A Computer‐Based Model for the Regulation of Mitogen Activated Protein Kinase (MAPK) Activation

Abstract

Computer simulations and mathematical modeling of biological processes are becoming increasingly popular, and yet the complexity of the biochemical systems or the differences between experimental setups make it very difficult to establish a standard formula for these modeling projects. Before we can start using computer‐based models for predictions or targeted experiment designs, it is very important to establish a reliable model on which those predictions can be based and experimentally tested. Here we attempt to present a computer model for the mitogen‐activated protein kinase (MAPK) signaling cascade which is consistent with previously published experimental results. In this study we have focused our attention to a generic MAPK ERK (extracellular signal‐regulated kinase) pathway activated by epidermal growth factor (EGF) in an attempt to understand how receptors may achieve different activation kinetics of the MAPK signaling. We successfully show that the level of receptor expression is one key determinant in this regulation, and that the binding affinity of the active receptor to adaptor proteins can have a small but albeit direct effect on the downstream activation.     

Integrins regulate the linkage between upstream and downstream events in G protein-coupled receptor signaling to mitogen-activated protein kinase

Receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) can both activate mitogen-activated protein kinase (MAPK), a critical intermediate in the transduction of proliferative signals. Numerous observations have demonstrated that integrin-mediated cell anchorage can regulate the efficiency of signaling from RTKs to MAPK. Recently, a relationship between integrins and GPCR signaling has also emerged; however, little is understood concerning the mechanisms involved. Here, we investigate integrin regulation of GPCR signaling to MAPK, focusing on the P2Y class of GPCRs that function through activation of phospholipase Cbeta. P2Y receptor signaling to the downstream components mitogen-activated protein kinase kinase and MAPK is highly dependent on integrin-mediated cell anchorage. However, activation of upstream events, including inositol phosphate production and generation of calcium transients, is completely independent of cell anchorage. This indicates that integrins regulate the linkage between upstream and downstream events in this GPCR pathway, just as they do in some aspects of RTK signaling. However, the P2Y pathway does not involve cross-activation of a RTK, nor a role for Shc or c-Raf; thus, it is quite distinct from the classical RTK-Ras-Raf-MAPK cascade. Rather, integrin-modulated P2Y receptor stimulation of MAPK depends on calcium and on the activation of protein kinase C.     

Analysis of CD95 threshold signaling: triggering of CD95 (FAS/APO-1) at low concentrations primarily results in survival signaling

Recently we generated a mathematical model (Bentele, M., Lavrik, I., Ulrich, M., Stosser, S., Heermann, D. W., Kalthoff, H., Krammer, P. H., and Eils, R. (2004) J. Cell Biol. 166, 839-851) of signaling in CD95(Fas/APO-1)-mediated apoptosis. Mathematical modeling in combination with experimental data provided new insights into CD95-mediated apoptosis and allowed us to establish a threshold mechanism of life and death. Here, we further assessed the predictability of the model experimentally by a detailed analysis of the threshold behavior of CD95 signaling. Using the model predictions for the mechanism of the threshold behavior we found that the CD95 DISC (death-inducing signaling complex) is formed at the cell membrane upon stimulation with low concentrations of agonistic anti-APO-1 monoclonal antibodies; however, activation of procaspase-8 at the DISC is blocked due to high cellular FLICE-inhibitory protein recruitment into the DISC. Given that death signaling does not occur upon CD95 stimulation at low (threshold) anti-APO-1 concentrations, we also analyzed survival signaling, focusing on mitogen-activated protein kinase activation. Interestingly, we found that mitogen-activated protein kinase activation takes place under threshold conditions. These findings show that triggering of CD95 can signal both life or death, depending on the strength of the stimulus.     

Cross Talk between β-Adrenergic and Bradykinin B2 Receptors Results in Cooperative Regulation of Cyclic AMP Accumulation and Mitogen-Activated Protein Kinase Activity

Costimulation of G protein-coupled receptors (GPCRs) may result in cross talk interactions between their downstream signaling pathways. Stimulation of GPCRs may also lead to cross talk regulation of receptor tyrosine kinase signaling and thereby to activation of mitogen-activated protein kinase (MAPK). In COS-7 cells, we investigated the interactions between two particular mitogenic receptor pathways, the endogenously expressed beta-adrenergic receptor (beta-AR) and the transiently transfected human bradykinin (BK) B(2) receptor (B(2)R). When beta-AR and B(2)R are costimulated, we found two different cross talk mechanisms. First, the predominantly G(q) protein-coupled B(2)R is enabled to activate a G(i) protein and, subsequently, type II adenylate cyclase. This results in augmentation of beta-AR-mediated cyclic AMP (cAMP) accumulation by BK, which alone is unable to increase the cAMP level. Second, independently of BK-induced superactivation of the cAMP system, costimulation of beta-AR leads to protein kinase A-mediated blockade of phospholipase C activation by BK. Thereby, the pathway from B(2)R to MAPK, which essentially involves protein kinase C activation, is selectively switched off. The MAPK activation in response to isoproterenol was not affected due to costimulation. Furthermore, in the presence of isoproterenol, BK lost its ability to stimulate DNA synthesis in COS-7 cells. Thus, our findings might establish a novel paradigm: cooperation between simultaneously activated mitogenic pathways may prevent multiple stimulation of MAPK activity and increased cell growth.     

Angiotensin II activation of focal adhesion kinase and pp60c-Src in relation to mitogen-activated protein kinases in hepatocytes

We have investigated signaling pathways leading to angiotensin II (Ang II) activation of mitogen-activated protein kinase (MAPK) in hepatocytes. MAPK activation by Ang II was abolished by the Ang II type 1 (AT1) receptor antagonist losartan, but not by the Ang II type 2 (AT2) receptor antagonist PD123319. Ang II (100 nM) induced a rapid phosphorylation of Src (peak approximately 2 min) and focal adhesion kinase (FAK, peak approximately 5 min) followed by a decrease to basal levels in 30 min. An increased association between FAK and Src in response to Ang II was detected after 1 min, which declined to basal levels after 30 min. Treatment with the Src kinase inhibitor PP-1 inhibited FAK phosphorylation. Downregulation of PKC, intracellular Ca2+ chelator BAPTA or inhibitors of PKC, Src kinase, MAPK kinase (MEK), Ca2+/calmodulin dependent protein kinase, phosphatidylinositol 3-kinase all blocked Ang II-induced MAPK phosphorylation. In contrast to other cells, there was no evidence for the role of EGF receptor transactivation in the activation of MAPK by Ang II. However, PDGF receptor phosphorylation is involved in the Ang II stimulated MAPK activation. Furthermore, Src/FAK and Ca/CaM kinase activation serve as potential links between the Ang II receptor and MAPK activation. These studies offer insight into the signaling network upstream of MAPK activation by AT1 receptor in hepatocytes.     

High density lipoprotein-induced signaling of the MAPK pathway involves scavenger receptor type BI-mediated activation of Ras

High density lipoprotein (HDL) stimulates multiple signaling pathways. HDL-induced activation of the mitogen-activated protein kinase (MAPK) pathway can be mediated by protein kinase C (PKC) and/or pertussis toxin-sensitive G-proteins. Although HDL-induced activation of MAPK involves Raf-1, Mek, and Erk1/2, the upstream contribution of p21(ras) (Ras) on the activation of Raf-1 and MAPK remains elusive. Here we examine the effect of HDL on Ras activity and demonstrate that HDL induces PKC-independent activation of Ras that is completely blocked by pertussis toxin, thus implicating heterotrimeric G-proteins. In addition, the HDL-induced activation of Ras is inhibited by a neutralizing antibody against scavenger receptor type BI. We conclude that the binding of HDL to scavenger receptor type BI activates Ras in a PKC-independent manner with subsequent induction of the MAPK signaling cascade.     

Mechanisms and Constraints on Yeast MAPK Signaling Specificity

The survival of cells relies on their ability to respond specifically to diverse environmental signals. Surprisingly, intracellular signaling pathways often share the same or homologous protein components, yet undesirable crosstalk is, in general, suppressed. This signaling specificity has been well studied in the yeast model system Saccharomyces cerevisiae, where the mitogen-activated protein kinase (MAPK) cascades are repeatedly employed in mediating distinct biological processes including pheromone-induced mating and filamentous growth under starvation. Although various mechanisms have been proposed to interpret the yeast MAPK signaling specificity, a consistent theory is still lacking. Here, we present a mathematical model that shows signaling specificity can arise through asymmetric hierarchical inhibition. The parameters of our model are, where possible, based on experimental data that allow us to determine the constraints imposed by signaling specificity on these parameters. Our model is in broad agreement with experimental observations to date and generates testable predictions that may stimulate further research.     

The human sef-a isoform utilizes different mechanisms to regulate receptor tyrosine kinase signaling pathways and subsequent cell fate

Negative feedback is among the key mechanisms for regulating receptor tyrosine kinase (RTK) signaling. Human Sef, a recently identified inhibitor of RTK signaling, encodes different isoforms, including a membrane spanning (hSef-a) and a cytosolic (hSef-b) isoform. Previously, we reported that hSef-b inhibited fibroblast proliferation and prevented the activation of mitogen-activated protein kinase (MAPK), without affecting protein kinase B/Akt or p38 MAPK. Conflicting results were reported concerning hSef-a inhibition of MAPK activation, and the effect of hSef-a on other RTK-induced signaling pathways is unknown. Here we show that, in fibroblasts, similar to hSef-b, ectopic expression of hSef-a inhibited fibroblast growth factor-induced cell proliferation. Unlike hSef-b, however, the growth arrest was mediated via a MAPK-independent mechanism, and was accompanied by elevated p38 MAPK phosphorylation and inhibition of protein kinase B/Akt. In addition, hSef-a, but not hSef-b, mediated apoptosis in fibroblast growth factor-stimulated cells. Chemical inhibitor of p38 MAPK abrogated the effect of hSef-a on apoptosis. In epithelial cells, ectopic expression of hSef-a inhibited the activation of MAPK, whereas down-regulation of endogenous hSef-a significantly increased MAPK activation and accelerated growth factor-dependent cell proliferation. These results indicate that hSef-a is a multifunctional negative modulator of RTK signaling and clearly demonstrate that hSef-a can inhibit the activation of MAPK, although in a cell type-specific manner. Moreover, the differences between the activities of hSef-a and hSef-b suggest that hSef isoforms can control signal specificity and subsequent cell fate by utilizing different mechanisms to modulate RTK signaling.     

Vav transformation requires activation of multiple GTPases and regulation of gene expression

Although Vav can act as a guanine nucleotide exchange factor for RhoA, Rac1, and Cdc42, its transforming activity has been ascribed primarily to its ability to activate Rac1. However, because activated Vav, but not Rac-specific guanine nucleotide exchange factors, exhibits very potent focus-forming transforming activity when assayed in NIH 3T3 cells, Vav transforming activity must also involve activation of Rac-independent pathways. In this study, we determined the involvement of other Rho family proteins and their signaling pathways in Vav transformation. We found that RhoA, Rac1, and Cdc42 functions are all required for Vav transforming activity. Furthermore, we determined that Vav activation of nuclear factor-kappaB and the Jun NH2-terminal kinase mitogen-activated protein kinase (MAPK) is necessary for full transformation by Vav, whereas p38 MAPK does not seem to play an important role. We also determined that Vav is a weak activator of Elk-1 via a Ras- and MAPK/extracellular signal-regulated kinase kinase-dependent pathway, and this activity was essential for Vav transformation. Thus, we conclude that full Vav transforming activation is mediated by the activation of multiple small GTPases and their subsequent activation of signaling pathways that regulate changes in gene expression. Because Vav is activated by the epidermal growth factor receptor and other tyrosine kinases involved in cancer development, defining the role of aberrant Vav signaling may identify activities of receptor tyrosine kinases important for human oncogenesis.     

An activating mutation in sos-1 identifies its Dbl domain as a critical inhibitor of the epidermal growth factor receptor pathway during Caenorhabditis elegans vulval development

Proper regulation of receptor tyrosine kinase (RTK)-Ras-mitogen-activated protein kinase (MAPK) signaling pathways is critical for normal development and the prevention of cancer. SOS is a dual-function guanine nucleotide exchange factor (GEF) that catalyzes exchange on Ras and Rac. Although the physiologic role of SOS and its CDC25 domain in RTK-mediated Ras activation is well established, the in vivo function of its Dbl Rac GEF domain is less clear. We have identified a novel gain-of-function missense mutation in the Dbl domain of Caenorhabditis elegans SOS-1 that promotes epidermal growth factor receptor (EGFR) signaling in vivo. Our data indicate that a major developmental function of the Dbl domain is to inhibit EGF-dependent MAPK activation. The amount of inhibition conferred by the Dbl domain is equal to that of established trans-acting inhibitors of the EGFR pathway, including c-Cbl and RasGAP, and more than that of MAPK phosphatase. In conjunction with molecular modeling, our data suggest that the C. elegans mutation, as well as an equivalent mutation in human SOS1, activates the MAPK pathway by disrupting an autoinhibitory function of the Dbl domain on Ras activation. Our work suggests that functionally similar point mutations in humans could directly contribute to disease.     

Caspase-8 activity has an essential role in CD95/Fas-mediated MAPK activation

Stimulation of CD95/Fas/APO-1 results in the induction of both apoptotic and non-apoptotic signaling pathways. The processes regulating these two opposing pathways have not been thoroughly elucidated to date. In this study, using quantitative immunoblots, imaging, and mathematical modeling, we addressed the dynamics of the DED proteins of the death-inducing signaling complex (DISC), procaspase-8, and cellular FLICE inhibitory proteins (c-FLIPs) to the onset of CD95-mediated ERK1/2 and p38 mitogen-activated protein kinase (MAPK) activation. We found that CD95 DISC-induced caspase-8 activity is important for the initiation of ERK1/2 and p38 MAPK activation. The long c-FLIP isoform, c-FLIP_L, and the short c-FLIP isoform, c-FLIP_R, inhibited MAPK induction by blocking caspase-8 processing at the DISC. Furthermore, we built a mathematical model describing CD95 DISC-mediated MAPK activation and apoptosis. The model quantitatively defined the dynamics of DED proteins, procaspase-8, and c-FLIP, which lead to caspase-8 activation and induction of apoptotic and non-apoptotic signaling pathways. In conclusion, the combination of biochemical analysis with mathematical modeling provides evidence for an important role of caspase-8 in CD95-mediated activation of MAPKs, while c-FLIP exerts a regulatory function in this process.     

Inhibition of Mitogen-Activated Kinase Signaling Sensitizes HeLa Cells to Fas Receptor-Mediated Apoptosis

The Fas receptor (FasR) is an important physiological mediator of apoptosis in various tissues and cells. However, there are also many FasR-expressing cell types that are normally resistant to apoptotic signaling through this receptor. The mitogen-activated protein kinase (MAPK) signaling cascade has, apart from being a growth-stimulating factor, lately received attention as an inhibitory factor in apoptosis. In this study, we examined whether MAPK signaling could be involved in protecting FasR-insensitive cells. To this end, we used different approaches to inhibit MAPK signaling in HeLa cells, including treatment with the MAPK kinase inhibitor PD 98059, serum withdrawal, and expression of dominant-interfering MAPK kinase mutant protein. All of these treatments were effective in sensitizing the cells to FasR-induced apoptosis, demonstrating that MAPK indeed is involved in the control of FasR responses. The MAPK-mediated control seemed to occur at or upstream of caspase 8, the initiator caspase in apoptotic FasR responses. Transfection with the constitutively active MAPK kinase abrogated FasR-induced apoptosis also in the presence of cycloheximide, indicating that the MAPK-generated suppression of FasR-mediated apoptotic signaling is protein synthesis independent. In cells insensitive to FasR-induced apoptosis, stimulation of the FasR with an agonistic antibody resulted in significant MAPK activation, which was inhibited by PD 98059. When different cell types were compared, the FasR-mediated MAPK activation seemed proportional to the degree of FasR insensitivity. These results suggest that the FasR insensitivity is likely to be a consequence of FasR-induced MAPK activation, which in turn interferes with caspase activation.     

Autocrine loops with positive feedback enable context-dependent cell signaling

We describe a mechanism for context-dependent cell signaling mediated by autocrine loops with positive feedback. We demonstrate that the composition of the extracellular medium can critically influence the intracellular signaling dynamics induced by extracellular stimuli. Specifically, in the epidermal growth factor receptor (EGFR) system, amplitude and duration of mitogen-activated protein kinase (MAPK) activation are modulated by the positive-feedback loop formed by the EGFR, the Ras-MAPK signaling pathway, and a ligand-releasing protease. The signaling response to a transient input is short-lived when most of the released ligand is lost to the cellular microenvironment by diffusion and/or interaction with an extracellular ligand-binding component. In contrast, the response is prolonged or persistent in a cell that is efficient in recapturing the endogenous ligand. To study functional capabilities of autocrine loops, we have developed a mathematical model that accounts for ligand release, transport, binding, and intracellular signaling. We find that context-dependent signaling arises as a result of dynamic interaction between the parts of an autocrine loop. Using the model, we can directly interpret experimental observations on context-dependent responses of autocrine cells to ionizing radiation. In human carcinoma cells, MAPK signaling patterns induced by a short pulse of ionizing radiation can be transient or sustained, depending on cell type and composition of the extracellular medium. On the basis of our model, we propose that autocrine loops in this, and potentially other, growth factor and cytokine systems may serve as modules for context-dependent cell signaling.     

Interaction of the CC-chemokine RANTES with glycosaminoglycans activates a p44/p42 mitogen-activated protein kinase-dependent signaling pathway and enhances human immunodeficiency virus type 1 infectivity

The interaction of the CC-chemokine RANTES with its cell surface receptors transduces multiple intracellular signals: low concentrations of RANTES (1 to 10 nM) stimulate G-protein-coupled receptor (GPCR) activity, and higher concentrations (1 microM) activate a phosphotyrosine kinase (PTK)-dependent pathway. Here, we show that the higher RANTES concentrations induce rapid tyrosine phosphorylation of multiple proteins. Several src-family kinases (Fyn, Hck, Src) are activated, as is the focal adhesion kinase p125 FAK and, eventually, members of the p44/p42 mitogen-activated protein kinase (MAPK) family. This PTK signaling pathway can be activated independently of known seven-transmembrane GPCRs for RANTES because it occurs in cells that lack any such RANTES receptors. Instead, activation of the PTK signaling pathway is dependent on the expression of glycosaminoglycans (GAGs) on the cell surface, in that it could not be activated by RANTES in GAG-deficient cells. We have previously demonstrated that RANTES can both enhance and inhibit infection of cells with human immunodeficiency virus type 1 (HIV-1). Here we show that activation of both PTK and MAPK is involved in the enhancement of HIV-1 infectivity caused by RANTES in cells that lack GPCRs for RANTES but which express GAGs.