/Akt) pathway, a cascade involved in cell survival. Hence, acute slices pretreated with the Akt antagonist MK2206 in combination with BDNF lost the capability to resist the damage inflicted with OGD-RP. Consistently, with these results, CA3 pyramidal cells exhibited reduced propidium iodide uptake and caspase-3 activity in slices pretreated with BDNF and exposed to OGD-RP. We propose that PI-3-K/Akt downstream activation mediated by TrkB represents an endogenous mechanism responsible for the resistance of area CA3 to ischemic damage.

FES-related tyrosine kinase activates the insulin-like growth factor-1 receptor at sites of cell adhesion

IGF-1 receptor (IGF-1R) and integrin cooperative signaling promotes cancer cell survival, proliferation, and motility, but whether this influences cancer progression and therapy responses is largely unknown. Here we investigated the non-receptor tyrosine adhesion kinase FES-related (FER), following its identification as a potential mediator of sensitivity to IGF-1R kinase inhibition in a functional siRNA screen. We found that FER and the IGF-1R co-locate in cells and can be co-immunoprecipitated. Ectopic FER expression strongly enhanced IGF-1R expression and phosphorylation on tyrosines 950 and 1131. FER phosphorylated these sites in an IGF-1R kinase-independent manner and also enhanced IGF-1-mediated phosphorylation of SHC, and activation of either AKT or MAPK-signaling pathways in different cells. The IGF-1R, β1 Integrin, FER, and its substrate cortactin were all observed to co-locate in cell adhesion complexes, the disruption of which reduced IGF-1R expression and activity. High FER expression correlates with phosphorylation of SHC in breast cancer cell lines and with a poor prognosis in patient cohorts. FER and SHC phosphorylation and IGF-1R expression could be suppressed with a known anaplastic lymphoma kinase inhibitor (AP26113) that shows high specificity for FER kinase. Overall, we conclude that FER enhances IGF-1R expression, phosphorylation, and signaling to promote cooperative growth and adhesion signaling that may facilitate cancer progression.

Differential effects of epidermal growth factor (EGF) receptor ligands on receptor binding, downstream signalling pathways and DNA synthesis in hepatocytes

Hepatocytes are responsive to mitogenic effects of several ligands acting via EGFR. Studying primary cultures of rat hepatocytes, we found that, as compared to EGF, HB-EGF had a markedly higher affinity of the EGFR, while AR and TGFα had lower affinity. HB-EGF was also more potent compared to the other growth factors regarding phosphorylation of EGFR, Shc, ERK1/2 and Akt. All ligands induced phosphorylation of ErbB2, indicating receptor heterodimerization. TGFα, despite having much lower receptor affinity, was about equally potent and efficacious as HB-EGF as a stimulator of DNA synthesis. In contrast, EGF had relatively high affinity but markedly lower efficacy in stimulation of DNA synthesis. The results suggest that amplifying and/or inhibitory mechanisms may modulate the mitogenic responses downstream of the initial signalling steps, and that this may affect the effects of the EGFR ligands differentially.

Abstract A31: Early signaling dynamics of the epidermal growth factor receptor

Abstract Despite extensive study of the EGF receptor (EGFR) signaling network, the immediate posttranslational changes that occur in response to growth factor stimulation remain poorly characterized; as a result, the biological mechanisms underlying signaling initiation remain obscured. To address this deficiency, we have used a mass spectrometry-based approach to measure system-wide phosphorylation changes throughout the network with 10-s resolution in the 80 s after stimulation in response to a range of eight growth factor concentrations. Significant changes were observed on proteins far downstream in the network as early as 10 s after stimulation, indicating a system capable of transmitting information quickly. Meanwhile, canonical members of the EGFR signaling network fall into clusters with distinct activation patterns. Src homology 2 domain containing transforming protein (Shc) and phosphoinositol 3-kinase (PI3K) phosphorylation levels increase rapidly, but equilibrate within 20 s, whereas proteins such as Grb2-associated binder-1 (Gab1) and SH2-containing tyrosine phosphatase (SHP2) show slower, sustained increases. Proximity ligation assays reveal that Shc and Gab1 phosphorylation patterns are representative of separate timescales for physical association with the receptor. Inhibition of phosphatases with vanadate reveals site-specific regulatory mechanisms and also uncovers primed activating components in the network, including Src family kinases, whose inhibition affects only a subset of proteins within the network. The results presented highlight the complexity of signaling initiation and provide a window into exploring mechanistic hypotheses about receptor tyrosine kinase (RTK) biology. Note: This abstract was not presented at the conference. Citation Format: Raven J. Reddy. Early signaling dynamics of the epidermal growth factor receptor. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A31.

Human Protein Kinases and Obesity.

The action of protein kinases and protein phosphatases is essential for multiple physiological responses. Each protein kinase displays its own unique substrate specificity, and a regulatory mechanism that may be modulated by association with other proteins. Protein kinases are classified by the target amino acid in their substrates. Some protein kinases can phosphorylate both serine/threonine, as well as tyrosine residues. This group of kinases has been known as dual specificity kinases. Unlike the dual specificity kinases, a heterogeneous group of protein phosphatases are known as dual-specificity phosphatases. These phosphatases remove phosphate groups from tyrosine and serine/threonine residues on their substrate. Dual-specificity phosphatases are important signal transduction enzymes that regulate various cellular processes in coordination with protein kinases. The protein kinase-phosphoproteins interactions play an important role in obesity . In obesity, the pro- and anti-inflammatory effects of adipokines and cytokines through intracellular signaling pathways mainly involve the nuclear factor kappa B (NF-kappaB) and the c-Jun N-terminal kinase (JNK) systems as well as the inhibitor of kappaB-kinase beta (IKK beta). Impairment of insulin signaling in obesity is largely mediated by the activation of the IKKbeta and the JNK. Furthermore, oxidative stress and endoplasmic reticulum (ER) stress activate the JNK pathway which suppresses insulin biosynthesis. Additionally, obesity-activated calcium/calmodulin dependent-protein kinase II/p38 suppresses insulin-induced protein kinase B phosphorylation by activating the ER stress effector, activating transcription factor-4. Obese adults with vascular endothelial dysfunction have greater endothelial cells activation of unfolded protein response stress sensors, RNA-dependent protein kinase-like ER eukaryotic initiation factor-2alpha kinase (PERK) and activating transcription factor-6. The transcriptional regulation of adipogenesis in obesity is influenced by AGC (protein kinase A (PKA), PKG, PKC) family signaling kinases. Obesity may induce systemic oxidative stress and increase reactive oxygen species in adipocytes. Increase in intracellular oxidative stress can promote PKC-beta activation. Activated PKC-beta induces growth factor adapter Shc phosphorylation. Shc-generated peroxides reduce mitochondrial oxygen consumption and enhances triglyceride accumulation. Obesity is fundamentally caused by cellular energy imbalance and dysregulation. Like adenosine monophosphate (AMP)-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR), N-terminal Per-ARNT-Sim (PAS) kinase are nutrient responsive protein kinases and important for proper regulation of glucose metabolism in mammals at both the hormonal and cellular level. Defective responses of AMPK to leptin may contribute to resistance to leptin action on food intake and energy expenditure in obese states.

Matrine suppresses the growth of human chronic myeloid leukemia K562 cells via inhibiting bcr-abl-mediated MEK-ERK pathway

Objective To investigate the mechanism of matrine in inhibition of proliferation the proliferation of human chronic myeloid leukemia (CML) K562 cells via MEK-ERK signaling pathway. Methods Western blot was used to detect the expression of MEK1, ERK1/2, Shc and SHP2 (the signal effect molecules of MEK-ERK pathway) in K562 cells. The transcription and translation of bcr-abl and target protein (bcl-xL, Cyclin D1, c-myc and p27) were detected by RT-PCR and Western blot. Results Matrine was able to significantly inhibit the phosphorylation of MEK1, ERK1/2, Shc and SHP2 in K562 cells and suppress the protein and mRNA expression of bcr-abl. Moreover, the expressions of bcl-xL, Cyclin D1 and c-myc were down-regulated significantly, while the expression level of p27 (a negative regulator of cell cycle progression) was increased markedly after matrine treatment. Conclusions Suppression of the growth of human CML K562 cells is related to the inhibition of bcr-abl-mediated MEK-ERK pathway activity. The down-regulation of phosphorylated proteins or protein kinases activity in signaling pathways might be an important molecular mechanism in control the activity of MEK-ERK pathway. Key words: Matrine; Leukemia, myeloid, chronic; MEK-ERK signaling pathway; bcr-abl

Tyrosine 599 Plays an Essential Role and Cooperates with Tyrosine 604 in MPL W515L-Indiced Myeloproliferative Neoplasms

The somatic mutation MPL W515L constitutively activates signaling pathways including JAK/STATs, MAPK, and PI3K/AKT, leading to myeloproliferative neoplasm (MPN). How each of these signaling pathways contributes to MPL W515L-induced MPN has not been well defined. Moreover, the requirement for the tyrosine residues 599 and 604, which have been shown to be critical for normal MPL signaling, in MPL W515L-induced disease remains uncharacterized. Thus, we created W515L/Y599F double mutant (abbreviated as MPL 515/599), W515L/Y604F double mutant (MPL 515/604), and the MPL W515L/Y599F/Y604F triple mutant (MPL 515/599/604) and tested their activities in the TPO-dependent G1ME cell line. Interestingly, MPL 515/604 supported TPO-independent proliferation similar to MPL W515L whereas MPL 515/599 exhibited significantly impaired ability to do so. In contrast, the triple mutant completely lost the ability to support TPO-independent proliferation. Consistent with their effect on cell proliferation, MPL W515L and MPL 515/604 significantly increased CFU-Mk in murine bone marrow while MPL 515/599 and the triple mutant did not. Moreover, MPL 515/599 exhibited impaired phosphorylation of Shc, STAT3, STAT5, AKT, and ERK while MPL 515/604 barely had any effect on their phosphorylation. Notably, MPL 515/599/604 further reduced STAT5 phosphorylation although it retained STAT3 phosphorylation as compared to MPL 515/599. Together, these results reveal that Y599 is critical in mediating MPL W515L-induced megakaryocyte hyperproliferation and signaling.To further test which downstream signaling pathway is critical for MPL W515L-induced megakaryocyte hyperproliferation, we restored each signaling in MPL 515/599/604-transduced G1ME cells by overexpressing constitutively active forms of downstream signaling molecules and GFP. Cells overexpressing constitutively active STAT5 or AKT showed an increased GFP percentage over time with TPO depletion. However, constitutively active STAT3 or Ras did not affect cell growth. We verified the requirement for STAT5 in MPL W515L-induced megakaryocyte hyperproliferation by overexpressing MPL W515L in murine bone marrow progenitors from STAT5-null and littermate control mice. Deletion of STAT5 significantly reduced CFU-Mk and impaired CD41 expression. Notably, MPL W515L-induced expansion of CFU-Mk observed in littermate control cells was dramatically impaired in STAT5-null cells. These findings suggest that STAT5 is critical in MPL W515L-induced megakaryocyte hyperproliferation.To further test the in vivo role of Y599 and Y604 in MPL W515L-induced MPN, we performed bone marrow transplantation. Consistent with the in vitro phenotype, MPL W515L and MPL 515/604 caused MPN characterized by hypercellularity of bone marrow, leukocytosis, thrombocytosis, splenomegaly, hepatomegaly, and infiltration of blood cells to liver and lung. Interestingly, the disease burden of MPL 515/604 was more severe, with higher WBC and platelet counts than that seen with MPL W515L. Surprisingly, MPL 515/599 and MPL 515/599/604 caused hypercellularity of bone marrow, although they did not cause any other significant features of MPN. MPL 515/599 also caused splenomegaly without other symptoms. Unexpectedly, all MPL mutant alleles supported expansion of LSK cells in bone marrow. Despite the finding that MPL W515L and MPL 515/604 caused thrombocytosis, they did not increase MEP numbers. In fact, all mutant MPL alleles except the triple mutant increased HPC, CMP, and GMP cells. In addition, mature myeloid and megakaryocyte lineages were expanded, possibly at the expense of erythrocytes. In the spleen, we also observed expansion of megakaryocytes in the double mutant alleles and a huge expansion of myeloid lineage cells, but a decrease of erythrocytes in MPL 515/604. Thus our study has revealed distinct features of Y599 and Y604 in mediating MPL W515L-induced signaling, megakaryocyte hyperproliferation, and MPN. Y599 plays a critical role and cooperates with Y604. Our study also suggests that MPL cytosolic phosphorylated Y599 and flanking sequences could become targets for pharmacologic inhibition in MPNs. DisclosuresNo relevant conflicts of interest to declare.

Abstract P6-04-01: Protein pathway activation mapping of LCM tumor epithelium from I-SPY 1 TRIAL surgical specimens reveals activation of receptor tyrosine kinase drug target signaling networks in the non-responding patient cohort

Abstract Background: Identification of alternative therapeutic strategies is critically needed for patients who do not achieve complete clinical response in the neoadjuvant setting, and in particular, from that cohort with the most aggressive disease (shortest relapse-free survival (RFS)). The I-SPY 1 TRIAL (CALGB 150007/150012, ACRIN 6657) is a trial of neoadjuvant anthracycline- and taxane-based chemotherapy that provides longitudinal biopsy specimens and extensive molecular and clinical/pathological characterization of non-responding patients. Methods: Tumor epithelium was procured by Laser Capture Microdissection (LCM) from frozen surgical specimens of 27 patients who did not achieve complete pathological response in the I-SPY1 TRIAL. Reverse Phase Protein Microarray (RPPA) technology was used to measure the levels of 120 key signaling proteins including drug targets for Phase I-III and FDA-approved therapeutics. Cox proportional hazard univariate and multivariate analyses were used to evaluate RFS correlates. Significance threshold was p = <0.05. Results: Univariate analysis of pathway activation revealed a number of actionable/druggable targets, particularly receptor tyrosine kinase (RTK) activation, where increased phosphorylation of ALK (Y1586), EGFR (Y1173), ERBB3 (Y1289), cABL (T735), and FAK (Y576/Y577) was significantly associated with aggressive disease (shorter RFS). Higher levels of SRC (Y527) inhibitory phosphorylation were also significantly associated with early recurrence. Coordinate activation of downstream targets of these RTKs, such as RAS-GRF S916, SHC (Y317), MEK (S217/221) and the Aurora kinases were also associated with early relapse in this study set. In multivariate analyses controlling for receptor subtype (HR+/HER2-, triple-negative (TN), HER2+), the associations between shorter RFS and RTK pathway activation including EGFR, ERBB3, cABL and FAK activation and SRC inhibition, as well as RAS-GRF and SHC phosphorylation remained significant. Conclusions: This unique functional protein pathway/proteomic analysis of LCM-isolated tumor epithelium by RPPA in surgical specimens taken from patients who did not achieve complete pathological response provides evidence for a significant number of druggable RTK pathway targets in patients with residual disease who are most at risk to die from breast cancer. Activation of ALK, EGFR, ERBB3, ABL, FAK, along with downstream RAS-ERK and Aurora kinase signaling activation in this cohort provide new insights into tumor progression along with distinct, actionable information on new therapeutic modalities to test in patients with progressing disease. Identification of new therapeutic strategies for patients who progress or recur early after neoadjuvant therapy is critical and, if validated in larger independent cohorts, could lead to rationalized personalized therapy-based trials. This strategy is being utilized in the I SPY 2 TRIAL as we introduce pathway-directed therapeutics in the neoadjuvant setting and are employing the RPPA platform to determine whether these agents are impacting their intended pathway. This work was partially supported by NIH grants CA31946 and CA33601. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-01.

The tyrosine kinase CSK associates with FLT3 and c-Kit receptors and regulates downstream signaling

Type III receptor tyrosine kinases (RTKs), FLT3 and c-Kit play important roles in a variety of cellular processes. A number of SH2-domain containing proteins interact with FLT3 and c-Kit and regulate downstream signaling. The SH2-domain containing non-receptor protein tyrosine kinase CSK is mainly studied in the context of regulating Src family kinases. Here we present an additional role of this kinase in RTK signaling. We show that CSK interacts with FLT3 and c-Kit in a phosphorylation dependent manner. This interaction is facilitated through the SH2-domain of CSK. Under basal conditions CSK is mainly localized throughout the cytosolic compartment but upon ligand stimulation it is recruited to the inner side of cell membrane. CSK association did not alter receptor ubiquitination or phosphorylation but disrupted downstream signaling. Selective depletion of CSK using siRNA, or inhibition with CSK inhibitor, led to increased phosphorylation of Akt and Erk, but not p38, upon FLT3 ligand (FL) stimulation. Stem cell factor (SCF)-mediated Akt and Erk activation was also elevated by CSK inhibition. However, siRNA mediated CSK knockdown increased SCF stimulated Akt phosphorylation but decreased Erk phosphorylation. CSK depletion also significantly increased both FL- and SCF-induced SHC, Gab2 and SHP2 phosphorylation. Furthermore, CSK depletion contributed to oncogenic FLT3- and c-Kit-mediated cell proliferation, but not to cell survival. Thus, the results indicate that CSK association with type III RTKs, FLT3 and c-Kit can have differential impact on receptor downstream signaling.

Dysregulation of PSTPIP2 Due to Loss of GATA-1 Contributes to Aberrant Megakaryopoiesis

AbstractAbstract 854GATA-1-deficiency causes dysregulation of multiple genes and leads to aberrant megakaryopoiesis. How the dysregulated genes may contribute to abnormal megakaryocyte development and differentiation is largely unknown. In this study, we focused on Pstpip2, a gene that was upregulated in GATA-1low and GATA-1s mutant megakaryocytes. A strong GATA-1 binding site in the intron 1 of Pstpip2 gene was previously identified by GATA-1 ChIP-Seq studies. These observations strongly suggest that Pstpip2 is a GATA-1 target gene. By using ChIP-PCR we showed that GATA-1 indeed bound to Pstpip2 intron 1 region in megakaryocytes. We also observed that Pstpip2 was expressed in myeloid cells and hematopoietic progenitors, but not lymphocytes. Noticeably, two cell lines with GATA-1 deficiencies, G1ME and CMK, which approximate the MEP and megakaryoblast stages, respectively, showed moderate expression of Pstpip2. Moreover, K562 cells displayed significant upregulation of PSTPIP2 and concomitant downregulation of GATA-1 during TPA-induced megakaryocytic differentiation. To study its function in megakaryocytic differentiation, we modulated PSTPIP2 expression by overexpression or shRNA knockdown. Ectopic expression of PSTPIP2 in K562 or CMK cells impaired TPA-induced megakaryocytic differentiation as evidenced by decreased CD41 expression. In contrast, downregulation of PSTPIP2 by shRNA promoted CD41 expression and increased polyploidy. These findings suggest that failure to suppress PSTPIP2 due to loss of GATA-1 may contribute to abnormal megakaryopoiesis.To probe the mechanism by which PSTPIP2 inhibits megakaryocytic differentiation, we tested whether the interaction with its partner PEST-PTP or phosphorylation of key tyrosine residues in the protein by Src kinase is required. We created two mutants, W232A and Y323/333/F, which block its interaction with PEST-PTP or prevent tyrosine phosphorylation, respectively. Similar to WT PSTPIP2, both mutants retained the ability to inhibit megakaryocytic differentiation suggesting that inhibition of megakaryocytic differentiation does not depend on the PEST-PTP interaction or Src kinase phosphorylation. Separately, we have noted that PSTPIP2 also interacts with Fyn, Grb2, and Shc, suggesting that PSTPIP2 may affect signal transduction. Indeed, we found that ERK phosphorylation was downregulated by PSTPIP2. Since THPO/MPL signaling via JAK/STAT is the physiological pathway in megakaryocyte maturation rather than BCR-ABL signaling seen in K562 cells, we next analyzed the effect of PSTPIP2 on MPL signaling in G1ME cells. Consistent with our findings in K562 and CMK cells, both WT and mutant PSTPIP2 inhibited CD41 expression. Interestingly, ectopic expression of WT PSTPIP2 also caused cell cycle arrest at S phase in G1ME cells. PSTPIP2 also suppressed Erk and Shc phosphorylation. Considering the important role of MAPK/ERK signaling in megakaryocyte development, we believe that PSTPIP2 acts as a negative regulator of THPO-MPL signaling to suppress TPO-stimulated MAPK/ERK activation and subsequently inhibit megakaryopoiesis. Finally, we confirmed the function of PSTPIP2 in megakaryocyte differentiation using mouse bone marrow progenitor cells. Ectopic expression of PSTPIP2 significantly downregulated CD41 expression, reduced CFU-GM and CFU-Mk numbers, and reduced polyploidy, while knockdown of PSTPIP2 exhibited the opposite effects. Taken together, we have identified a new negative regulator of THPO-MPL signaling that is inhibited by GATA-1 in normal megakaryocyte development. Upregulation of PSTPIP2 may contribute to abnormal megakaryopoiesis observed in GATA-1-deficient megakaryocytes. Disclosures:No relevant conflicts of interest to declare.

Requirements of Gαi1 and Gαi3 for BDNF and NGF induced PI3K/AKT and MAPK signaling, neuronal survival and migration

Our previous study has indicated an unexpected and critical role of Gαi proteins in mediating receptor tyrosine kinase signaling (Cao et al., Science Signaling, 2009). Here we discovered a novel function of Gαi proteins in BDNF/NGF signaling. Gαi proteins are required for Brain‐Derived Neurotrophic Factor (BDNF) and Nerve Growth Factor (NGF) to transduce signals from Trk receptor to stimulate MAPK (p38, JNK and ERK) and PI3K/AKT/mTORC1 signaling. Knockout of Gαi1 and Gαi3 completely blocked BDNF and NGF induced PI3K/AKT/mTORC1 and MAPK activation in MEFs (mouse embryonic fibroblasts). Both Gαi1 and Gαi3 associated with the Grb2‐associated binding protein 1 (Gab1), and were required for the interaction of Gab1 with SHP2 as well as the interactions of Grb2 with SHP2, Shc, MEKK1 and MEKK2, in response to BDNF and NGF. Loss of Gαi1 and Gαi3 impaired the phosphorylation of Gab1, SHP2 and Shc in response to BDNF and NGF. Importantly, siRNA knockdown of both Gαi1 and Gαi3 inhibited BDNF and NGF induced AKT/mTORC1 and MAPK activation in cerebellar granule neuron, and BDNF induced neuronal survival, neuronal migration and neurite outgrowth were also largely impaired in cerebellar neurons with Gαi1 and Gαi3 deficiency. Overall, these results showed that Gαi1 and Gαi3 lie downstream of Trk receptor, but upstream of Gab1‐Shc‐Grb2‐Shp2, mediating MAPK and PI3K/AKT/mTORC1 signaling as well neuronal survival and migration.

Characterization of major elements of insulin signaling cascade in chicken adipose tissue: Apparent insulin refractoriness

The role of insulin in chicken adipose tissue appears weak or questionable. In a first study, proximal and distal components of the insulin signaling cascade were characterized in abdominal adipose tissue of fasted or fed chickens for the first time. Similar measurements were performed on epididymal adipose tissue from fasted or fed rats for comparison. Tyrosine phosphorylation of IR beta subunit, IRS-1 and Shc and phosphorylation of downstream components (Akt and MAPK ERK1/2) were significantly reduced as expected by fasting in rat, but not in chicken. Phosphorylation of MAPK P38 was increased by fasting in chicken but not in rat. Phosphorylation of AMPK was not affected in the conditions investigated in either species. Whatever the nutritional state, the protein levels of IR and IRS-1 were lower in chicken than in rat, whereas those of Shc, Akt, AMPK, MAPK ERK2 and MAPK P38 were similar in both species. In fed state, PI3K activity was higher in chicken than in rat. Insulin sensitivity of insulin cascade was further investigated in chicken adipose tissue following in vivo insulin neutralization for 1 or 5h in fed chickens. Insulin privation did not alter early insulin signaling steps (IRβ, IRS-1 and Shc) or downstream elements (Akt, P70S6K, S6 ribosomal protein, AMPK, MAPK ERK2 and MAPK P38). Finally, phosphorylation of the transcription factor Creb was increased by 2-fold by 5h fasting or 5h insulin privation, most likely in response to an increase in plasma glucagon levels. Thus, insulin signaling is markedly different in chicken abdominal adipose tissue from that operating in mammals making chicken an interesting model of insulin resistance or refractoriness.