Abstract 273: Phosphoinositide 3-Kinase γ Regulates Cardio-protective ERK by Kinase Independent Mechanism

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Maradumane L Mohan ◽  
George Jolly ◽  
Rohit Anand ◽  
Sathyamangla V Naga Prasad
Keyword(s):  
Tyrosine Kinases ◽  
Hek293 Cells ◽  
Independent Function ◽  
Signaling Mechanism ◽  
Erk Activation ◽  
Cellular Processes ◽  
Erk Phosphorylation ◽  
Phosphoinositide 3 Kinase ◽  
Sirna Knockdown

Phosphoinositide 3-kinase (PI3K) enzymes are critical in many cellular processes including survival. PI3Kγ, a member of the PI3K family activated by G-protein coupled receptor (GPCR), is known to be a critical player in activation of extracellular regulated kinase (ERK) signal transduction cascade, a cell survival pathway. However, the exact mechanism by which PI3Kγ plays a role in ERK activation is not clearly understood. Our studies show that PI3Kγ plays a crucial role in enhancing the tone of ERK activation as use of PI3K inhibitors reduced GPCR stimulated ERK phosphorylation in HEK293 cells. siRNA knockdown of PI3Kγ resulted in loss of ERK phosphorylation through GPCRs (β-adrenergic) as well as receptor tyrosine kinases. The role of PI3Kγ in ERK activation was further corroborated by loss of insulin stimulated ERK phosphorylation in PI3Kγ-knockout (KO) mouse embryonic fibroblasts (MEFs). Surprisingly, ERK activation in KO MEFs post-insulin stimulation was completely rescued by expression of kinase-dead PI3Kγ mutant in KO MEFs suggesting a kinase-independent role of PI3Kγ in regulating ERK function. Indepth mechanistic studies showed that PI3Kγ mediated activation of ERK by inhibiting ERK dephosphorylation following stimulation, thus stabilizing the ERK phosphorylation. PI3Kγ physically disrupts the interaction between ERK and ERK dephosphorylating phosphatase PP2A as evidenced by increase in phosphatase association with ERK in KO MEFs. Consistent with this observation, ERK activation was completely abolished in KO MEFs following carvedilol suggesting an essential role for PI3Kγ in cardio-protective ERK activation pathway. In this context, it is known that transverse aortic constriction (TAC) in mice leads to increase in ERK activation in the hearts and is also associated with concurrent up-regulation of PI3Kγ suggesting a key role for kinase-independent function of PI3Kγ in activating and maintaining the ERK signaling cascade. These indepth cellular studies and observation from our TAC studies led us to believe that kinase-dependent function of PI3Kγ may contribute to pathology while kinase-independent function may be cardio-protective through inhibition of PP2A by PI3Kγ. This novel signaling mechanism by PI3Kγ will be presented.

Abstract 425: Protein Kinase Activity of Pi3kγ Regulates Insulin-βArs Cross-talk

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Anita SAHU ◽  
Sromona Mukherjee ◽  
Kate Stenson ◽  
Maradumane L Mohan ◽  
Sathyamangla V Prasad
Keyword(s):  
Hek293 Cells ◽  
Protein Kinase Activity ◽  
Pi3k Inhibitors ◽  
Pp2a Activity ◽  
Phosphatase 2A ◽  
Β Blocker ◽  
Sirna Knockdown ◽  
G Protein Coupled ◽  
Diabetes And Obesity

β adrenergic receptor (βAR) function is regulated by G-protein coupled receptor (GPCR) kinase (GRK) driven desensitization and protein phosphatase 2A (PP2A) mediated resensitization to its classical agonist. Surprisingly, Insulin modulates (βARs) function thus regulating cardiac function. Although insulin is known to modulate βAR function through GRKs, less is known about insulin mediated resensitization mechanisms. PI3Kγ is activated by GPCRs regulates resensitization by inhibiting PP2A activity. Therefore, we tested whether insulin could mediate βAR dysfunction through inhibition of resensitization. Co-immunoprecipitation in cardiac lysates and surface plasmon resonance (SPR) with purified proteins show that PI3Kγ interacts with GRK-2 resulting in recruitment of GRK2-PI3Kγ to the β2AR complex upon insulin. Furthermore, use of PI3K inhibitors significantly reduced Insulin-stimulated β2ARs phosphorylation in HEK293 cells. The role of PI3Kγ was further validated by the loss of insulin stimulated β 2 AR phosphorylation in PI3Kγ-knockout (KO) mouse embryonic fibroblasts (MEFs), shRNA and CRIPSR knockdown of PI3Kγ. Data shows that PI3Kγ inhibits PP2A activity at the βAR complex upon insulin while loss of PI3Kγ unravels this inhibition resulting in increased PP2A activity leading to β2AR dephosphorylation and resensitization. Mechanistically, PI3Kγ inhibits PP2A activity at the β2AR complex by phosphorylating an endogenous inhibitor of PP2A (I2PP2A). CRISPR knockout and siRNA knockdown of endogenous I2PP2A in HEK293 cells restored PP2A activity resulting in β2AR dephosphorylation despite PI3Kγ. Furthermore, β blocker (propranolol) pretreatment did not affect β2AR phosphorylation and there was no β-arrestin recruitment to the βAR complex with Insulin. Together these studies show that Insulin mediates β2AR desensitization through β-agonist and β-arrestin independent mechanisms wherein, PI3Kγ-mediated regulation of PP2A activity plays a pivotal role in cardiac βAR function in hyperinsulemic conditions like diabetes and obesity.

scholarly journals FSH receptor-specific residues L501 and I505 in extracellular loop 2 are essential for its function

2015 ◽  
Vol 54 (3) ◽  
pp. 193-204 ◽  
Author(s):  
Antara A Banerjee ◽  
Madhavi Dupakuntla ◽  
Bhakti R Pathak ◽  
Smita D Mahale
Keyword(s):  
Amino Acids ◽  
Hek293 Cells ◽  
Fsh Receptor ◽  
Receptor Function ◽  
Extracellular Loop ◽  
Camp Production ◽  
Transfected Cells ◽  
Erk Phosphorylation ◽  
Loop 2

The extracellular loop 2 (EL2) of FSH receptor (FSHR) plays a pivotal role in various events downstream of FSH stimulation. Because swapping the six FSHR-specific residues in EL2 (chimeric EL2M) with those from LH/choriogonadotropin receptor resulted in impaired internalization of FSH–FSHR complex and low FSH-induced cAMP production, six substitution mutants of EL2 were generated to ascertain the contribution of individual amino acids to the effects shown by chimeric EL2M. Results revealed that L501F mainly and I505V to a lesser extent contribute to the diminished receptor function in chimeric EL2M. HEK293 cells stably expressing WT and chimeric EL2M FSHR were generated to track the fate of the receptors post FSH induction. The chimeric EL2M FSHR stable clone showed weak internalization and cAMP response similar to transiently transfected cells. Furthermore, reduced FSH-induced ERK phosphorylation was also observed. The interaction of activated chimeric EL2M and L501F FSHR with β-arrestins was weak compared with WT FSHR, thus explaining the impaired internalization of chimeric EL2M and corroborating the indispensable role of EL2 in receptor function.

scholarly journals ERK: A Double-Edged Sword in Cancer. ERK-Dependent Apoptosis as a Potential Therapeutic Strategy for Cancer

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2509
Author(s):  
Reiko Sugiura ◽  
Ryosuke Satoh ◽  
Teruaki Takasaki
Keyword(s):  
Cell Proliferation ◽  
Cell Death ◽  
Tumor Cell Death ◽  
Cancer Treatments ◽  
Erk Activation ◽  
Cellular Processes ◽  
Dual Specificity ◽  
Anti Cancer ◽  
Dual Specificity Phosphatases

The RAF/MEK/ERK signaling pathway regulates diverse cellular processes as exemplified by cell proliferation, differentiation, motility, and survival. Activation of ERK1/2 generally promotes cell proliferation, and its deregulated activity is a hallmark of many cancers. Therefore, components and regulators of the ERK pathway are considered potential therapeutic targets for cancer, and inhibitors of this pathway, including some MEK and BRAF inhibitors, are already being used in the clinic. Notably, ERK1/2 kinases also have pro-apoptotic functions under certain conditions and enhanced ERK1/2 signaling can cause tumor cell death. Although the repertoire of the compounds which mediate ERK activation and apoptosis is expanding, and various anti-cancer compounds induce ERK activation while exerting their anti-proliferative effects, the mechanisms underlying ERK1/2-mediated cell death are still vague. Recent studies highlight the importance of dual-specificity phosphatases (DUSPs) in determining the pro- versus anti-apoptotic function of ERK in cancer. In this review, we will summarize the recent major findings in understanding the role of ERK in apoptosis, focusing on the major compounds mediating ERK-dependent apoptosis. Studies that further define the molecular targets of these compounds relevant to cell death will be essential to harnessing these compounds for developing effective cancer treatments.

scholarly journals The Dual Role of TAM Receptors in Autoimmune Diseases and Cancer: An Overview

Cells ◽  
2018 ◽  
Vol 7 (10) ◽  
pp. 166 ◽  
Author(s):  
Martha Wium ◽  
Juliano Paccez ◽  
Luiz Zerbini
Keyword(s):  
Autoimmune Diseases ◽  
Lupus Erythematosus ◽  
Tyrosine Kinases ◽  
Therapeutic Potential ◽  
Cellular Processes ◽  
Tam Receptors ◽  
Different Types ◽  
Extracellular Environment ◽  
Migration And Development

Receptor tyrosine kinases (RTKs) regulate cellular processes by converting signals from the extracellular environment to the cytoplasm and nucleus. Tyro3, Axl, and Mer (TAM) receptors form an RTK family that plays an intricate role in tissue maintenance, phagocytosis, and inflammation as well as cell proliferation, survival, migration, and development. Defects in TAM signaling are associated with numerous autoimmune diseases and different types of cancers. Here, we review the structure of TAM receptors, their ligands, and their biological functions. We discuss the role of TAM receptors and soluble circulating TAM receptors in the autoimmune diseases systemic lupus erythematosus (SLE) and multiple sclerosis (MS). Lastly, we discuss the effect of TAM receptor deregulation in cancer and explore the therapeutic potential of TAM receptors in the treatment of diseases.

scholarly journals Noncanonical regulation of insulin-mediated ERK activation by phosphoinositide 3-kinase γ

2017 ◽  
Vol 28 (22) ◽  
pp. 3112-3122 ◽  
Author(s):  
Maradumane L. Mohan ◽  
Arunachal Chatterjee ◽  
Swetha Ganapathy ◽  
Sromona Mukherjee ◽  
Sowmya Srikanthan ◽  
...  
Keyword(s):  
Cardiac Fibroblasts ◽  
Knock Out ◽  
Erk Activation ◽  
Cellular Processes ◽  
Gpcr Activation ◽  
Extracellular Signal ◽  
Phosphatase 2A ◽  
Epidermal Growth ◽  
Phosphoinositide 3 Kinase ◽  
Knock Out Mice

Classically Class IB phosphoinositide 3-kinase (PI3Kγ) plays a role in extracellular signal–regulated kinase (ERK) activation following G-protein coupled receptor (GPCR) activation. Knock-down of PI3Kγ unexpectedly resulted in loss of ERK activation to receptor tyrosine kinase agonists such as epidermal growth factor or insulin. Mouse embryonic fibroblasts (MEFs) or primary adult cardiac fibroblasts isolated from PI3Kγ knock-out mice (PI3KγKO) showed decreased insulin-stimulated ERK activation. However, expression of kinase-dead PI3Kγ resulted in rescue of insulin-stimulated ERK activation. Mechanistically, PI3Kγ sequesters protein phosphatase 2A (PP2A), disrupting ERK–PP2A interaction, as evidenced by increased ERK–PP2A interaction and associated PP2A activity in PI3KγKO MEFs, resulting in decreased ERK activation. Furthermore, β-blocker carvedilol-mediated β-arrestin-dependent ERK activation is significantly reduced in PI3KγKO MEF, suggesting accelerated dephosphorylation. Thus, instead of classically mediating the kinase arm, PI3Kγ inhibits PP2A by scaffolding and sequestering, playing a key parallel synergistic step in sustaining the function of ERK, a nodal enzyme in multiple cellular processes.

The PI3K Pathway at the Crossroads of Cancer and the Immune System: Strategies for Next Generation Immunotherapy Combinations

2018 ◽  
Vol 18 (4) ◽  
pp. 355-364 ◽  
Author(s):  
Dearbhaile C. Collins ◽  
Maxime Chenard-Poirier ◽  
Juanita S. Lopez
Keyword(s):  
Immune System ◽  
Pi3k Pathway ◽  
Growth And Survival ◽  
Cellular Processes ◽  
Combination Strategies ◽  
Integral Role ◽  
Phosphoinositide 3 Kinase ◽  
Growth Factor Pathways

Immunotherapy has led to a paradigm shift in the treatment of some malignancies, providing long-term, durable responses for a subset of patients with advanced cancers. Increasingly, research has identified links between the immune system and critical oncogenic growth factor pathways. The phosphoinositide 3-kinase (PI3K)-AKT-mTOR cascade is frequently hyperactivated in cancer, and plays an integral role in many cellular processes including tumour growth and survival and can underlie resistance to therapies. In this review, we first summarize two key learnings from the initial studies of inhibitors of this pathway, including the profile of immune-related adverse events such as colitis, transaminitis and pneumonitis and the increased incidence of infections with the majority of agents that target the PI3K-AKT-mTOR pathway. We then discuss recent advances in our understanding of the role of this pathway in the tumour micro-environment, and in the regulation of innate and adaptive immune responses, and propose synergistic combination strategies with PI3K-network inhibitors and cancer immunotherapy.

scholarly journals Extracellular Vesicles From SDHB Deficient hPheo1 Cells Activate STAT3 in Wild-Type Cells

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A68-A69
Author(s):  
Hans K Ghayee ◽  
Sujal Patel ◽  
Kubra M Tuna ◽  
Lauren Liu ◽  
Yiling Xu ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Extracellular Vesicles ◽  
Tyrosine Kinases ◽  
Wild Type ◽  
Cellular Processes ◽  
Transmission Electron ◽  
Different Types ◽  
Inactivating Mutations ◽  
Human Wild Type

Abstract Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors that originate from the adrenal medulla and extra-adrenal paraganglia, respectively. Inactivating mutations in succinate dehydrogenase (SDHx) genes leads to succinate accumulation, increased HIF1-α levels, and uncontrollable growth of PPGLs. We hypothesized that small extracellular vesicles (EVs) released from progenitor cells derived from pheochromocytoma (hPheo1) with a shRNA mediated knockdown of SDHB are enriched in succinate metabolites that play a key role in the activation of various tyrosine dependent signaling pathways that are involved in turmorigenesis and proliferation. We isolated EVs from the conditioned media of human wild-type hPheo1 cells and hPheo1 cells with shRNA SDHB knockdown. The EVs from three separate preparations of each group were characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blotting using antibodies against different types of EV and one non-EV marker. Our results show small EVs from the SDHB knockdown hPheo1 cells increased the activation of phosphotyrosine residues in wild-type cells compared to cells treated with control EVs from the same cell type. Additionally, our data show these EVs increase phospho-STAT3 compared to the control EVs (3843.10 +/- 1138.89 vs. 213.65+/- 40.75; p<0.05; n=3) in cultured wild-type hPheo1 cells. Protein tyrosine kinases (PTKs) control various cellular processes including growth, differentiation, and metabolism by activating various signaling pathways including STAT3. The significance of these findings is that in some cancers, elevated succinate from a SDHx mutation has been shown to activate STAT3 which may explain a possible pathway for tumorigenesis. Studies from other investigators have shown that STAT3 expression is elevated in malignant PPGL tissues. Through enriched EV analysis our findings have confirmed the role of STAT3 in SDHB deficient cells. Additional studies are needed to identify other metabolites that are enriched in EVs that regulate phosphorylation of tyrosine residues and STAT3 activation.

scholarly journals Phosphoinositide 3-kinase signalling pathways

2001 ◽  
Vol 114 (8) ◽  
pp. 1439-1445 ◽  
Author(s):  
D.A. Cantrell
Keyword(s):  
Tyrosine Kinases ◽  
Cell Cycle Progression ◽  
Subcellular Localisation ◽  
Nucleotide Exchange ◽  
Cellular Processes ◽  
Signalling Molecule ◽  
Inositol Phospholipids ◽  
Wide Range ◽  
Ph Domains ◽  
Phosphoinositide 3 Kinase

Phosphoinositide 3-kinases (PI3Ks) phosphorylate the 3′-OH position of the inositol ring of inositol phospholipids, producing three lipid products: PtdIns(3)P, PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3). These lipids bind to the pleckstrin homology (PH) domains of proteins and control the activity and subcellular localisation of a diverse array of signal transduction molecules. Three major classes of signalling molecule are regulated by binding of D-3 phosphoinositides to PH domains: guanine-nucleotide-exchange proteins for Ρ family GTPases, the TEC family tyrosine kinases such as BTK and ITK in B and T lymphocytes, respectively, and the AGC superfamily of serine/threonine protein kinases. These molecules are activated by a variety of extracellular stimuli and have been implicated in a wide range of cellular processes, including cell cycle progression, cell growth, cell motility, cell adhesion and cell survival.

scholarly journals Overexpression of miR-210 is associated with SDH-related pheochromocytomas, paragangliomas, and gastrointestinal stromal tumours

2014 ◽  
Vol 21 (3) ◽  
pp. 415-426 ◽  
Author(s):  
V H M Tsang ◽  
T Dwight ◽  
D E Benn ◽  
G Y Meyer-Rochow ◽  
A J Gill ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multivariate Analysis ◽  
Cell Lines ◽  
Fold Increase ◽  
Cellular Responses ◽  
Hek293 Cells ◽  
Gastrointestinal Stromal Tumours ◽  
Sirna Knockdown ◽  
Normal Controls

miR-210 is a key regulator of response to hypoxia. Pheochromocytomas (PCs) and paragangliomas (PGLs) with germlineSDHxorVHLmutations have pseudohypoxic gene expression signatures. We hypothesised that PC/PGLs containingSDHxorVHLmutations, and succinate dehydrogenase (SDH)-deficient gastrointestinal stromal tumours (GISTs), would overexpress miR-210 relative to non-SDHor -VHL-mutated counterparts. miR-210 was analysed by quantitative PCR in i) 39 PC/PGLs, according to genotype (oneSDHA, fiveSDHB, sevenVHL, threeNF1, sevenRET, 15 sporadic, one unknown) and pathology (18 benign, eight atypical, 11 malignant, two unknown); ii) 18 GISTs, according to SDHB immunoreactivity (nine SDH-deficient and nine SDH-proficient) and iii) two novelSDHB-mutant neurosphere cell lines. miR-210 was higher inSDHx- orVHL-mutated PC/PGLs (7.6-fold) compared with tumours withoutSDHxorVHLmutations (P=0.0016). miR-210 was higher in malignant than in unequivocally benign PC/PGLs (P=0.05), but significance was lost when benign and atypical tumours were combined (P=0.08). In multivariate analysis, elevated miR-210 was significantly associated withSDHxorVHLmutation, but not with malignancy. In GISTs, miR-210 was higher in SDH-deficient (median 2.58) compared with SDH-proficient tumours (median 0.60;P=0.0078). miR-210 was higher in patient-derived neurosphere cell lines containingSDHBmutations (6.5-fold increase) compared with normal controls, in normoxic conditions (P<0.01). Furthermore, siRNA-knockdown of SDHB in HEK293 cells increased miR-210 by 2.7-fold (P=0.001) under normoxia. Overall, our results suggest that SDH deficiency in PC, PGL and GISTs induces miR-210 expression and substantiates the role of aberrant hypoxic-type cellular responses in the development of these tumours.

The regulation of cell migration by PTEN

2005 ◽  
Vol 33 (6) ◽  
pp. 1507-1508 ◽  
Author(s):  
N.R. Leslie ◽  
X. Yang ◽  
C.P. Downes ◽  
C.J. Weijer
Keyword(s):  
Cell Migration ◽  
Phosphatase Activity ◽  
Tumour Suppressor ◽  
Chromosome 10 ◽  
Cellular Processes ◽  
Lipid Phosphatase ◽  
Directed Cell Migration ◽  
Phosphoinositide 3 Kinase ◽  
Phosphatase And Tensin Homologue

In vertebrates, the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) regulates many cellular processes through its PtdIns(3,4,5)P3 lipid phosphatase activity, antagonizing PI3K (phosphoinositide 3-kinase) signalling. Given the important role of PI3Ks in the regulation of directed cell migration and the role of PTEN as an inhibitor of migration, it is somewhat surprising that data now indicate that PTEN is able to regulate cell migration independent of its lipid phosphatase activity. Here, we discuss the role of PTEN in the regulation of cell migration.

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