SUMMARY Cells contain multiple F-actin assembly pathways including the Arp2/3 complex, formins, and Ena/VASP, which have largely been analyzed separately. They collectively generate the bulk of F-actin from a common pool of G-actin; however, the interplay/competition between these pathways remains poorly understood. Using fibroblast lines derived from an Arpc2 conditional knockout mouse, we established matched-pair cells with and without the Arp2/3 complex. Arpc2−/− cells lack lamellipodia and migrate slower than WT cells, but have F-actin levels indistinguishable from controls. Actin assembly in Arpc2−/− cells was resistant to cytochalasin-D and was highly dependent on profilin-1 and Ena/VASP, but not formins. Profilin-1 depletion in WT cells increased F-actin and Arp2/3 complex in lamellipodia. Conversely, addition of exogenous profilin-1 inhibited Arp2/3 complex actin nucleation in vitro and in vivo. These observations suggest that antagonism of the Arp2/3 complex by profilin-1 in cells maintains actin homeostasis by balancing Arp2/3 complex-dependent and independent actin assembly pathways.

The directed movement of fibroblasts towards locally released platelet-derived growth factor (PDGF) is a critical event in wound healing. Although recent studies have implicated polarized activation of phosphoinositide (PI) 3-kinase in G protein-mediated chemotaxis, the role of 3′ PI lipids in tyrosine kinase-triggered chemotaxis is not well understood. Using evanescent wave microscopy and green fluorescent protein–tagged Akt pleckstrin homology domain (GFP–AktPH) as a molecular sensor, we show that application of a shallow PDGF gradient triggers a markedly steeper gradient in 3′ PI lipids in the adhesion zone of fibroblasts. Polar GFP–AktPH gradients, as well as a new type of radial gradient, were measured from front to rear and from the periphery to the center of the adhesion zone, respectively. A strong spatial correlation between polarized 3′ PI production and rapid membrane spreading implicates 3′ PI lipids as a direct mediator of polarized migration. Analysis of the temporal changes of 3′ PI gradients in the adhesion zone revealed a fast diffusion coefficient (0.5 μm2/s) and short lifetime of 3′ PIs of <1 min. Together, this study suggests that the tyrosine kinase-coupled directional movement of fibroblasts and their radial membrane activity are controlled by local generation and rapid degradation of 3′ PI second messengers.

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.

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

Ras is a key signal transduction protein in the cell. Mutants of Gly 12 and Gln 61 impair GTPase activity and are found prominently in cancers. In wild type Ras-GTP, an allosteric switch promotes disorder to order transition in switch II, placing Gln 61 in the active site. We show that the "on" and "off" conformations of the allosteric switch can also be attained in RasG12V and RasQ61L. Although both mutants have similarly impaired active sites in the on state, RasQ61L stabilizes an anti-catalytic conformation of switch II in the off state of the allosteric switch when bound to Raf. This translates into more potent activation of the MAPK pathway involving Ras, Raf kinase, MEK, and ERK (Ras/Raf/MEK/ERK) in cells transfected with RasQ61L relative to RasG12V. This differential is not observed in the Raf-independent pathway involving Ras, phosphoinositide 3-kinase (PI3K), and Akt (Ras/PI3K/Akt). Using a combination of structural analysis, hydrolysis rates, and experiments in NIH-3T3 cells, we link the allosteric switch to the control of signaling in the Ras/Raf/MEK/ERK pathway, supporting a GTPase-activating protein-independent model for duration of the Ras-Raf complex.Ras GTPase is a central protein in signal transduction pathways in the cell that mediate the control of cell proliferation as well as many other functions (1, 2). It is isoprenylated at the C terminus through which it is anchored to the cell membrane (3). When bound to GTP, Ras propagates its signal by interacting with effector proteins such as Raf (4) and phosphoinositide 3-kinase (PI3K) (5) among others (6, 7). Once GTP is hydrolyzed to GDP on Ras, complexes with effectors are no longer favored, and signaling is turned off. The signal is thus directly connected to the level of Ras-GTP, which in turn is kept in check by the opposing actions of guanine nucleotide exchange factors that catalyze the loading of GTP (8), and of GTPase-activating proteins (GAPs) 2 that increase the intrinsically slow GTPase activity of Ras for timely depletion of Ras-GTP (9). We have recently discovered a mechanism through which the active site of Ras can be modulated by ligand binding at an allosteric site near the Ras-membrane interface, suggesting a GAP-independent mechanism through which hydrolysis rates could be increased (10). The binding of calcium and a negatively charged ligand, mimicked in our Ras-GppNHp crystal structure by an acetate molecule, promote a network of Hbonding interactions that connect the allosteric site to switch II, resulting in a disorder to order transition that positions key residues for catalysis (10). Thus, an "on" state of the allosteric switch implies increased hydrolysis rates associated with signaling being turned off. When the allosteric switch is "off," GTP hydrolysis is impaired and signaling remains on.The active site residues are situated primarily in the socalled switch I, switch II, and the phosphate-binding loop (Ploop) composed of residues 30 -40, 60 -76, and 10 -17, respectively (11). In particular, mutants of residues Gly 1...

Regulated activation of the highly conserved RasGTPase 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 Shc⅐Grb2⅐Sos complexes, which increases the rate of Ras guanine nucleotide exchange. In order to control Ras-mediated cell responses, this activity is regulated by receptor downregulation 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-␣, 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.The 170-kDa epidermal growth factor receptor (EGFR) 1 exerts its biological effects in response to binding of specific polypeptide ligands, including epidermal growth factor (EGF) and transforming growth factor-␣ (TGF␣). This leads to activation of the EGFR catalytic tyrosine kinase domain, autophosphorylation of specific residues in its carboxyl terminus, and recruitment and phosphorylation of heterologous signaling proteins (1). The EGFR can also transactivate other members of the erbB receptor family via heterodimerization, enhancing the diversity of potential signaling interactions (2). Overexpression and activating mutations of EGFR and other erbB family members, in conjunction with other permissive mutations, have been widely implicated in transformation and tumorigenesis.Increased ligand secretion and autocrine signaling through the EGFR can also contribute to uncontrolled cell proliferation. Secretion of TGF␣ in particular is potently mitogenic, because its dissociation from EGFR after endocytosis promotes receptor recycling; the sparing of receptors from proteolysis allows unabated si...

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.

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 p110␣ catalytic subunit of PI 3-kinase contributes most to PDGFstimulated 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.Platelet-derived growth factor (PDGF) 1 is a polypeptide mitogen of broad specificity, one of the earliest and most potent serum factors to be isolated (1). Beyond signaling of proliferation, PDGF acts as a strong chemoattractant during wound healing and can mediate protection from apoptosis in response to serum withdrawal and certain stress stimuli (2, 3). Three forms of PDGF have been studied extensively. They are composed of disulfide-bonded homo-and heterodimers of A and B chains, of which PDGF-BB is the best characterized. There are two structurally related PDGF receptors, ␣ and ␤, which exhibit different affinities for the A chain but roughly equivalent affinities for the B chain (4, 5). More recently, PDGF-C and -D isoforms, which form homodimers, have also been identified; these too exhibit different affinities for PDGF ␣-and ␤-receptors (6 -8).PDGF receptors belong to the well studied class of signal transducers collectively known as receptor-tyrosine kinases (9, 10). As with other receptors of this class, ligand-induced dimerization of PDGF receptors activates their intrinsic kinase domains, which catalyze the autophosphorylation of the receptors on multiple intracellular tyrosine residues in trans (11-13). The phosphorylated receptor may then act as a scaffold for specific binding interactions with cytosolic signal transduction enzymes and adaptor proteins (14). Among the most important of these are isoforms of phosphoinositide (PI) 3-kinase, which phosphorylate phosphatidylinositol (PtdIns) lipids in cell membranes to produce the 3Ј-PI second messengers PtdI...