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.

scholarly journals Role of Phosphoinositide 3-Kinase and Endocytosis in Nerve Growth Factor-Induced Extracellular Signal-Regulated Kinase Activation via Ras and Rap1

2000 ◽  
Vol 20 (21) ◽  
pp. 8069-8083 ◽  
Author(s):  
Randall D. York ◽  
Derek C. Molliver ◽  
Savraj S. Grewal ◽  
Paula E. Stenberg ◽  
Edwin W. McCleskey ◽  
...  
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Map Kinase ◽  
Pc12 Cells ◽  
Erk Signaling ◽  
Nerve Growth ◽  
Erk Activation ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Phosphoinositide 3 Kinase

ABSTRACT Neurotrophins promote multiple actions on neuronal cells including cell survival and differentiation. The best-studied neurotrophin, nerve growth factor (NGF), is a major survival factor in sympathetic and sensory neurons and promotes differentiation in a well-studied model system, PC12 cells. To mediate these actions, NGF binds to the TrkA receptor to trigger intracellular signaling cascades. Two kinases whose activities mediate these processes include the mitogen-activated protein (MAP) kinase (or extracellular signal-regulated kinase [ERK]) and phosphoinositide 3-kinase (PI3-K). To examine potential interactions between the ERK and PI3-K pathways, we studied the requirement of PI3-K for NGF activation of the ERK signaling cascade in dorsal root ganglion cells and PC12 cells. We show that PI3-K is required for TrkA internalization and participates in NGF signaling to ERKs via distinct actions on the small G proteins Ras and Rap1. In PC12 cells, NGF activates Ras and Rap1 to elicit the rapid and sustained activation of ERKs respectively. We show here that Rap1 activation requires both TrkA internalization and PI3-K, whereas Ras activation requires neither TrkA internalization nor PI3-K. Both inhibitors of PI3-K and inhibitors of endocytosis prevent GTP loading of Rap1 and block sustained ERK activation by NGF. PI3-K and endocytosis may also regulate ERK signaling at a second site downstream of Ras, since both rapid ERK activation and the Ras-dependent activation of the MAP kinase kinase kinase B-Raf are blocked by inhibition of either PI3-K or endocytosis. The results of this study suggest that PI3-K may be required for the signals initiated by TrkA internalization and demonstrate that specific endocytic events may distinguish ERK signaling via Rap1 and Ras.

scholarly journals The Class II Phosphoinositide 3-Kinase C2β Is Not Essential for Epidermal Differentiation

2005 ◽  
Vol 25 (24) ◽  
pp. 11122-11130 ◽  
Author(s):  
Kazutoshi Harada ◽  
Amy B. Truong ◽  
Ti Cai ◽  
Paul A. Khavari
Keyword(s):  
Class Ii ◽  
Epidermal Cells ◽  
Epidermal Differentiation ◽  
Class I ◽  
Differentiation Markers ◽  
Cellular Processes ◽  
Targeted Gene Deletion ◽  
Epidermal Growth ◽  
Phosphoinositide 3 Kinase

ABSTRACT Phosphoinositide 3-kinases (PI3Ks) regulate an array of cellular processes and are comprised of three classes. Class I PI3Ks include the well-studied agonist-sensitive p110 isoforms; however, the functions of class II and III PI3Ks are less well characterized. Of the three class II PI3Ks, C2α and C2β are widely expressed in many tissues, including the epidermis, while C2γ is confined to the liver. In contrast to the class I PI3K p110α, which is expressed throughout the epidermis, C2β was found to be localized in suprabasal cells, suggesting a potential role for C2β in epidermal differentiation. Overexpressing C2β in epidermal cells in vitro induced differentiation markers. To study a role for C2β in tissue, we generated transgenic mice overexpressing C2β in both suprabasal and basal epidermal layers. These mice lacked epidermal abnormalities. Mice deficient in C2β were then generated by targeted gene deletion. C2β knockout mice were viable and fertile and displayed normal epidermal growth, differentiation, barrier function, and wound healing. To exclude compensation by C2α, RNA interference was then used to knock down both C2α and C2β in epidermal cells simultaneously. Induction of differentiation markers was unaffected in the absence of C2α and C2β. These findings indicate that class II PI3Ks are not essential for epidermal differentiation.

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 218: PI3Kγ Regulates Age-Dependent Cardiac Hypertrophy Through Kinase-Independent GSK-3-PP2A Axis

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Sathyamangla V Naga Prasad ◽  
Maradumane L Mohan ◽  
Elizabeth E Martelli ◽  
Manveen K Gupta ◽  
Neelakantan T Vasudevan
Keyword(s):  
Glycogen Synthase ◽  
Growth Factor Receptor ◽  
Methyl Transferase ◽  
Knock Out ◽  
Akt Activation ◽  
Pp2a Activity ◽  
Phosphatase 2A ◽  
Heart Size ◽  
Phosphoinositide 3 Kinase

Activation of phosphoinositide 3-kinase α (PI3Kα) by Receptor Tyrosine Kinase (RTK) or PI3Kγ by G-protein coupled receptor (GPCR) inhibits glycogen synthase kinase-3 (GSK-3) via protein kinase B (Akt). We show that in addition to promoting GSK-3 phosphorylation through Akt, PI3Kγ in parallel suppresses PP2A dependent GSK-3 dephosphorylation. This is evidenced by accelerated GSK-3 dephosphorylation in PI3Kγ knock out (PI3Kγ-KO) mice downstream of RTK-PI3Kα-Akt axis despite robust Akt activation by insulin. Confocal microscopy and immunoblotting show marked reduction of steady state GSK-3 phosphorylation in PI3Kγ-KO compared to littermate controls. Assessment of GSK-3 dephosphorylating enzyme protein phosphatase 2A (PP2A) showed significant elevation in PP2A and GSK-associated phosphatase activity in PI3Kγ-KO mice compared to controls. Mechanistically, we found that elevated PP2A activity in PI3Kγ-KO was due to PP2A methylation mediated by elevated PP2A methyl transferase (PPMT-1) activity. Consistent with the elevated anti-hypertrophic GSK-3 activity, we observed reduced heart size in PI3Kγ-KO mice at 6, 12, and 18 months compared to age matched littermate controls. To test in vivo whether PI3Kγ activity regulates cardiac GSK-3 function through PP2A, we bred transgenic mice with cardiac overexpression of inactive PI3Kγ (PI3Kγ inact ) with PI3Kγ-KO mice. Surprisingly, cardiac overexpression of PI3Kγ inact transgene in PI3Kγ-KO background completely normalized cardiac PPMT-1 activity resulting in reduced PP2A activity and increased GSK-3 phosphorylation. Expression of PI3Kγ inact transgene in PI3Kγ-KO resulted in normalization of heart size compared to PI3Kγ-KO littermates consistent with the increased GSK-3 phosphorylation and consequent inhibition of GSK-3 activity suggesting a novel kinase independent role of PI3Kγ downstream of growth factor receptor in regulating cardiac growth with age.

scholarly journals An Uncleavable uPAR Mutant Allows Dissection of Signaling Pathways in uPA-dependent Cell Migration

2006 ◽  
Vol 17 (1) ◽  
pp. 367-378 ◽  
Author(s):  
Roberta Mazzieri ◽  
Silvia D'Alessio ◽  
Richard Kamgang Kenmoe ◽  
Liliana Ossowski ◽  
Francesco Blasi
Keyword(s):  
Cell Migration ◽  
Signaling Pathways ◽  
Egf Receptor ◽  
Alternative Pathway ◽  
Erk Activation ◽  
Type Plasminogen Activator ◽  
Extracellular Signal ◽  
Urokinase Type Plasminogen Activator ◽  
Dependent Cell ◽  
Epidermal Growth

Urokinase-type plasminogen activator (uPA) binding to uPAR induces migration, adhesion, and proliferation through multiple interactions with G proteins-coupled receptor FPRL1, integrins, or the epidermal growth factor (EGF) receptor (EGFR). At least two forms of uPAR are present on the cell surface: full-length and cleaved uPAR, each specifically interacting with one or more transmembrane proteins. The connection between these interactions and the effects on the signaling pathways activation is not clear. We have exploited an uPAR mutant (hcr, human cleavage resistant) to dissect the pathways involved in uPA-induced cell migration. This mutant is not cleaved by proteases, is glycosylphosphatidylinositol anchored, and binds uPA with a normal Kd. Both wild-type (wt) and hcr-uPAR are able to mediate uPA-induced migration, are constitutively associated with the EGFR, and associate with α3β1 integrin upon uPA binding. However, they engage different pathways in response to uPA. wt-uPAR requires both integrins and FPRL1 to mediate uPA-induced migration, and association of wt-uPAR to α3β1 results in uPAR cleavage and extracellular signal-regulated kinase (ERK) activation. On the contrary, hcr-uPAR does not activate ERK and does not engage FPRL1 or any other G protein-coupled receptor, but it activates an alternative pathway initiated by the formation of a triple complex (uPAR-α3β1-EGFR) and resulting in the autotyrosine phosphorylation of EGFR.

scholarly journals Signal Strength Dictates Phosphoinositide 3-Kinase Contribution to Ras/Extracellular Signal-Regulated Kinase 1 and 2 Activation via Differential Gab1/Shp2 Recruitment: Consequences for Resistance to Epidermal Growth Factor Receptor Inhibition

2007 ◽  
Vol 28 (2) ◽  
pp. 587-600 ◽  
Author(s):  
Carla Sampaio ◽  
Marie Dance ◽  
Alexandra Montagner ◽  
Thomas Edouard ◽  
Nicole Malet ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Growth Factor Receptor ◽  
Signal Strength ◽  
Extracellular Signal Regulated Kinase ◽  
Egfr Activation ◽  
Extracellular Signal ◽  
Epidermal Growth ◽  
Phosphoinositide 3 Kinase

ABSTRACT Phosphoinositide 3-kinase (PI3K) participates in extracellular signal-regulated kinase 1 and 2 (ERK1-2) activation according to signal strength, through unknown mechanisms. We report herein that Gab1/Shp2 constitutes a PI3K-dependent checkpoint of ERK1-2 activation regulated according to signal intensity. Indeed, by up- and down-regulation of signal strength in different cell lines and through different methods, we observed that Gab1/Shp2 and Ras/ERK1-2 in concert become independent of PI3K upon strong epidermal growth factor receptor (EGFR) stimulation and dependent on PI3K upon limited EGFR activation. Using Gab1 mutants, we observed that this conditional role of PI3K is dictated by the EGFR capability of recruiting Gab1 through Grb2 or through the PI3K lipid product PIP3, according to a high or weak level of receptor stimulation, respectively. In agreement, Grb2 siRNA generates, in cells with maximal EGFR stimulation, a strong dependence on PI3K for both Gab1/Shp2 and ERK1-2 activation. Therefore, Ras/ERK1-2 depends on PI3K only when PIP3 is required to recruit Gab1/Shp2, which occurs only under weak EGFR mobilization. Finally, we show that, in glioblastoma cells displaying residual EGFR activation, this compensatory mechanism becomes necessary to efficiently activate ERK1-2, which could probably contribute to tumor resistance to EGFR inhibitors.

scholarly journals Reevaluation of the role of LIP-1 as an ERK/MPK-1 dual specificity phosphatase in the C. elegans germline

2022 ◽  
Vol 119 (3) ◽  
pp. e2113649119
Author(s):  
Debabrata Das ◽  
Jacob Seemann ◽  
David Greenstein ◽  
Tim Schedl ◽  
Swathi Arur
Keyword(s):  
Failure To Rescue ◽  
Gain Of Function ◽  
Erk Activation ◽  
C Elegans ◽  
Cellular Processes ◽  
Dual Specificity ◽  
Extracellular Signal ◽  
Dual Specificity Phosphatases ◽  
Organismal Development

The fidelity of a signaling pathway depends on its tight regulation in space and time. Extracellular signal-regulated kinase (ERK) controls wide-ranging cellular processes to promote organismal development and tissue homeostasis. ERK activation depends on a reversible dual phosphorylation on the TEY motif in its active site by ERK kinase (MEK) and dephosphorylation by DUSPs (dual specificity phosphatases). LIP-1, a DUSP6/7 homolog, was proposed to function as an ERK (MPK-1) DUSP in the Caenorhabditis elegans germline primarily because of its phenotype, which morphologically mimics that of a RAS/let-60 gain-of-function mutant (i.e., small oocyte phenotype). Our investigations, however, reveal that loss of lip-1 does not lead to an increase in MPK-1 activity in vivo. Instead, we show that loss of lip-1 leads to 1) a decrease in MPK-1 phosphorylation, 2) lower MPK-1 substrate phosphorylation, 3) phenocopy of mpk-1 reduction-of-function (rather than gain-of-function) allele, and 4) a failure to rescue mpk-1–dependent germline or fertility defects. Moreover, using diverse genetic mutants, we show that the small oocyte phenotype does not correlate with increased ectopic MPK-1 activity and that ectopic increase in MPK-1 phosphorylation does not necessarily result in a small oocyte phenotype. Together, these data demonstrate that LIP-1 does not function as an MPK-1 DUSP in the C. elegans germline. Our results caution against overinterpretation of the mechanistic underpinnings of orthologous phenotypes, since they may be a result of independent mechanisms, and provide a framework for characterizing the distinct molecular targets through which LIP-1 may mediate its several germline functions.

scholarly journals GP78 Cooperates with Dual-Specificity Phosphatase 1 To Stimulate Epidermal Growth Factor Receptor-Mediated Extracellular Signal-Regulated Kinase Signaling

2019 ◽  
Vol 39 (11) ◽  
Author(s):  
Dhong Hyo Kho ◽  
Mohammed Hafiz Uddin ◽  
Madhumita Chatterjee ◽  
Andreas Vogt ◽  
Avraham Raz ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Growth Factor Receptor ◽  
Erk Activation ◽  
Extracellular Signal Regulated Kinase ◽  
Dual Specificity ◽  
Extracellular Signal ◽  
Epidermal Growth ◽  
Proliferation And Invasion

ABSTRACT GP78 is an autocrine motility factor (AMF) receptor (AMFR) with E3 ubiquitin ligase activity that plays a significant role in tumor cell proliferation, motility, and metastasis. Aberrant extracellular signal-regulated kinase (ERK) activation via receptor tyrosine kinases promotes tumor proliferation and invasion. The activation of GP78 leads to ERK activation, but its underlying mechanism is not fully understood. Here, we show that GP78 is required for epidermal growth factor receptor (EGFR)-mediated ERK activation. On one hand, GP78 interacts with and promotes the ubiquitination and subsequent degradation of dual-specificity phosphatase 1 (DUSP1), an endogenous negative regulator of mitogen-activated protein kinases (MAPKs), resulting in ERK activation. On the other hand, GP78 maintains the activation status of EGFR, as evidenced by the fact that EGF fails to induce EGFR phosphorylation in GP78-deficient cells. By the regulation of both EGFR and ERK activation, GP78 promotes cell proliferation, motility, and invasion. Therefore, this study identifies a previously unknown signaling pathway by which GP78 stimulates ERK activation via DUSP1 degradation to mediate EGFR-dependent cancer cell proliferation and invasion.

scholarly journals Differential activation of the Ras/extracellular-signal-regulated protein kinase pathway is responsible for the biological consequences induced by the Axl receptor tyrosine kinase.

1996 ◽  
Vol 16 (1) ◽  
pp. 135-145 ◽  
Author(s):  
Y W Fridell ◽  
Y Jin ◽  
L A Quilliam ◽  
A Burchert ◽  
P McCloskey ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Egf Receptor ◽  
Kinase Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Erk Activation ◽  
Interleukin 3 ◽  
Extracellular Signal ◽  
Axl Receptor Tyrosine Kinase ◽  
Epidermal Growth ◽  
Kinase Pathway

To understand the mechanism of Axl signaling, we have initiated studies to delineate downstream components in interleukin-3-dependent 32D cells by using a chimeric receptor containing the recombinant epidermal growth factor (EGF) receptor extracellular and transmembrane domains and the Axl kinase domain (EAK [for EGF receptor-Axl kinase]). We have previously shown that upon exogenous EGF stimulation, 32D-EAK cells are capable of proliferation in the absence of interleukin-3. With this system, we determined that EAK-induced cell survival and mitogenesis are dependent upon the Ras/extracellular-signal-regulated protein kinase (ERK) cascade. Although the phosphatidylinositol-3 kinase pathway is activated upon EAK signaling, it appears to be dispensable for the biological actions of the Axl kinase. Furthermore, we demonstrated that different threshold levels of Ras/ERK activation are needed to induce a block to apoptosis or proliferation in 32D cells. Recently, we have identified an Axl ligand, GAS6. Surprisingly, GAS6-stimulated 32D-Axl cells exhibited no blockage to apoptosis or mitogenic response which is correlated with the absence of Ras/ERK activation. Taken together, these data suggest that different extracellular domains dramatically alter the intracellular response of the Axl kinase. Furthermore, our data suggest that the GAS6-Axl interaction does not induce mitogenesis and that its exact role remains to be determined.

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