In the present paper, we describe multiple levels of cross-talk between the PI3K (phosphoinositide 3-kinase)/Akt and Ras/MAPK (mitogen-activated protein kinase) signalling pathways. Experimental data and computer simulations demonstrate that cross-talk is context-dependent and that both pathways can activate or inhibit each other. Positive influence of the PI3K pathway on the MAPK pathway is most effective at sufficiently low doses of growth factors, whereas negative influence of the MAPK pathway on the PI3K pathway is mostly pronounced at high doses of growth factors. Pathway cross-talk endows a cell with emerging capabilities for processing and decoding signals from multiple receptors activated by different combinations of extracellular cues.
- mitogen-activated protein kinase (MAPK)
- phosphoinositide 3-kinase (PI3K)
A cell in an organism is immersed in an ocean of growth factors and hormones, yet how signalling networks integrate multiple cues is largely unknown. Although individual signalling pathways have been studied extensively, the processing and integration of pathway responses by cross-talk is less understood. One plausible role of cross-talk is to achieve robust activation of key downstream targets by low physiological doses of external stimuli. For instance, if a signal propagates through different branches that converge at a common target, the signalling responses along these branches will add to the overall target response . Likewise, feedforward and feedback loops embracing interacting pathways make the input–output response of one pathway depend on the activity of the other, thereby creating context-dependent signalling output. Finally, pathway cross-talk generates a plethora of distinct spatiotemporal response patterns, which may facilitate an effective discrimination between combinations of extracellular cues and lead to different cell-fate decisions .
A diverse family of growth factors and other stimuli activate the MAPK (mitogen-activated protein kinase) and the PI3K (phosphoinositide 3-kinase)/Akt cascades (Figure 1A). Signalling by these pathways govern fundamental physiological processes, such as cell proliferation, differentiation, metabolism, cytoskeleton reorganization and cell death and survival [3–6]. The MAPK and PI3K/Akt pathways are often mutated in cancer. Growth and survival of many cancer cells critically depends on aberrant signalling by these pathways, which are also involved in intensive crosstalk. In the present paper, we describe multiple levels of cross-talk interactions between the PI3K/Akt and MAPK signalling pathways. We present experimental data and computer simulations that show that cross-talk is context-dependent and mostly operational at low physiological doses of growth factors, such as EGF (epidermal growth factor).
Organization and functions of Ras/MAPK signalling pathway
There are at least six distinct MAPK signalling pathways, which are named according to their terminal tier kinases: the extracellular-signal-related kinases (ERK1/2), the c-Jun N-terminal kinases/stress-activated protein kinases (JNK1/2/3 or SAPKs), the p38 MAPK, ERK3/4, ERK5 and ERK7/8 [5,6]. In the best-studied Ras/MAPK pathway, growth-factor-induced ERK1/2 (hereafter referred to as ERK) activation involves the recruitment of the cytosolic Grb (growth-factor-receptor-bound protein) 2 to the plasma membrane. Grb2 binds to tyrosine-phosphorylated receptors directly or via the docking protein Shc (Src homology and collagen protein). A subset of Grb2 molecules is constitutively associated with the proline-rich domain of the SOS (Son of Sevenless) protein, which is a GEF (guanine-nucleotide-exchange factor) for the small G-protein Ras . In addition, Grb2 molecules can be involved in activation of PLD2 (phospholipase D2), which generates phosphatidic acid that interacts with the PH (pleckstrin homology) domain of SOS . This further facilitates SOS recruitment to the plasma membrane where SOS catalyses a transformation of inactive GDP-bound Ras into active GTP-bound Ras. Active RasGTP stimulates multiple downstream effectors, including PI3Ks (thereby creating one point of Ras/MAPK→PI3K cross-talk), RalGDS (Ral guanine-nucleotide-dissociation stimulator) and serine/threonine kinase Raf, which is the first kinase of the three-tiered MAPK/ERK cascade. The major substrates of Raf are cytosolic kinases MEK (MAPK/ERK kinase) 1/2, which subsequently phosphorylate ERK on two conserved threonine and tyrosine residues within the ERK activation loop. Activated ERK then phosphorylates a plethora of nuclear and cytoplasmic substrates [5,6]. The temporal activation patterns of the MAPK cascade is also modulated by scaffolding proteins [e.g. RKIP (Raf kinase inhibitor protein), KSR (kinase suppressor of Ras), MP1 (MEK partner 1)], phosphatases [e.g. MKPs (MAPK phosphatases)] and various feedback pathways [5,6,8]. In many cell types, the localization, strength and duration of ERK signalling control cellular programmes of embryogenesis, proliferation, differentiation and apoptosis. Aberrant activation of the Ras/MAPK pathway correlates with cancer progression and metastatic tumour cell growth [5,6].
Organization and functions of the PI3K/Akt signalling pathway
The p85 regulatory subunit of PI3K stabilizes and protects the p110α subunit from degradation, but, at the same time, inhibits its catalytic activity. Consequently, in resting cells, PI3K is inactive. Upon extracellular stimuli, interactions of the SH (Src homology) 2 domains of p85 with phosphorylated tyrosine residues of receptors, non-receptor tyrosine kinases and/or adaptor proteins relieve autoinhibition of PI3K and recruit it to the inner surface of the plasma membrane, where PI3K activity is modulated further by RasGTP and SFKs (Src family kinases) [4,9]. Activated class I PI3Ks generate PtdIns(3,4,5)P3, a second messenger, which is subsequently converted into PtdIns(3,4)P2 or PtdIns(4,5)P2 by PI5Ps (phosphoinositide 5-phosphatases) or PI3Ps (phosphoinositide 3-phosphatases) SHIP (SH2-domain-containing inositol phosphatase) 2, PTEN (phosphatase and tensin homologue deleted on chromosome 10) and others .
Generation of membrane-associated phospholipids recruits a battery of signalling molecules that have lipid-binding domains, such as the PH domain, including non-receptor tyrosine kinases (e.g. Btk family kinases), GEFs (e.g. Vav, Tiam-1 and SOS), GAPs (GTPase-activating proteins) (e.g. RasGAP), lipid kinases [e.g. PLCγ (phospholipase Cγ)], adaptor/docking/scaffolding proteins [e.g. IRS (insulin receptor substrate), Grb7 and intersectin families] and serine/threonine kinases such as PDK1 (phosphoinositide-dependent kinase 1) and PKB (protein kinase B)/Akt [10–12]. PDK1 acts upstream of a major downstream effector of PI3K (Akt), PKC (protein kinase C) isoforms, SGK (serum- and glucocorticoid-induced protein kinase) and p70S6K (p70 ribosomal S6 kinase) [11,13]. In addition, PDK1 activates PAK (p21-activated kinase) 1  and PKN (protein kinase N)/PRK (PKC-related kinase) that are effectors of Rho family GTPases. As described below, the phospholipid-mediated recruitment of multiple proteins not only facilitates signal propagation through the PI3K/Akt pathway, but also affects complex processes of Ras and Raf activation, thereby creating another point of PI3K→Ras/MAPK cross-talk.
Full Akt activation requires dual phosphorylation on Ser473 by mTORC2 [mTOR (mammalian target of rapamycin)–rictor (rapamycin-insensitive companion of mTOR) complex], ILK-1 (integrin-linked kinase 1) or DNA-PK (DNA-dependent protein kinase) and on Thr308 by PDK1 . Active Akt dissociates from the membrane and translocates to the cytoplasm and the nucleus where it phosphorylates multiple target proteins, implicated in the regulation of apoptosis, DNA repair, metabolism, protein synthesis and cell division [3,4,13,16]. Activation of Akt stimulates angiogenesis and induces epithelial–mesenchymal transition characterized by the morphological changes, production of metalloproteinases, loss of cell–cell adhesion and increased invasive cell migration [3,16,17].
Cross-talk between Ras/MAPK and PI3K/Akt signalling pathways
Traditional schemes of growth factor, hormone and cytokine receptor signalling networks display PI3K/Akt and Ras/MAPK as two independent parallel pathways. However, there are multiple cross-talk points between these two pathways, whose co-ordinated action determines the cell fate (Figure 1B). There are 802 interactive proteins involved in PI3K-mediated signalling  and more than 2000 interactions related to the MAPK family kinases , where at least 284 proteins are the components of endogenous ERK1 complexes . In view of such broad interactomes, it is not surprising that the PI3K/Akt and Ras/MAPK pathways influence each other at different stages of signal propagation, both negatively and positively, resulting in dynamic and complex cross-talk.
PI3K-mediated regulation of ERK responses
PI3K affects MAPK signalling at multiple nodes of the Ras/ERK pathway (Figure 2). A major cross-talk node is at the level of PtdIns(3,4,5)P3-mediated RasGEF and RasGAP signalling. Generation of PtdIns(3,4,5)P3 by PI3K induces the plasma membrane recruitment of the PH-domain-containing GAB (Grb2-associated binding partner), IRS and Grb7 scaffolding proteins, which are subsequently phosphorylated on multiple tyrosine residues by membrane receptors and non-receptor tyrosine kinases. Tyrosine-phosphorylated GAB and IRS bind p85 PI3K, thereby bringing additional PI3K molecules to the plasma membrane and creating positive feedback [21,22]. Phosphorylated GAB interacts with a number of molecules, including Shc, PLCγ, Grb2, Crk, SHIP, STAT (signal transducer and activator of transcription) 3, STAT5, c-Src, ERK and SHP2 (SH2-domain-containing tyrosine phosphatase 2) . The recruitment of Grb2–SOS complexes to the plasma membrane by GAB, IRS and SHP2 amplifies Ras activation [24–26]. GAB1 association with SHP2 increases SHP2 phosphatase activity, manifested by dephosphorylation of SHP2 substrates, such as RasGAP, c-Src (on the inhibitory Tyr527 site) and the binding partners of c-Src-inactivating kinase CSK (C-terminal Src kinase). Resulting activation of c-Src and Ras leads to up-regulation of ERK signalling [23,27].
Other PI3K→ERK cross-talk nodes include PI3K-induced Raf and MEK stimulation, although some of these interactions also amplify Ras signalling. Positive PI3K/GAB/PI3K feedback further increases PtdIns(3,4,5)P3 production and stimulates the Rac/Cdc42/PAK signalling pathway [3,28,29]. PAK then phosphorylates Raf on Ser388, which is required for Raf activation, and also increases Raf association with MEK [30,31]. In addition, PAK-mediated MEK1 phosphorylation on Ser289 increases the association between MEK1 and ERK2 .
Similarly, Grb7 is recruited to the plasma membrane in a PI3K-dependent manner. Subsequent association of Grb7 (via the SH2 domain) with activated FAK (focal adhesion kinase) or with RasGTP [via the RA (Ras-associating) domain of Grb7], promotes Rac/Cdc42/PAK and MAPK signalling [33,34]. Interactions with tyrosine-phosphorylated receptors, GAB1, PtdIns(3,4,5)P3 and Rac GTPases activate PLCγ . PLCγ produces the second messenger DAG (diacylglycerol), leading to the activation of PKC isoforms that influence the MAPK/ERK cascade at the levels of Raf, MEK and ERK [36–38]. PDK1 can increase the MAPK/ERK responses by activation of PKCs  and PAK1 . In addition, PDK1 phosphorylates MEK on Ser222 and Ser226, which is essential for full activation .
Whereas cross-talk interactions resulting from PI3K activation and mediated by GAB/IRS/Grb7, PAK and PDK1 activate the Ras/MAPK pathway, Akt and its downstream effectors, mTOR and p70S6K, negatively affect ERK signalling (Figure 2). Upstream or at the level of Ras, a negative-feedback control occurs via serine phosphorylation of GAB and IRS proteins, resulting in a decrease of their tyrosine phosphorylation levels and impaired ability to sustain or amplify ERK phosphorylation [21,40,41]. In addition, Akt-dependent phosphorylation of c-Raf on Ser259 and Rac/Cdc42 on Ser71 can interfere with Raf membrane recruitment and Raf activation by PAK respectively [42,43].
Interestingly, Akt-mediated transcriptional controls of ERK activation can be positive. This regulation involves multiple protein phosphatases (MKPs) that dephosphorylate ERK. MKP expression and activities are positively regulated by ERK, p38 MAPK and GSK3 (glycogen synthase kinase 3) [44,45]. Suppression of p38 MAPK and GSK3 by Akt [4,46] down-regulates MKP expression and can therefore prolong the period of time when ERK is phosphorylated.
ERK-mediated regulation of the PI3K/Akt pathway
Active ERK can influence the PI3K/Akt pathway via several interaction routes (Figure 2). One mechanism involves the modulation of the tyrosine phosphorylation level and/or half-life of GAB and IRS scaffolding proteins by ERK-mediated phosphorylation at serine and threonine residues. For instance, ERK phosphorylates GAB1 on several serine residues that are adjacent to p85 PI3K-binding sites (three YXXM motifs) . Depending on the type of stimulation, this results in decreased (e.g. EGF signalling) or increased [e.g. HGF (hepatocyte growth factor) signalling] levels of GAB–p85 PI3K complexes, which correlates with PI3K activity due to positive PI3K–PtdIns(3,4,5)P3–GAB–PI3K feedback [21,48–50]. Likewise, IRS-1 and FRS (fibroblast growth factor receptor substrate)-2 phosphorylation on serine and threonine residues mediated by ERK or its downstream kinases, such as p70S6K, decreases their binding to p85 PI3K and thereby decreases PI3K activity by disrupting positive feedback [51,52].
Both ERK and its kinase substrate p90RSK (90 kDa ribosomal protein S6 kinase) phosphorylate and inhibit GSK3 . Since GSK3 is a negative regulator of PI3K antagonist PTEN , activation of ERK alleviates PTEN inhibition, thereby decreasing PtdIns(3,4,5)P3 levels. This disrupts positive feedback loops via PtdIns(3,4,5)P3-binding proteins, thereby decreasing PI3K activity. Additional cross-talk mechanisms include FAK dephosphorylation on Tyr397 following ERK-mediated FAK phosphorylation on Ser910 . This can disrupt the formation of the FAK complexes with p85 PI3K, c-Src, Grb7 and Grb2, and eventually decrease the activation levels of c-Src, PI3K, Rac/Cdc42/PAK and Ras/MAPK [9,34].
Whereas ERK-mediated phosphorylation of GAB/IRS/FRS scaffolding proteins and kinase substrates serves as negative-feedback loops to PI3K, RasGTP-induced stimulation of PI3K creates a positive growth-factorinduced loop [4,9]. The E3 ubiquitin ligase and multifunctional scaffolding protein c-Cbl can also serve as a point of cross-talk between PI3K/Akt and Ras/MAPK pathways (Figure 2). Serine/threonine phosphorylation of c-Cbl by PKCs  inhibits c-Cbl tyrosine phosphorylation, thus affecting its interactions with numerous SH2-domain-containing proteins . This can result in delayed ubiquitylation and proteasomal degradation of receptors and other c-Cbl-associated-proteins (e.g. PI3K, Vav and SFKs), consequently enhancing Ras/ERK signalling. On the other hand, c-Cbl binding to both the SH2 and SH3 domains of p85 subunit of PI3K facilitates PI3K activation [57,58]. This process may be hindered due to c-Cbl sequestration by Sprouty proteins , whose expression levels, in turn, are positively regulated by ERK .
The experimental findings reviewed above suggest that, in most cellular systems, PI3K positively regulates the Ras/MAPK cascade, facilitating maximal ERK responses to physiological stimuli, whereas activated ERK, in turn, negatively controls the PI3K/Akt pathway [21,48] (Figure 3). Yet, multiple signalling routes and nodes involved in Ras/MAPK→PI3K/Akt cross-talk make it context-dependent, and, in some cells, activation of Raf, MEK and/or ERK is enhanced by PI3K inhibition [61–63], and PI3K activity is decreased following MAPK inhibition . Experimental data and mathematical modelling demonstrate that cross-talk depends dramatically on the concentrations of growth factors and the levels of receptors and scaffolding proteins, such as GAB and IRS [21,64].
For instance, at saturating EGF doses, PI3K inhibition by wortmannin only slightly attenuates ERK phosphorylation in A431 human epidermoid carcinoma and MCF7 human mammary carcinoma cells (over the 2–60 min response period), whereas PI3K inhibition dramatically decreases ERK phosphorylation at low physiological EGF doses (Figure 3A, top and middle panels). In MCF10A normal mammary epithelial cells stimulated with moderate EGF doses, wortmannin decreases ERK phosphorylation in the early and late time points of signal propagation, but the peak of activation (7.5–10 min) does not differ from that of control cells (Figure 3A, bottom panel). This decreasing sensitivity of ERK phosphorylation to PI3K inhibition with increasing EGF doses is also observed in HEK (human embryonic kidney)-293, HeLa, T47D, BT-474 and other cell lines (Figure 3B and E. Aksamitiene, unpublished work). Thus the positive control exerted by PI3K activation on the Ras/MAPK pathway is most effective at low EGF doses, and these findings are also supported by computational modelling [21,64] (Figures 3D and 3E).
In contrast, the negative influence of the Ras/MAPK pathway on the PI3K/Akt pathway is more pronounced at high EGF doses (Figure 3D). In fact, inhibition of ERK phosphorylation by the same concentration of the specific MEK inhibitor U0126 causes larger increases in Akt activity at higher EGF doses  (Figure 3C). Importantly, the Ras/MAPK→PI3K/Akt cross-talk effects depend on both signal strength (EGF dose) and the stimulation time. Thus the analysis of the entire time-course of ERK and Akt activation at various signal strength conditions and respective inhibitors is required to characterize interactions between the pathways. Judging cross-talk on the basis of a single EGF dose and one time point can be misleading, since it might suggest that the Ras/MAPK pathway is uncoupled from the PI3K/Akt pathway (Figure 3). However, in some cases, cross-talk cannot be detected due to activating mutations within different signalling branches. For example, wortmannin treatment does not suppress EGF-induced ERK phosphorylation in Ras mutant PL-5, A549 and T24 cells (Figure 3B and E. Aksamitiene, unpublished work).
Pathway cross-talk endows a cell with emerging capabilities for signal processing and decoding, thereby adapting the cellular behaviours to the combinatorial variety of external cues and conditions. In the present paper, we have shown that interactions between mitogenic (Ras/ERK) and survival (PI3K/Akt) pathways, which involve multiple signalling nodes and routes, generate context-dependent responses to growth factor stimulation. Cross-talk changes the dynamic topologies of signal propagation networks downstream of cell-surface receptors, leading to amplification or attenuation of key target protein activities. It was recently shown that Ras/ERK→PI3K/Akt cross-talk can mediate insulin–EGF interactions, leading to amplification of mitogenic signalling by insulin at physiological concentrations of growth factors . Importantly, cross-talk leads to activation of compensatory signalling, allowing cancer cells to evade apoptosis if only the ERK cascade or the PI3K pathway are targeted therapeutically. Combined inhibition of PI3K and MAPK proved to be more efficient in suppressing cancer cell growth and viability than targeting the components of each pathway alone [46,62].
Our work is supported by Science Foundation Ireland [grant number 06/CE/B1129] and the National Institutes of Health [grant number GM059570].
Signalling 2011: a Biochemical Society Centenary Celebration: A Biochemical Society Focused Meeting held at the University of Edinburgh, U.K., 8–10 June 2011. Organized and Edited by Nicholas Brindle (Leicester, U.K.), Simon Cook (The Babraham Institute, U.K.), Jeff McIlhinney (Oxford, U.K.), Simon Morley (University of Sussex, U.K.), Sandip Patel (University College London, U.K.), Susan Pyne (University of Strathclyde, U.K.), Colin Taylor (Cambridge, U.K.), Alan Wallace (AstraZeneca, U.K.) and Stephen Yarwood (Glasgow, U.K.).
Abbreviations: EGF, epidermal growth factor; ERK, extracellular-signal-related kinase; FAK, focal adhesion kinase; FRS, fibroblast growth factor receptor substrate; GAP, GTPase-activating protein; GEF, guanine-nucleotide-exchange factor; Grb, growth-factor-receptor-bound protein; GAB, Grb2-associated binding partner; GSK3, glycogen synthase kinase 3; IRS, insulin receptor substrate; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; MKP, MAPK phosphatase; mTOR, mammalian target of rapamycin; p70S6K, p70 ribosomal S6 kinase; PAK, p21-activated kinase; PDK1, phosphoinositide-dependent kinase 1; PH, pleckstrin homology; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PLCγ, phospholipase Cγ; PTEN, phosphatase and tensin homologue deleted on chromosome 10; SFK, Src family kinase; SH, Src homology; Shc, Src homology and collagen protein; SHIP, SH2-domain-containing inositol phosphatase; SHP2, SH2-domain-containing tyrosine phosphatase 2; SOS, Son of Sevenless; STAT, signal transducer and activator of transcription
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