Quantitative phosphoproteomic analysis of the PI3K‐regulated signaling network

The PI3K pathway is commonly activated in cancer. Only a few studies have attempted to explore the spectrum of phosphorylation signaling downstream of the PI3K cascade. Such insight, however, is imperative to understand the mechanisms responsible for oncogenic phenotypes. By applying MS‐based phosphoproteomics, we mapped 2509 phosphorylation sites on 1096 proteins, and quantified their responses to activation or inhibition of PIK3CA using isogenic knock‐in derivatives and a series of targeted inhibitors. We uncovered phosphorylation changes in a wide variety of proteins involved in cell growth and proliferation, many of which have not been previously associated with PI3K signaling. A significant update of the posttranslational modification database PHOSIDA ( http://www.phosida.com ) allows efficient use of the data. All MS data have been deposited in the ProteomeXchange with identifier PXD003899 ( http://proteomecentral.proteomexchange.org/dataset/PXD003899 ).     

GRK2-Dependent Desensitization Downstream of G Proteins

G protein-coupled receptor kinases (GRKs) are serine/threonine kinases first discovered by its role in receptor desensitization. Phosphorylation of the C-terminal tail of GPCRs by GRKs triggers the docking of β-arrestins and the functional uncoupling of G proteins and receptors. In addition, we and others have uncovered new direct ways by which GRKs could impinge into intracellular signalling pathways independently of receptor phosphorylation. In particular, we have characterized that elevated GRK2 levels can reduce CCR2-mediated activation of the ERK MAPK route in a manner that is independent of kinase activity and also of G proteins. This inhibition of ERK occurred in the absence of any reduction on MEK phosphorylation, what implicates that GRK2 is acting at the level of MEK or at the MEK-ERK interface to achieve a downregulation of ERK phosphorylation. In fact, we describe here that a direct association between GRK2 and MEK proteins can be detected in vitro. p38 MAPK pathway also appears to be regulated directly by GRK2 in a receptor-independent manner. p38 can be phosphorylated by GRK2 in threonine 123, a residue sitting at the entrance of a docking groove by which this MAPK associates to substrates and upstream activators. The T123phospho-mimetic mutant of p38 shows a reduced ability to bind to MKK6, concomitant with an impaired p38 activation, and a decreased phosphorylation of downstream substrates such as MEF2, MK2 and ATF2. Elevated levels of GRK2 downregulate p38-dependent cellular responses, such as differentiation of preadipocytic cells, while LPS-induced cytokine release is enhanced in macrophages from GRK2 (+/?) mice. In sum, we describe in this article different ways by which GRK2 directly regulates MAPK-mediated cellular events. This regulation of the MAPK modules by GRK2 could be relevant in pathological situations where the levels of this kinase are altered, such as during inflammatory diseases or cardiovascular pathologies.     

GRK2-dependent desensitization downstream of G proteins

G protein-coupled receptor kinases (GRKs) are serine/threonine kinases first discovered by its role in receptor desensitization. Phosphorylation of the C-terminal tail of GPCRs by GRKs triggers the docking of beta-arrestins and the functional uncoupling of G proteins and receptors. In addition, we and others have uncovered new direct ways by which GRKs could impinge into intracellular signalling pathways independently of receptor phosphorylation. In particular, we have characterized that elevated GRK2 levels can reduce CCR2-mediated activation of the ERK MAPK route in a manner that is independent of kinase activity and also of G proteins. This inhibition of ERK occurred in the absence of any reduction on MEK phosphorylation, what implicates that GRK2 is acting at the level of MEK or at the MEK-ERK interface to achieve a downregulation of ERK phosphorylation. In fact, we describe here that a direct association between GRK2 and MEK proteins can be detected in vitro. p38 MAPK pathway also appears to be regulated directly by GRK2 in a receptor-independent manner. p38 can be phosphorylated by GRK2 in threonine 123, a residue sitting at the entrance of a docking groove by which this MAPK associates to substrates and upstream activators. The T123phospho-mimetic mutant of p38 shows a reduced ability to bind to MKK6, concomitant with an impaired p38 activation, and a decreased phosphorylation of downstream substrates such as MEF2, MK2 and ATF2. Elevated levels of GRK2 downregulate p38-dependent cellular responses, such as differentiation of preadipocytic cells, while LPS-induced cytokine release is enhanced in macrophages from GRK2 (+/-) mice. In sum, we describe in this article different ways by which GRK2 directly regulates MAPK-mediated cellular events. This regulation of the MAPK modules by GRK2 could be relevant in pathological situations where the levels of this kinase are altered, such as during inflammatory diseases or cardiovascular pathologies.     

A mitogen-activated protein kinase-dependent signaling pathway in the activation of platelet integrin alpha IIbbeta3

We have recently shown that the platelet integrin alpha(IIb)beta(3) is activated by von Willebrand factor (vWF) binding to its platelet receptor, glycoprotein Ib-IX (GPIb-IX), via the protein kinase G (PKG) signaling pathway. Here we show that GPIb-IX-mediated activation of integrin alpha(IIb)beta(3) is inhibited by dominant negative mutants of Raf-1 and MEK1 in a reconstituted integrin activation model in Chinese hamster ovary (CHO) cells and that the integrin-dependent platelet aggregation induced by either vWF or low dose thrombin is inhibited by MEK inhibitors PD98059 and U0126. Thus, mitogen-activated protein kinase (MAPK) pathway is important in GPIb-IX-dependent activation of platelet integrin alpha(IIb)beta(3). Furthermore, vWF binding to GPIb-IX induces phosphorylation of Thr-202/Tyr-204 of extracellular signal-regulated kinase 2 (ERK2). GPIb-IX-induced ERK2 phosphorylation is inhibited by PKG inhibitors and enhanced by overexpression of recombinant PKG. PKG activators also induce ERK phosphorylation, indicating that activation of MAPK pathway is downstream from PKG. Thus, our data delineate a novel integrin activation pathway in which ligand binding to GPIb-IX activates PKG that stimulates MAPK pathway, leading to integrin activation.     

PKC isoenzymes differentially modulate the effect of thrombin on MAPK-dependent RPE proliferation

Thrombin signalling through PAR (protease-activated receptor)-1 is involved in cellular processes, such as proliferation, differentiation and cell survival. Following traumatic injury to the eye, thrombin signalling may participate in disorders, such as PVR (proliferative vitreoretinopathy), a human eye disease characterized by the uncontrolled proliferation, transdifferentiation and migration of otherwise quiescent RPE (retinal pigment epithelium) cells. PARs activate the Ras/Raf/MEK/ERK MAPK pathway (where ERK is extracellular-signal-regulated kinase, MAPK is mitogen-activated protein kinase and MEK is MAPK/ERK kinase) through the activation of G(alpha) and G(betagamma) heterotrimeric G-proteins, and the downstream stimulation of the PLC (phospholipase C)-beta/PKC (protein kinase C) and PI3K (phosphoinositide 3-kinase) signalling axis. In the present study, we examined the molecular signalling involved in thrombin-induced RPE cell proliferation, using rat RPE cells in culture as a model system for PVR pathogenesis. Our results showed that thrombin activation of PAR-1 induces RPE cell proliferation through Ras-independent activation of the Raf/MEK/ERK1/2 MAPK signalling cascade. Pharmacological analysis revealed that the activation of 'conventional' PKC isoforms is essential for proliferation, although thrombin-induced phosphorylation of ERK1/2 requires the activation of atypical PKCzeta by PI3K. Consistently, thrombin-induced ERK1/2 activation and RPE cell proliferation were prevented completely by PI3K or PKCzeta inhibition. These results suggest that thrombin induces RPE cell proliferation by joint activation of PLC-dependent and atypical PKC isoforms and the Ras-independent downstream stimulation of the Raf/MEK/ERK1/2 MAPK cascade. The present study is the first report demonstrating directly thrombin-induced ERK phosphorylation in the RPE, and the involvement of atypical PKCzeta in this process.     

Oligonol, an oligomerized lychee fruit-derived polyphenol, activates the Ras/Raf-1/MEK1/2 cascade independent of the IL-6 signaling pathway in rat primary adipocytes

Oligonol is a lychee fruit-derived low-molecular form of polyphenol. In this study, the effect of Oligonol on the mitogen activated-protein kinase (MAPK) signaling pathway in primary adipocytes was investigated to examine the mechanism underlying the enhanced levels of phosphorylated extracellular-signaling regulatory kinase1/2 (ERK1/2) that accompany an in vitro increase in lipolysis. Oligonol significantly elevated the levels of activated Ras and the phosphorylation of Raf-1 and MAPK/ERK kinase1/2 (MEK1/2) with no increase in pan-Raf-1 and -MEK1/2 proteins. The increase in phosphorylation of Raf-1 and MEK1/2 with Oligonol was inhibited completely by pretreatment with GW5074, a selective Raf-1 inhibitor, or PD98059, a selective MEK1/2 inhibitor. IL-6 also activated the MAPK signaling pathway in adipocytes through the association with its receptor. IL-6-induced phosphorylation of Raf-1 and MEK1/2 was significantly inhibited by pretreatment with the IL-6 receptor antibody. Under such a condition, however, the levels of phosphorylated Raf-1 and MEK1/2 with Oligonol still remained significantly higher, and there was a significant decrease in secretion of IL-6 from adipocytes, compared with untreated control cells. These results suggest that Oligonol activates the Ras/Raf-1/MEK1/2 signaling pathway, independent of the IL-6 signaling pathway, leading to activation of ERK1/2 proteins in primary adipocytes.     

MEK/ERK-dependent uPAR expression is required for motility via phosphorylation of P70S6K in human hepatocarcinoma cells

Motility and invasiveness events require specific intracellular signaling cascade activations. In cancer liver cells, one of these mechanisms could involve the MAPK MEK/ERK cascade activation which has been shown over expressed and activated in hepatocellular carcinoma. To study whether the MEK/ERK cascade is involved in the motility of HCC, we examined the effect of MEK inhibitor and ERK2 silencing using monolayer wound-healing assays and fluoroblock invasion systems. Evidence was provided that the MAPK cascade is a key transduction pathway which controls HCC cells motility and invasiveness. We could disconnect proliferation to motility using mitomycin C and we established that RNAi-mediated inhibition of ERK2 led to strongly reduced cell motility. To improve our understanding, we analysed the regulation and the role of urokinase receptor (uPAR) in this process. We provided evidence that uPAR was under a MEK/ERK dependent mechanism and blocking uPAR activity using specific antagonist or inhibiting its expression by RNA interference which resulted in complete inhibition of motility. Moreover, we found in MAPK inhibited cultures and in uPAR silencing cells that p70S6K phosphorylation on residue Thr-389 was significantly reduced, whereas Ser-421/Thr-424 phosphorylation did not change. We highlighted that the FRAP/mTOR pathway did not affect motility and Thr-389 phosphorylation. Furthermore, we demonstrated that p70S6K inhibition by RNA interference completely inhibited hepatocarcinoma cell motility. Therefore, targeting uPAR and/or MEK/ERK/S6K by RNA interference could be a major therapeutic strategy for the future treatment of invasive hepatocarcinoma cells.     

Activation of several MAP kinases upon stimulation of rat alveolar macrophages: role of the NADPH oxidase.

Zymosan-activated serum (ZAS), a source of C5a, stimulates the rat alveolar macrophages (AM) to release superoxide anion. Here we show that treatment of rat AM with ZAS induced a time-dependent increase in the tyrosine phosphorylation of several proteins (116, 105-110, 82-78, 66-72, 62, 45, 42, and 38 kDa). This increase was sensitive to genistein, a tyrosine kinase inhibitor. ZAS stimulated the tyrosine phosphorylation and activation of three members of a family of serine/threonine kinases known as the mitogen-activated protein kinases (MAPK), i.e., ERK1 and ERK2, as assessed by immunoblotting, immunoprecipitation, and phosphotransferase activity, and p38 MAPK, as determined by immunoblotting with phospho-specific antibodies. In addition, ZAS induced the tyrosine phosphorylation of the SHC proteins and their association with GRB2, suggesting a role for this complex in the activation of the ERK pathway. Addition of extracellular catalase during ZAS stimulation significantly reduced the tyrosine phosphorylation response and the activation of ERK1 and ERK2 and their activator MEK1/2 while it did not affect that of p38 MAPK and MKK3/MKK6. Superoxide dismutase marginally increased the response to ZAS, supporting a role for hydrogen peroxide. In contrast to the results with AM, stimulation of human neutrophils with ZAS in the presence of catalase minimally altered the activation of ERK1 and ERK2. These data show that, in ZAS-stimulated rat AM, activation of the respiratory burst and production of hydrogen peroxide via superoxide dismutation are largely responsible for the activation of the ERK pathway through an upstream target.     

The Mos/MAP kinase pathway stabilizes c-Fos by phosphorylation and augments its transforming activity in NIH 3T3 cells.

The c-mos proto-oncogene product, Mos, is a serine/threonine kinase that can activate ERK1 and 2 mitogen-activated protein (MAP) kinases by direct phosphorylation of MAPK/ERK kinase (MEK). ERK activation is essential for oncogenic transformation of NIH 3T3 cells by Mos. In this study, we examined how mitogenic and oncogenic signalling from the Mos/MEK/ERK pathway reaches the nucleus to activate downstream target genes. We show that c-Fos (the c-fos protooncogene product), which is an intrinsically unstable nuclear protein, is metabolically highly stabilized, and greatly enhances the transforming efficiency of NIH 3T3 cells, by Mos. This stabilization of c-Fos required Mos-induced phosphorylation of its C-terminal region on Ser362 and Ser374, and double replacements of these serines with acidic (Asp) residues markedly increased the stability and transforming efficiency of c-Fos even in the absence of Mos. Moreover, activation of the ERK pathway was necessary and sufficient for the c-Fos phosphorylation and stabilization by Mos. These results indicate that c-Fos undergoes stabilization, and mediates at least partly the oncogenic signalling, by the Mos/MEK/ERK pathway. The present findings also suggest that, in general, the ERK pathway may regulate the cell fate and function by affecting the metabolic stability of c-Fos.     

Tumor Suppressor PTEN Inhibits Integrin- and Growth Factor–mediated Mitogen-activated Protein (MAP) Kinase Signaling Pathways

The tumor suppressor PTEN dephosphorylates focal adhesion kinase (FAK) and inhibits integrin-mediated cell spreading and cell migration. We demonstrate here that expression of PTEN selectively inhibits activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. PTEN expression in glioblastoma cells lacking the protein resulted in inhibition of integrin-mediated MAP kinase activation. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)- induced MAPK activation were also blocked. To determine the specific point of inhibition in the Ras/Raf/ MEK/ERK pathway, we examined these components after stimulation by fibronectin or growth factors. Shc phosphorylation and Ras activity were inhibited by expression of PTEN, whereas EGF receptor autophosphorylation was unaffected. The ability of cells to spread at normal rates was partially rescued by coexpression of constitutively activated MEK1, a downstream component of the pathway. In addition, focal contact formation was enhanced as indicated by paxillin staining. The phosphatase domain of PTEN was essential for all of these functions, because PTEN with an inactive phosphatase domain did not suppress MAP kinase or Ras activity. In contrast to its effects on ERK, PTEN expression did not affect c-Jun NH₂-terminal kinase (JNK) or PDGF-stimulated Akt. Our data suggest that a general function of PTEN is to down-regulate FAK and Shc phosphorylation, Ras activity, downstream MAP kinase activation, and associated focal contact formation and cell spreading.     

Automated oncogene detection in complex protein networks with applications to the MAPK signal transduction pathway

Activation of the extracellular signal-regulated kinases (ERK1/2; p42/p44 mitogen-activated protein kinase (MAPK)) is one of the most extensively studied signaling pathways not least because it occurs downstream of oncogenic RAS. Here, we take advantage of the wealth of experimental data available on the canonical RAS/RAF/MEK/ERK pathway of Bhalla et al. to test the utility of a newly developed nonlinear analysis algorithm designed to predict likelihood of cellular transformation. By using ERK phosphorylation as an "output signal", the method analyzes experimentally determined kinetic data and predicts putative oncogenes and tumor suppressor gene products impacting the RAS/MAPK module using a purely theoretical approach. This analysis identified several modifiers of ERK/MAPK activation described previously. In addition, several novel enzymes are identified which are not previously described to affect ERK/MAPK phosphorylation. Importantly, the nonlinear analysis enables a ranking of modifiers of MAPK activation predicting their relative importance in RAS-dependent oncogenesis. The results are compared with a linearized analysis based on sensitivity analysis about the steady state or metabolic control analysis (MCA). The results are favorable, pointing to the utility of first-order sensitivity analysis and MCA in the analysis of complex signaling networks for oncogenes.     

Raf-1 kinase activation is uncoupled from downstream MEK/ERK pathway in cells treated with Src tyrosine kinase inhibitor PP2

Recently we demonstrated that PP2 (4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a potent and selective inhibitor of the Src-family tyrosine kinase, markedly enhanced Ras-independent activation of Raf-1 by the combination of phorbol myristate acetate (PMA) and hydrogen peroxide (H(2)O(2)). We report here that Raf-1 knockdown cells were significantly more sensitive to treatment of PP2 than control cells. This PP2-induced growth inhibition was found to be linked to decreased ERK and p38 activity. Interestingly, the growth of Sprouty knockdown cells appeared to be inhibited at earlier time points of PP2 treatment when compared with control cells. Unexpectedly, siRNA-mediated knockdown of Spry2, which is known to modulate the Ras/Raf/MAPK signal through feedback regulation, resulted in decreased Raf-1 kinase activity. PP2 had limited effect on the ability of PMA/H(2)O(2) to induce significant phosphorylation of MEK/ERK proteins in both Spry2 knockdown and control cells, indicating that PP2-mediated activation of Raf-1 did not potentiate signaling through the downstream MEK/ERK pathway. Taken together our results suggest that Raf-1 signaling may be bypassed in PP2-treated cells by uncoupling from downstream MEK/ERK pathway.     

PBK/TOPK在乳腺癌细胞中介导MEK非依赖性ERK激活中作用机制分析

该文探讨了乳腺癌细胞中表皮生长因子(EGF)介导的MEK非依赖性ERK激活通路。Western blot检测EGF刺激下,siRNA抑制MEK1/2后的T47D细胞的p-ERK水平,以验证T47D细胞中存在EGF介导的MEK非依赖性ERK激活的通路。接着使用可能参与MEK非依赖性ERK激活的激酶的小分子抑制剂抑制相关激酶(AC、PKC、Src、PI3K、PDK1和Akt)活性后,检测T47D细胞EGF介导ERK的磷酸化水平。siRNA抑制MEK1/2表达后,T47D细胞在EGF刺激后的仍保留部分p-ERK,即在T47D细胞中,存在EGF介导的MEK非依赖性的ERK磷酸化通路。小分子抑制剂抑制AC、PKC、Src对MEK非依赖性ERK激活途径影响不大。而使用小分子抑制剂抑制PI3K、PDK1和Akt后,ERK的磷酸化水平显著降低,提示PI3K/Akt通路下游的激酶参与T47D中EGF介导的MEK非依赖性ERK激活途径。siRNA干扰PI3K/Akt通路下游PBK/TOPK后并使用U0126抑制MEK功能后,几乎检测不到p-ERK,提示PBK/TOPK参与T47D细胞中EGF介导的MEK非依赖性ERK激活途径。乳腺癌抗雌激素药物耐药株T47D细胞存在EGF介导的MEK非依赖性ERK激活途径,且该途径受PI3K/Akt下游的PBK/TOPK调控。     

Effects of osmotic stress on the activity of MAPKs and PDGFR-beta-mediated signal transduction in NIH-3T3 fibroblasts

Signaling in cell proliferation, cell migration, and apoptosis is highly affected by osmotic stress and changes in cell volume, although the mechanisms underlying the significance of cell volume as a signal in cell growth and death are poorly understood. In this study, we used NIH-3T3 fibroblasts in a serum- and nutrient-free inorganic medium (300 mosM) to analyze the effects of osmotic stress on MAPK activity and PDGF receptor (PDGFR)-beta-mediated signal transduction. We found that hypoosmolarity (cell swelling at 211 mosM) induced the phosphorylation and nuclear translocation of ERK1/2, most likely via a pathway independent of PDGFR-beta and MEK1/2. Conversely, hyperosmolarity (cell shrinkage at 582 mosM) moved nuclear and phosphorylated ERK1/2 to the cytoplasm and induced the phosphorylation and nuclear translocation of p38 and phosphorylation of JNK1/2. In a series of parallel experiments, hypoosmolarity did not affect PDGF-BB-induced activation of PDGFR-beta, whereas hyperosmolarity strongly inhibited ligand-dependent PDGFR-beta activation as well as downstream mitogenic signal components of the receptor, including Akt and the MEK1/2-ERK1/2 pathway. Based on these results, we conclude that ligand-dependent activation of PDGFR-beta and its downstream effectors Akt, MEK1/2, and ERK1/2 is strongly modulated (inhibited) by hyperosmotic cell shrinkage, whereas cell swelling does not seem to affect the activation of the receptor but rather to activate ERK1/2 via a different mechanism. It is thus likely that cell swelling via activation of ERK1/2 and cell shrinkage via activation of the p38 and JNK pathway and inhibition of the PDGFR signaling pathway may act as key players in the regulation of tissue homeostasis.     

Inhibition of caspase-9 through phosphorylation at Thr 125 by ERK MAPK

Many pro-apoptotic signals activate caspase-9, an initiator protease that activates caspase-3 and downstream caspases to initiate cellular destruction. However, survival signals can impinge on this pathway and suppress apoptosis. Activation of the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) pathway is associated with protection of cells from apoptosis and inhibition of caspase-3 activation, although the targets are unknown. Here, we show that the ERK MAPK pathway inhibits caspase-9 activity by direct phosphorylation. In mammalian cell extracts, cytochrome c-induced activation of caspases-9 and -3 requires okadaic-acid-sensitive protein phosphatase activity. The opposing protein kinase activity is overcome by treatment with the broad-specificity kinase inhibitor staurosporine or with inhibitors of MEK1/2. Caspase-9 is phosphorylated at Thr 125, a conserved MAPK consensus site targeted by ERK2 in vitro, in a MEK-dependent manner in cells stimulated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Phosphorylation at Thr 125 is sufficient to block caspase-9 processing and subsequent caspase-3 activation. We suggest that phosphorylation and inhibition of caspase-9 by ERK promotes cell survival during development and tissue homeostasis. This mechanism may also contribute to tumorigenesis when the ERK MAPK pathway is constitutively activated.