Browsing by Subject "Proto-Oncogene Proteins c-akt"
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Item B-Cell Adapter for Phosphoinositide 3-Kinase Is a Signaling Adapter in the Toll-Like Receptor/Interleukin-1 Receptor Superfamily(2014-02-17) Troutman, Ty Dale; van Oers, Nicolai S. C.; Pasare, Chandrashekhar; Hooper, Lora V.; Chen, Zhijian J.; Krämer, HelmutToll-like receptor (TLR)/Interleukin-1 receptor (IL1R) superfamily members share signaling components and (with the exception of TLR3) depend on the adapter myeloid differentiation primary response gene 88 (MyD88) for engagement of downstream pathways. Signals from the receptor to the adapter are transmitted through homotypic interaction of TIR (Toll-Interleukin-1 receptor) homology domains found in all TLR/IL1R family members and their adapters. The present work defines a novel TLR/IL1R signaling adapter, termed BCAP (B-cell adapter for PI3K), which was identified based on the presence of a cryptic N-terminal TIR domain. I show here that BCAP (B-cell adapter for PI3K) contains a functional TIR domain enabling its participation in the TLR signaling pathway. Through its TIR domain, BCAP associates with the TLR/IL1R signaling adapter MyD88, as well as the TLR signaling adapter toll-interleukin 1 receptor domain containing adapter protein (TIRAP). Importantly, BCAP plays an obligate role in linking TLRs to activation of phosphoinositide 3-kinase (PI3K) through recruitment of PI3K to the signaling complex and relief of inhibitory influences on PI3K activity. Importantly, BCAP selectively mediates TLR signaling towards the PI3K branch without affecting signaling to NFκB nor MAP kinases. In this capacity, BCAP inhibits secretion of inflammatory cytokines and regulates susceptibility to inflammatory colitis. Because the TLR/IL1R family shares signaling components, BCAP may also function in IL1R family signaling. To test this hypothesis, T cells were chosen as a model cell type responding to IL1R family signals. T helper cells utilize IL18 and IL1 (which engage the IL18R or the IL1R respectively, both IL1R family members) cytokines provided by myeloid cells to achieve optimal Th1 and Th17 effector capacities. I show here that BCAP intrinsically regulates differentiation of naïve T cells towards Th1 and Th17 effector lineages by participation in the IL1R family signaling pathways. Further, BCAP intrinsically regulates both T cell proliferation and survival during priming. The significance of this work lies in the revelation of a TLR signaling adapter serving as a node connecting TLRs to PI3K. Further, the findings here will increase the understanding of key signaling pathways involved in disease and inflammation.Item Context-Selective Support of the AKT/mTOR Regulatory Axis by Tank-Binding Kinase 1 (TBK1)(2016-11-28) Cooper, Jonathan Mark; Brugarolas, James B.; Cobb, Melanie H.; Brekken, Rolf A.; White, Michael A.Oncogenic mutation of Ras or Ras effector signaling characterizes roughly thirty percent of all cancers. Persistent obstacles to the treatment of these diseases by direct Ras inhibition prompt alternative strategies aimed at leveraging signaling networks downstream of Ras. Tank-Binding Kinase 1 (TBK1) is downstream of the RalGEF/RalB arm of Ras effector signaling and supports Ras-driven oncogenic transformation via direct regulation of AKT. While TBK1 has been nominated as a therapeutic target, the field lacks knowledge of the mechanisms whereby TBK1 inhibitors mediate lethality and of the preferential context(s) for their application. We therefore leveraged toxicity profiles for TBK1 inhibitors in 100 NSCLC cell lines and identified robust correlation between TBK1 inhibitors and a cadre of mTOR direct and upstream regulatory network signaling inhibitors. This observation, along with orthogonal phosphoproteomics data, suggested an intersection exists between TBK1 and mTOR regulation and mechanistic target space. We identified that TBK1 is required for AKT/mTOR activation during the nutrient starved-to-fed state transition. Furthermore, we established that TBK1 physically intersects with the AKT/mTOR regulatory axis signaling at multiple nodes and can follow permissive and instructive mechanistic routes to regulate mTORC1 activation in response to nutrients. In parallel, we utilized a bioinformatics approach to identify that "Ras-mutant/mesenchymal" status serves as a molecular indicator of TBK1 inhibitor sensitivity in NSCLC. Concordantly, signaling through the AKT/mTOR regulatory axis was acutely attenuated by TBK1 inhibition in Ras-mutant/mesenchymal but remained unresponsive in Ras-mutant/epithelial NSCLC, indicating TBK1-resistant NSCLC may have uncoupled AKT/mTOR signaling from substantive TBK1 regulation. We furthermore demonstrated that TBK1 inhibition synergizes with Transforming Growth Factor-beta (TGF-beta)-mediated induction of the epithelial-to-mesenchymal transition (EMT) to reduce cancer cell viability. Together, these observations suggest that TBK1 supports pro-survival signaling downstream of Ras and EMT/TGF-beta signaling through the AKT/mTOR regulatory axis. Our findings, therefore, reveal novel mechanistic contributions of TBK1 in the regulation of AKT/mTOR signaling, and also nominate Ras-mutant/mesenchymal NSCLC as the preferential context for therapeutic interventions targeting TBK1.Item Evaluation of Chronic RalGTPase Activation as a Core Specifier of Oncogenic Transformation(2009-01-08) Cheng, Tzuling; White, Michael A.Ral (RAS-Like) GTPases, RalA and RalB, were originally identified based on sequence similarity to Ras and are directly activated via the Ras effector family Ral guanine nucleotide exchange factors (RalGEFs). Previous studies have demonstrated that RalA and RalB collaborate to maintain tumorigenicity through regulating both proliferation and survival. Remarkably, RalB is specifically required for survival in Ras-dependent tumor cells rather than normal cells, while RalA is required for anchorage-independent proliferation but dispensable for survival. However, the spectrum of cancer cell lineages dependent upon Ral functions for tumor formation is currently unknown. We examined whether Ral pathway activation is required for proliferation of cancer cells with activated Ras, Raf, or PI3K. Our data indicate that the Ral pathway is aberrantly activated and required for maintaining tumorigenicity of cancers that are driven by oncogenes other than Ras. In order to begin to understand how the Ral pathway may be chronically engaged in diverse oncogenic backgrounds, we further examined the expression of RalGEFs in a variety of cells derived from different tissue origin. Our results showed a divergent and complex distribution of RalGEFs among different cell types. In addition, through examination of historical tumor resequenceing efforts, we found several somatic mutations in RalGEFs, including RalGDS and RGL1. Through biochemical and cell biological studies, we find that the RGL1 mutations identified in human breast cancers are gain-of -function mutations, and found the mutations contribute to tumor cell survival through RalB pathway. Furthermore, we showed that chronic activation of RGL1 is sufficient to transform immortalized human mammary epithelial cells. Together, our data suggest RGL1 is a bona fide oncogene. These studies broaden our knowledge about RalGEF-Ral contributions in tumorigenicity, and provide a potential target for cancer therapeutic interventions.Item Molecular Mechanisms Underlying Innate Immune Kinase TBK1-Driven Oncogenic Transformation(2013-04-16) Ou, Yi-Hung 1977-; Lum, Lawrence; White, Michael A.; Cobb, Melanie H.; Minna, John D.An essential kinase in innate immune signaling, TBK1 couples pathogen surveillance to induction of host defense mechanisms. The pathological activation of TBK1 in cancer can overcome programmed cell death cues, enabling cells to survive oncogenic stress. The mechanistic basis of TBK1 prosurvival signaling, however, has been enigmatic. Here we show that TBK1 directly activates AKT by phosphorylation of the canonical activation loop and hydrophobic motif sites independently of PDK1 and mTORC2. A population of AKT is bound to components of the exocyst complex. Upon mitogen stimulation, triggering of the innate immune response, re-exposure to glucose, or oncogene activation, TBK1 is recruited to the exocyst, where it activates AKT. In cells lacking TBK1, insulin activates AKT normally, but AKT activation by these exocyst-dependent mechanisms is impaired. Discovery and characterization of a 6-aminopyrazolopyrimidine derivative, as a selective low nanomolar TBK1 inhibitor, indicates this regulatory arm can be pharmacologically perturbed independently of canonical PI3K/PDK1 signaling. Thus, AKT is a direct TBK1 substrate that connects TBK1 to prosurvival signaling. Additionally, biochemical and cell biological evidence indicates critical roles of TBK1 and its analog IKKε in the amino acid-dependent activation of mTORC1. TBK1 and IKKε are activated by amino acids and both proteins interact with mTORC1. In TBK1 and/or IKKε-deficient cells, mTORC1 activation by amino acids is impaired. Of note, we also discovered a set of TBK1 substrates and interacting proteins participating in amino acid-dependent mTORC1 signaling. In conclusion, our results suggest that TBK1 not only supports physiological and oncogenic activation of AKT, but also plays a central role in the regulation of mTORC1 activation in response to amino acids. In addition, our studies reveal novel mTORC1 components and provide new insights into the regulation of the mTORC1 signaling network.Item Molecular Regulation of Direct Cardiac Reprogramming(2017-08-14) Zhou, Huanyu; Zhang, Chun-Li; Hill, Joseph A.; Cleaver, Ondine; Olson, Eric N.A heart attack (also known as myocardial infarction, MI) happens when the flow of blood to the heart is blocked. A massive heart attack can kill billions of cardiomyocytes. The heart has limited regenerative potential because adult mammalian cardiomyocytes cannot proliferate, therefore lost cardiomyocytes cannot be replaced. This causes permanent heart damage and results in decreased contraction properties to a large portion of the heart muscle. Therapeutic treatments for heart attack patients have improved dramatically over the past decades. However, due to the inability to replenish lost cardiomyocytes, heart failure is still the primary cause of death in the world. Cardiac fibroblasts (CFs) constitute ~50% of the cells in the heart and form scar tissue following heart injury. Reprogramming CFs to induced-cardiomyocytes (iCMs) by forced expression of cardiac specific transcription factors holds promise for enhancing cardiac repair by reducing scar tissue while simultaneously generating new cardiomyocytes. However, low efficiency as well as a lack of understanding of molecular mechanism of the reprogramming process have significantly hampered its clinical application. The two goals of my PhD study were 1) to optimize the cardiac reprogramming protocol by increasing the efficiency; and 2) to decipher molecular mechanisms of cardiac reprogramming using the information obtained from the optimization process. To improve the efficiency of reprogramming fibroblasts to iCMs by cardiac transcription factors [Gata4, Hand2, Mef2c, and Tbx5 (GHMT)], we screened 192 protein kinases and discovered that Akt/protein kinase B dramatically accelerates and amplifies this process in three different types of fibroblasts (mouse embryo, adult cardiac, and tail tip). Approximately 50% of the reprogrammed mouse embryo fibroblasts displayed spontaneous beating after 3 weeks of induction by AKT1 plus GHMT (AGHMT). Furthermore, AGHMT evoked a more mature cardiac phenotype for iCMs, as seen by enhanced polynucleation, cellular hypertrophy, gene expression, and metabolic reprogramming. Insulin-like growth factor 1 (IGF1) and phosphoinositol 3-kinase (PI3K) acted upstream of AKT1 whereas the mitochondrial target of rapamycin complex 1 (mTORC1) and forkhead box o3a (Foxo3a) acted downstream of AKT1 to influence fibroblast-to-cardiomyocyte reprogramming. Addition of AGHMT converted 50% of mouse embryo fibroblasts to beating cardiomyocytes. However, only 1% of adult fibroblasts displayed spontaneous beating after three weeks of induction by AGHMT. This indicates that there are "barriers" in adult fibroblasts that hinder cardiac reprogramming. We continued to optimize methods for reprogramming fibroblasts to cardiomyocytes in vitro and in vivo. To identify additional regulators of this reprogramming process, we carried out an unbiased screen of ~1,100 open reading frames (ORFs) encoding transcription factors and cytokines for the ability to enhance reprogramming by AGHMT in adult tail-tip fibroblasts (ATTFs). One of the strongest activators of cardiac reprogramming was Krüppel-Type Zinc-Finger Transcription Factor 281 (ZNF281). Adding ZNF281 in AGHMT converted ~30% of ATTFs to iCMs which is comparable to AGHMT reprogrammed MEFs. We showed that ZNF281 enhanced cardiac reprogramming by associating with GATA4 on cardiac enhancers and by inhibiting inflammatory signaling, which antagonizes cardiac reprogramming. Our findings not only identify AKT1 and ZNF281 as robust and efficient activators of adult cardiac reprogramming, but also provide new insights into the molecular mechanisms underlying direct cardiac reprogramming.Item Proteomic Discovery of Functionally Important Pathways in Myocardial Ischemia-Reperfusion Injury(2014-02-04) Ahmed, Kamran; Luo, Yang; Taneja, Shikha; Keshishian, Hasmik; Carr, Steven; Rosenzweig, AnthonyBACKGROUND: Coronary heart disease, a source of myocardial ischemia-reperfusion injury (IRI), is the world's leading cause of death and disability. Insulin-like growth factor 1 (IGF1) transgenic (Tg) mouse hearts are protected from IRI, whereas Akt-Tg mouse hearts recover poorly from IRI. Surprisingly, Akt is a downstream component of IGF1 signaling. The Akt-Tg phenotype can be rescued by cardiac gene transfer of activated PI3-kinase (PI3K), another component of the IGF1 pathway, suggesting that PI3K-dependent but Akt-independent pathways are key determinants of IRI. To discern such pathways, we analyzed the proteomic and phosphoproteomic changes in wild-type (WT), IGF1-Tg, and Akt-Tg mouse hearts, identified 20 differentially regulated candidates as potential modifiers of IRI, and began testing their functional roles in an in vitro model. We hypothesize that the cardioprotection observed in IGF1 overexpression is a result of PI3K-dependent but Akt-independent signaling pathways. METHODS: WT hearts were collected at 4 time points of ex-vivo Langendorff IRI and analyzed with liquid chromatography-tandem mass spectrometry to determine protein expression and phosphorylation changes. IGF1-Tg and Akt-Tg hearts were analyzed at baseline. Protein network analysis was performed using Cytoscape software. The functional effects of candidates with expression or phosphorylation differences ≥2-fold were assessed in rat neonatal ventricular myocytes using in vitro redox-based viability assays and cell proliferation studies. RESULTS: In the WT IRI studies, 6403 proteins and 22833 phosphopeptides were quantified. During IRI, no proteins changed in expression, 45 phosphopeptides were upregulated, and 975 phosphopeptides were downregulated. In the IGF1-Tg and Akt-Tg hearts, 6700 proteins and 23000 phosphopeptides were quantified. In vitro knockdown of rho-associated protein kinase 2 (ROCK2) increased the viability signal by 17% in normoxia and 33% in simulated IRI (p<0.05) and increased EdU incorporation from 28.9% to 40.15% (p<0.00001). Network analysis of Akt-Tg hearts revealed significant downregulation of 23 out of 45 subunits of Complex I (p<0.05). CONCLUSIONS: Dephosphorylation of the cardiac phosphoproteome is the dominant pattern in IRI, which may reflect phosphatase activation or reduced ATP levels inhibiting kinase activity. ROCK2 knockdown increased the viability signal by stimulating proliferation in vitro. Whether ROCK2 is involved in cardiomyogenesis in the adult heart will be addressed in future studies. Akt-Tg hearts may be susceptible to IRI due to a reduced ATP reserve caused by Complex I downregulationItem Proteomic Discovery of Functionally Important Pathways in Myocardial Ischemia-Reperfusion Injury(2016-04-01) Ahmed, Kamran; Hill, Joseph A.; Rosenzweig, Anthony; Munshi, Nikhil; Sadek, Hesham A.BACKGROUND: Coronary heart disease, a source of myocardial ischemia-reperfusion injury (IRI), is the world's leading cause of death and disability. Insulin-like growth factor 1 (IGF1) transgenic (Tg) mouse hearts are protected from IRI, whereas Akt-Tg mouse hearts recover poorly from IRI. Surprisingly, Akt is a downstream component of IGF1 signaling. The Akt-Tg phenotype can be rescued by cardiac gene transfer of activated PI3-kinase (PI3K), another component of the IGF1 pathway, suggesting that PI3K-dependent but Akt-independent pathways are key determinants of IRI. To discern such pathways, we analyzed the proteomic and phosphoproteomic changes in wild-type (WT) mouse hearts subjected to IRI ex vivo, and IGF1-TG and Akt-Tg mouse hearts in order to identify 20 differentially regulated candidates as potential modifiers of IRI, and began testing their functional roles in an in vitro model. OBJECTIVE: We hypothesize that the cardioprotection observed in IGF1 overexpression is a result of PI3K-dependent but Akt-independent signaling pathways. METHODS: WT hearts were collected at 4 time points of ex-vivo Langendorff IRI and analyzed with liquid chromatography-tandem mass spectrometry to determine protein abundance and phosphorylation changes. IGF1-Tg and Akt-Tg hearts were analyzed at baseline. Protein network analysis was performed using Cytoscape software. The functional effects of candidates with abundance or phosphorylation differences ≥2-fold were assessed in rat neonatal ventricular myocytes using in vitro redox-based viability assays and cell proliferation studies. RESULTS: In the WT IRI studies, 6403 proteins and 22833 phosphopeptides were quantified. During IRI, no proteins changed in abundance, 10 phosphopeptides were upregulated, and 330 phosphopeptides were downregulated. In the IGF1-Tg and Akt-Tg hearts, 6700 proteins and 23000 phosphopeptides were quantified. Out of the significantly regulated proteins, in vitro knockdown of rho-associated protein kinase 2 (ROCK2) increased the viability signal by 17% in normoxia and 33% in simulated IRI (p<0.05) and increased EdU incorporation from 28.9% to 40.15% (p<0.00001). Network analysis of Akt-Tg hearts revealed significant downregulation of 24 out of 45 subunits of Complex I of the electron transport chain (p<0.05). CONCLUSION: Dephosphorylation of the cardiac phosphoproteome is the dominant pattern in IRI, which may reflect phosphatase activation or reduced ATP levels inhibiting kinase activity. ROCK2 knockdown increased the viability signal by stimulating proliferation in vitro. Whether ROCK2 is involved in cardiomyogenesis in the adult heart will be addressed in future studies. Akt-Tg hearts may be susceptible to IRI due to a reduced ATP reserve caused by Complex I downregulation.