Jason M. Haugh

Internalized Epidermal Growth Factor Receptors Participate in the Activation of p21 ras in Fibroblasts

Regulated activation of the highly conserved Ras GTPase is a central event in the stimulation of cell proliferation, motility, and differentiation elicited by receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR). In fibroblasts, this involves formation and membrane localization of Shc·Grb2·Sos complexes, which increases the rate of Ras guanine nucleotide exchange. In order to control Ras-mediated cell responses, this activity is regulated by receptor down-regulation and a feedback loop involving the dual specificity kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK). We investigated the role of EGFR endocytosis in the regulation of Ras activation. Of fundamental interest is whether activated receptors in endosomes can participate in the stimulation of Ras guanine nucleotide exchange, because the constitutive membrane localization of Ras may affect its compartmentalization. By exploiting the differences in postendocytic signaling of two EGFR ligands, epidermal growth factor and transforming growth factor-α, we found that activated EGFR located at the cell surface and in internal compartments contribute equally to the membrane recruitment and tyrosine phosphorylation of Shc in NR6 fibroblasts expressing wild-type EGFR. Importantly, both the rate of Ras-specific guanine nucleotide exchange and the level of Ras-GTP were depressed to near basal values on the time scale of receptor trafficking. Using the selective MEK inhibitor PD098059, we were able to block the feedback desensitization pathway and maintain activation of Ras. Under these conditions, the generation of Ras-GTP was not significantly affected by the subcellular location of activated EGFR. In conjunction with our previous analysis of the phospholipase C pathway in the same cell line, this suggests a selective continuation of specific signaling activities and cessation of others upon receptor endocytosis.

Effect of Epidermal Growth Factor Receptor Internalization on Regulation of the Phospholipase C-γ1 Signaling Pathway

The epidermal growth factor receptor (EGFR) ligands, epidermal growth factor (EGF), and transforming growth factor-α (TGFα) elicit differential postendocytic processing of ligand and receptor molecules, which impacts long-term cell signaling outcomes. These differences arise from the higher affinity of the EGF-EGFR interaction versus that of TGFα-EGFR in the acidic conditions of sorting endosomes. To determine whether EGFR occupancy in endosomes might also affect short-term signaling events, we examined activation of the phospholipase C-γ1 (PLC-γ1) pathway, an event shown to be essential for growth factor-induced cell motility. We found that EGF continues to stimulate maximal tyrosine phosphorylation of EGFR following internalization, while, as expected, TGFα stimulates markedly less. The resulting higher level of receptor activation by EGF, however, did not yield higher levels of phosphatidylinositol (4,5)-bisphosphate (PIP2) hydrolysis over those stimulated by TGFα. By altering the ratio of activated receptors between the cell surface and the internalized compartment, we found that only cell surface receptors effectively participate in PLC function. In contrast to PIP2 hydrolysis, PLC-γ1 tyrosine phosphorylation correlated linearly with the total level of Tyr(P)-EGFR stimulated by either ligand, indicating that the functional deficiency of internal EGFR cannot be attributed to an inability to interact with and phosphorylate signaling proteins. We conclude that EGFR signaling through the PLC pathway is spatially restricted at a point between PLC-γ1 phosphorylation and PIP2 hydrolysis, perhaps because of limited access of EGFR-bound PLC-γ1 to its substrate in endocytic trafficking organelles.

Quantitative elucidation of a distinct spatial gradient-sensing mechanism in fibroblasts.

Migration of eukaryotic cells toward a chemoattractant often relies on their ability to distinguish receptor-mediated signaling at different subcellular locations, a phenomenon known as spatial sensing. A prominent example that is seen during wound healing is fibroblast migration in platelet-derived growth factor (PDGF) gradients. As in the well-characterized chemotactic cells Dictyostelium discoideum and neutrophils, signaling to the cytoskeleton via the phosphoinositide 3-kinase pathway in fibroblasts is spatially polarized by a PDGF gradient; however, the sensitivity of this process and how it is regulated are unknown. Through a quantitative analysis of mathematical models and live cell total internal reflection fluorescence microscopy experiments, we demonstrate that PDGF detection is governed by mechanisms that are fundamentally different from those in D. discoideum and neutrophils. Robust PDGF sensing requires steeper gradients and a much narrower range of absolute chemoattractant concentration, which is consistent with a simpler system lacking the feedback loops that yield signal amplification and adaptation in amoeboid cells.

Kinetic Analysis of Platelet-derived Growth Factor Receptor/Phosphoinositide 3-Kinase/Akt Signaling in Fibroblasts

Isoforms of the serine-threonine kinase Akt coordinate multiple cell survival pathways in response to stimuli such as platelet-derived growth factor (PDGF). Activation of Akt is a multistep process, which relies on the production of 3′-phosphorylated phosphoinositide (PI) lipids by PI 3-kinases. To quantitatively assess the kinetics of PDGF receptor/PI 3-kinase/Akt signaling in fibroblasts, a systematic study of this pathway was performed, and a mechanistic mathematical model that describes its operation was formulated. We find that PDGF receptor phosphorylation exhibits positive cooperativity with respect to PDGF concentration, and its kinetics are quantitatively consistent with a mechanism in which receptor dimerization is initially mediated by the association of two 1:1 PDGF/PDGF receptor complexes. Receptor phosphorylation is transient at high concentrations of PDGF, consistent with the loss of activated receptors upon endocytosis. By comparison, Akt activation responds to lower PDGF concentrations and exhibits more sustained kinetics. Further analysis and modeling suggest that the pathway is saturated at the level of PI 3-kinase activation, and that the p110α catalytic subunit of PI 3-kinase contributes most to PDGF-stimulated 3′-PI production. Thus, at high concentrations of PDGF the kinetics of 3′-PI production are limited by the turnover rate of these lipids, while the Akt response is additionally influenced by the rate of Akt deactivation.

Systematic Quantification of Negative Feedback Mechanisms in the Extracellular Signal-regulated Kinase (ERK) Signaling Network

Cell responses are actuated by tightly controlled signal transduction pathways. Although the concept of an integrated signaling network replete with interpathway cross-talk and feedback regulation is broadly appreciated, kinetic data of the type needed to characterize such interactions in conjunction with mathematical models are lacking. In mammalian cells, the Ras/ERK pathway controls cell proliferation and other responses stimulated by growth factors, and several cross-talk and feedback mechanisms affecting its activation have been identified. In this work, we take a systematic approach to parse the magnitudes of multiple regulatory mechanisms that attenuate ERK activation through canonical (Ras-dependent) and non-canonical (PI3K-dependent) pathways. In addition to regulation of receptor and ligand levels, we consider three layers of ERK-dependent feedback: desensitization of Ras activation, negative regulation of MEK kinase (e.g. Raf) activities, and up-regulation of dual-specificity ERK phosphatases. Our results establish the second of these as the dominant mode of ERK self-regulation in mouse fibroblasts. We further demonstrate that kinetic models of signaling networks, trained on a sufficient diversity of quantitative data, can be reasonably comprehensive, accurate, and predictive in the dynamical sense.

PI3K‐dependent cross‐talk interactions converge with Ras as quantifiable inputs integrated by Erk

Although it is appreciated that canonical signal‐transduction pathways represent dominant modes of regulation embedded in larger interaction networks, relatively little has been done to quantify pathway cross‐talk in such networks. Through quantitative measurements that systematically canvas an array of stimulation and molecular perturbation conditions, together with computational modeling and analysis, we have elucidated cross‐talk mechanisms in the platelet‐derived growth factor (PDGF) receptor signaling network, in which phosphoinositide 3‐kinase (PI3K) and Ras/extracellular signal‐regulated kinase (Erk) pathways are prominently activated. We show that, while PI3K signaling is insulated from cross‐talk, PI3K enhances Erk activation at points both upstream and downstream of Ras. The magnitudes of these effects depend strongly on the stimulation conditions, subject to saturation effects in the respective pathways and negative feedback loops. Motivated by those dynamics, a kinetic model of the network was formulated and used to precisely quantify the relative contributions of PI3K‐dependent and ‐independent modes of Ras/Erk activation.

Directed migration of mesenchymal cells: Where signaling and the cytoskeleton meet

Cell migration directed by spatial cues, or taxis, is a primary mechanism for orchestrating concerted and collective cell movements during development, wound repair, and immune responses. Compared with the classic example of amoeboid chemotaxis, in which fast-moving cells such as neutrophils are directed by gradients of soluble factors, directed migration of slow-moving mesenchymal cells such as fibroblasts is poorly understood. Mesenchymal cells possess a distinctive organization of the actin cytoskeleton and associated adhesion complexes as its primary mechanical system, generating the asymmetric forces required for locomotion without strong polarization. The emerging hypothesis is that the molecular underpinnings of mesenchymal taxis involve distinct signaling pathways and diverse requirements for regulation.

Mechanisms of gradient sensing and chemotaxis: conserved pathways, diverse regulation.

Directed cell migration is critical for normal development, immune responses, and wound healing and plays a prominent role in tumor metastasis. In eukaryotes, cell orientation is biased by an external chemoattractant gradient through a spatial contrast in chemoattractant receptor-mediated signal transduction processes that differentially affect cytoskeletal dynamics at the cell front and rear. Mechanisms of spatial gradient sensing and chemotaxis have been studied extensively in the social amoeba Dictyostelium discoideum and mammalian leukocytes (neutrophils), which are similar in their remarkable sensitivity to shallow gradients and robustness of response over a broad range of chemoattractant concentration. Recently, we have quantitatively characterized a different gradient sensing system, that of platelet-derived growth factor-stimulated fibroblasts, an important component of dermal wound healing. The marked differences between this system and the others have led us to speculate on the diversity of gradient sensing mechanisms and their biological implications.

Migrating fibroblasts reorient directionality by a metastable, PI3K-dependent mechanism

Migrating fibroblasts reorient directionality by PI3K-dependent branching and pivoting of protrusions, a mechanism that allows fibroblasts to align with an external chemotactic gradient.

Spontaneous phosphoinositide 3-kinase signaling dynamics drive spreading and random migration of fibroblasts

During directed cell migration (chemotaxis), cytoskeletal dynamics are stimulated and spatially biased by phosphoinositide 3-kinase (PI3K) and other signal transduction pathways. Live-cell imaging using total internal reflection fluorescence (TIRF) microscopy revealed that, in the absence of soluble cues, 3′-phosphoinositides are enriched in a localized and dynamic fashion during active spreading and random migration of mouse fibroblasts on adhesive surfaces. Surprisingly, we found that PI3K activation is uncoupled from classical integrin-mediated pathways and feedback from the actin cytoskeleton. Inhibiting PI3K significantly impairs cell motility, both in the context of normal spreading and when microtubules are dissociated, which induces a dynamic protrusion phenotype as seen by TIRF in our cells. Accordingly, during random migration, 3′-phosphoinositides are frequently localized to regions of membrane protrusion and correlate quantitatively with the direction and persistence of cell movement. These results underscore the importance of localized PI3K signaling not only in chemotaxis but also in basal motility/migration of fibroblasts.