Regulation of cell contraction and membrane ruffling by distinct signals in migratory cells.

Cell migration and wound contraction requires assembly of actin into a functional myosin motor unit capable of generating force. However, cell migration also involves formation of actin-containing membrane ruffles. Evidence is provided that actin-myosin assembly and membrane ruffling are regulated by distinct signaling pathways in the migratory cell. Interaction of cells with extracellular matrix proteins or cytokines promote cell migration through activation of the MAP kinases ERK1 and ERK2 as well as the molecular coupling of the adaptor proteins p130CAS and c-CrkII. ERK signaling is independent of CAS/Crk coupling and regulates myosin light chain phosphorylation leading to actin-myosin assembly during cell migration and cell-mediated contraction of a collagen matrix. In contrast, membrane ruffling, but not cell contraction, requires Rac GTPase activity and the formation of a CAS/Crk complex that functions in the context of the Rac activating protein DOCK180. Thus, during cell migration ERK and CAS/Crk coupling operate as components of distinct signaling pathways that control actin assembly into myosin motors and membrane ruffles, respectively.     

The endosomal adaptor protein APPL1 impairs the turnover of leading edge adhesions to regulate cell migration

Cell migration is a complex process that requires the integration of signaling events that occur in distinct locations within the cell. Adaptor proteins, which can localize to different subcellular compartments, where they bring together key signaling proteins, are emerging as attractive candidates for controlling spatially coordinated processes. However, their function in regulating cell migration is not well understood. In this study, we demonstrate a novel role for the adaptor protein containing a pleckstrin-homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif 1 (APPL1) in regulating cell migration. APPL1 impairs migration by hindering the turnover of adhesions at the leading edge of cells. The mechanism by which APPL1 regulates migration and adhesion dynamics is by inhibiting the activity of the serine/threonine kinase Akt at the cell edge and within adhesions. In addition, APPL1 significantly decreases the tyrosine phosphorylation of Akt by the nonreceptor tyrosine kinase Src, which is critical for Akt-mediated cell migration. Thus, our results demonstrate an important new function for APPL1 in regulating cell migration and adhesion turnover through a mechanism that depends on Src and Akt. Moreover, our data further underscore the importance of adaptor proteins in modulating the flow of information through signaling pathways.     

Regulator of G protein signaling 1 (RGS1) markedly impairs Gi alpha signaling responses of B lymphocytes

Regulator of G protein signaling (RGS) proteins modulate signaling through pathways that use heterotrimeric G proteins as transducing elements. RGS1 is expressed at high levels in certain B cell lines and can be induced in normal B cells by treatment with TNF-alpha. To determine the signaling pathways that RGS1 may regulate, we examined the specificity of RGS1 for various G alpha subunits and assessed its effect on chemokine signaling. G protein binding and GTPase assays revealed that RGS1 is a Gi alpha and Gq alpha GTPase-activating protein and a potential G12 alpha effector antagonist. Functional studies demonstrated that RGS1 impairs platelet activating factor-mediated increases in intracellular Ca+2, stromal-derived factor-1-induced cell migration, and the induction of downstream signaling by a constitutively active form of G12 alpha. Furthermore, germinal center B lymphocytes, which are refractory to stromal-derived factor-1-triggered migration, express high levels of RGS1. These results indicate that RGS proteins can profoundly effect the directed migration of lymphoid cells.     

Bifurcation of cell migratory and proliferative signaling by the adaptor protein Shc

Cytokines and extracellular matrix proteins initiate signaling cascades that regulate cell migration and proliferation. Evidence is provided that the adaptor protein Shc can differentially regulate these processes. Specifically, under growth factor-limiting conditions, Shc stimulates haptotactic cell migration without affecting anchorage-dependent proliferation. However, when growth factors are present, Shc no longer influences cell migration; rather, Shc is crucial for DNA synthesis. Mutational analysis of Shc demonstrates that, while tyrosine phosphorylation is required for both DNA synthesis and cell migration, the switch in Shc signaling is associated with differential use of Shc's phosphotyrosine interacting domains; the PTB domain regulates haptotaxis, while the SH2 domain is selectively required for proliferation.     

FAK and paxillin: regulators of N-cadherin adhesion and inhibitors of cell migration?

FAK and paxillin are important components in integrin-regulated signaling. New evidence suggests that these two proteins function in crosstalk between cell-matrix and cell-cell adhesions. Further, new insight suggests that under some conditions these proteins inhibit cell migration, in contrast to their established roles in several cell systems as positive regulators of cell adhesion and migration.     

A phosphoproteomic analysis of the ErbB2 receptor tyrosine kinase signaling pathways

Overexpression of the ErbB2 receptor tyrosine kinase is common in human cancers and is associated with an increased level of metastasis. To better understand the cellular signaling networks activated by ErbB2, a phosphoproteomic analysis of tyrosine-phosphorylated proteins was carried out in ErbB2-overexpressing breast and ovarian cancer cell lines. A total of 153 phosphorylation sites were assigned on 78 proteins. Treatment of cells with Herceptin, a monoclonal antibody that inhibits ErbB2 activity, significantly reduced the number of detectable protein phosphorylation sites, suggesting that many of these proteins participate in ErbB2-driven cell signaling. Of the 71 proteins that were differentially phosphorylated, only 13 were previously reported to directly associate with ErbB2. The differentially phosphorylated proteins included kinases, adaptor/docking proteins, proteins involved in cell proliferation and migration, and several uncharacterized RNA binding proteins. Selective depletion of some of these proteins, including RNA binding proteins SRRM2, SFRS1, SFRS9, and SFRS10, by siRNAs reduced the rate of migration of ErbB2-overexpressing ovarian cancer cells.     

Regulation of cell adhesions and motility during initiation of neural crest migration

Accurate neural crest cell (NCC) migration requires tight control of cell adhesions, cytoskeletal dynamics and cell motility. Cadherins and RhoGTPases are critical molecular players that regulate adhesions and motility during initial delamination of NCCs from the neuroepithelium. Recent studies have revealed multiple functions for these molecules and suggest that a precise balance of their activity is crucial. RhoGTPase appears to regulate both cell adhesions and protrusive forces during NCC delamination. Increasing evidence shows that cadherins are multi-functional proteins with novel, adhesion-independent signaling functions that control NCC motility during both delamination and migration. These functions are often regulated by specific proteolytic cleavage of cadherins. After NCC delamination, planar cell polarity signaling acts via RhoGTPases to control NCC protrusions and migration direction.     

Germ cell migration in zebrafish is cyclopamine-sensitive but Smoothened-independent

Primordial germ cells (PGCs) are the progenitors of reproductive cells in metazoans and are an important model for the study of cell migration in vivo. Previous reports have suggested that Hedgehog (Hh) protein acts as a chemoattractant for PGC migration in the Drosophila embryo and that downstream signaling proteins such as Patched (Ptc) and Smoothened (Smo) are required for PGC localization to somatic gonadal precursors. Here we interrogate whether Hh signaling is required for PGC migration in vertebrates, using the zebrafish as a model system. We find that cyclopamine, an inhibitor of Hh signaling, causes strong defects in the migration of PGCs in the zebrafish embryo. However, these defects are not due to inhibition of Smoothened (Smo) by cyclopamine; rather, we find that neither maternal nor zygotic Smo is required for PGC migration in the zebrafish embryo. Cyclopamine instead acts independently of Smo to decrease the motility of zebrafish PGCs, in part by dysregulating cell adhesion and uncoupling cell polarization and translocation. These results demonstrate that Hh signaling is not required for zebrafish PGC migration, and underscore the importance of regulated cell-cell adhesion for cell migration in vivo.     

Regulation of integrin-mediated cellular responses through assembly of a CAS/Crk scaffold

The molecular coupling of CAS and Crk in response to integrin activation is an evolutionary conserved signaling module that controls cell proliferation, survival and migration. However, when deregulated, CAS/Crk signaling also contributes to cancer progression and developmental defects in humans. Here we highlight recent advances in our understanding of how CAS/Crk complexes assemble in cells to modulate the actin cytoskeleton, and the molecular mechanisms that regulate this process. We discuss in detail the spatiotemporal dynamics of CAS/Crk assembly and how this scaffold recruits specific effector proteins that couple integrin signaling networks to the migration machinery of cells. We also highlight the importance of CAS/Crk signaling in the dual regulation of cell migration and survival mechanisms that operate in invasive cells during development and pathological conditions associated with cancer metastasis.     

Endogenous RGS proteins attenuate Galpha(i)-mediated lysophosphatidic acid signaling pathways in ovarian cancer cells

Lysophosphatidic acid is a bioactive phospholipid that is produced by and stimulates ovarian cancer cells, promoting proliferation, migration, invasion, and survival. Effects of LPA are mediated by cell surface G-protein coupled receptors (GPCRs) that activate multiple heterotrimeric G-proteins. G-proteins are deactivated by Regulator of G-protein Signaling (RGS) proteins. This led us to hypothesize that RGS proteins may regulate G-protein signaling pathways initiated by LPA in ovarian cancer cells. To determine the effect of endogenous RGS proteins on LPA signaling in ovarian cancer cells, we compared LPA activity in SKOV-3 ovarian cancer cells expressing G(i) subunit constructs that are either insensitive to RGS protein regulation (RGSi) or their RGS wild-type (RGSwt) counterparts. Both forms of the G-protein contained a point mutation rendering them insensitive to inhibition with pertussis toxin, and cells were treated with pertussis toxin prior to experiments to eliminate endogenous G(i/o) signaling. The potency and efficacy of LPA-mediated inhibition of forskolin-stimulated adenylyl cyclase activity was enhanced in cells expressing RGSi G(i) proteins as compared to RGSwt G(i). We further showed that LPA signaling that is subject to RGS regulation terminates much faster than signaling thru RGS insensitive G-proteins. Finally, LPA-stimulated SKOV-3 cell migration, as measured in a wound-induced migration assay, was enhanced in cells expressing Galpha(i2) RGSi as compared to cells expressing Galpha(i2) RGSwt, suggesting that endogenous RGS proteins in ovarian cancer cells normally attenuate this LPA effect. These data establish RGS proteins as novel regulators of LPA signaling in ovarian cancer cells.     

Urokinase-type plasminogen activator binding to its receptor stimulates tumor cell migration by enhancing integrin-mediated signal transduction.

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) participate in matrix degradation and cell migration by focusing proteolysis and functioning as a signaling ligand/receptor complex. uPAR, anchored by a lipid moiety in the membrane, is thought to require a transmembrane adapter to transduce signals into the cytoplasm. To study uPAR signaling, we transfected the prostate carcinoma cell line LNCaP, which does not express endogenous uPA or uPAR, with a uPAR encoding cDNA, resulting in high-level surface expression. We studied migration of these cells on fibronectin, which is mediated by the integrin alpha5beta1. Ligation of uPAR with uPA or its amino-terminal fragment enhanced haptotactic migration to fibronectin. In cells on fibronectin, but not on poly-l-lysine, ligation of uPAR also resulted in tyrosine phosphorylation of several proteins, including two proteins involved in integrin signaling, focal adhesion kinase and the crk-associated substrate p130(Cas). Furthermore, after uPAR ligation, uPAR was co-immunoprecipitated with beta1 integrins from the detergent-insoluble fraction of cell lysates. Thus, our data suggest that uPAR occupancy results in an interaction between uPAR and integrins and a potentiation of integrin-mediated signaling, which leads to enhanced cell migration.     

Human cytomegalovirus-encoded G protein-coupled receptor US28 mediates smooth muscle cell migration through Galpha12

Coupling of G proteins to ligand-engaged chemokine receptors is the paramount event in G-protein-coupled receptor signal transduction. Previously, we have demonstrated that the human cytomegalovirus-encoded chemokine receptor US28 mediates human vascular smooth muscle cell (SMC) migration in response to either RANTES or monocyte chemoattractant protein 1. In this report, we identify the G proteins that couple with US28 to promote vascular SMC migration and identify other signaling molecules that play critical roles in this process. US28-mediated cellular migration was enhanced with the expression of the G-protein subunits Galpha12 and Galpha13, suggesting that US28 may functionally couple to these G proteins. In correlation with this observation, US28 was able to activate RhoA, a downstream effector of Galpha12 and Galpha13 in cell types with these G proteins but not in those without them and activation of RhoA was dependent on US28 stimulation with RANTES. In addition, inactivation of RhoA or the RhoA-associated kinase p160ROCK with a dominant-negative mutant of RhoA or the small molecule inhibitor Y27632, respectively, abrogated US28-induced SMC migration. The data presented here suggest that US28 functionally signals through Galpha12 family G proteins and RhoA in a ligand-dependent manner and these signaling molecules are important for the ability of US28 to induce cellular migration.     

Regulation of Cell Motility by Mitogen-activated Protein Kinase

Cell interaction with adhesive proteins or growth factors in the extracellular matrix initiates Ras/ mitogen-activated protein (MAP) kinase signaling. Evidence is provided that MAP kinase (ERK1 and ERK2) influences the cells' motility machinery by phosphorylating and, thereby, enhancing myosin light chain kinase (MLCK) activity leading to phosphorylation of myosin light chains (MLC). Inhibition of MAP kinase activity causes decreased MLCK function, MLC phosphorylation, and cell migration on extracellular matrix proteins. In contrast, expression of mutationally active MAP kinase kinase causes activation of MAP kinase leading to phosphorylation of MLCK and MLC and enhanced cell migration. In vitro results support these findings since ERK-phosphorylated MLCK has an increased capacity to phosphorylate MLC and shows increased sensitivity to calmodulin. Thus, we define a signaling pathway directly downstream of MAP kinase, influencing cell migration on the extracellular matrix.     

Membrane-binding/modification model of signaling protein activation and analysis of its control by cell morphology

A mechanism for cell shape control of intracellular signal transduction, whereby the average concentration of activated proteins in the cytosol increases as the height of the cell decreases, has been described recently. An important modification of this analysis is offered, recognizing that signaling proteins are not only activated at the plasma membrane but must first form complexes with signaling molecules that reside there, such as receptors and lipids. With these more realistic boundary conditions, it is shown that the region of parameter space where cell shape amplifies the average cytosolic activity is greatly expanded. Moreover, this model allows for amplification of the activated protein bound at the membrane, which is considered more relevant for certain, spatially driven signaling processes in cell migration.     

Matrix metalloproteinase (MMP) and TGF-β1-stimulated cell migration in skin and cornea wound healing

Cell migration during wound healing is a complex process that involves the expression of a number of growth factors and cytokines. One of these factors, transforming growth factor-beta (TGF-β) controls many aspects of normal and pathological cell behavior. It induces migration of keratinocytes in wounded skin and of epithelial cells in damaged cornea. Furthermore, this TGF-β-induced cell migration is correlated with the production of components of the extracellular matrix (ECM) proteins, and expression of integrins and matrix metalloproteinases (MMPs). MMP digests ECMs and integrins during cell migration, but the mechanisms regulating their expression and the consequences of their induction remain unclear. It has been suggested that MMP-14 activates cellular signaling processes involved in the expression of MMPs and other molecules associated with cell migration. Because of the manifold effects of MMP-14, it is important to understand the roles of MMP-14 not only the cleavage of ECM but also in the activation of signaling pathways.     

CCL2/CCR2 Chemokine Signaling Coordinates Survival and Motility of Breast Cancer Cells through Smad3 Protein- and p42/44 Mitogen-activated Protein Kinase (MAPK)-dependent Mechanisms

Increased cell motility and survival are important hallmarks of metastatic tumor cells. However, the mechanisms that regulate the interplay between these cellular processes remain poorly understood. In these studies, we demonstrate that CCL2, a chemokine well known for regulating immune cell migration, plays an important role in signaling to breast cancer cells. We report that in a panel of mouse and human breast cancer cell lines CCL2 enhanced cell migration and survival associated with increased phosphorylation of Smad3 and p42/44MAPK proteins. The G protein-coupled receptor CCR2 was found to be elevated in breast cancers, correlating with CCL2 expression. RNA interference of CCR2 expression in breast cancer cells significantly inhibited CCL2-induced migration, survival, and phosphorylation of Smad3 and p42/44MAPK proteins. Disruption of Smad3 expression in mammary carcinoma cells blocked CCL2-induced cell survival and migration and partially reduced p42/44MAPK phosphorylation. Ablation of MAPK phosphorylation in Smad3-deficient cells with the MEK inhibitor U0126 further reduced cell survival but not migration. These data indicate that Smad3 signaling through MEK-p42/44MAPK regulates CCL2-induced cell motility and survival, whereas CCL2 induction of MEK-p42/44MAPK signaling independent of Smad3 functions as an alternative mechanism for cell survival. Furthermore, we show that CCL2-induced Smad3 signaling through MEK-p42/44MAPK regulates expression and activity of Rho GTPase to mediate CCL2-induced breast cancer cell motility and survival. With these studies, we characterize an important role for CCL2/CCR2 chemokine signaling in regulating the intrinsic relationships between breast cancer cell motility and survival with implications on the metastatic process.     

GRASP and IPCEF Promote ARF-to-Rac Signaling and Cell Migration by Coordinating the Association of ARNO/cytohesin 2 with Dock180

ARFs are small GTPases that regulate vesicular trafficking, cell shape, and movement. ARFs are subject to extensive regulation by a large number of accessory proteins. The many different accessory proteins are likely specialized to regulate ARF signaling during particular processes. ARNO/cytohesin 2 is an ARF-activating protein that promotes cell migration and cell shape changes. We report here that protein–protein interactions mediated by the coiled-coil domain of ARNO are required for ARNO induced motility. ARNO lacking the coiled-coil domain does not promote migration and does not induce ARF-dependent Rac activation. We find that the coiled-coil domain promotes the assembly of a multiprotein complex containing both ARNO and the Rac-activating protein Dock180. Knockdown of either GRASP/Tamalin or IPCEF, two proteins known to bind to the coiled-coil of ARNO, prevents the association of ARNO and Dock180 and prevents ARNO-induced Rac activation. These data suggest that scaffold proteins can regulate ARF dependent processes by biasing ARF signaling toward particular outputs.     

Intracellular trafficking of raft/caveolae domains: insights from integrin signaling

Cells have a complex system for delivering and compartmentalizing proteins and lipids in order to achieve spatio-temporal coordination of signaling. Rafts/caveolae are plasma membrane microdomains that regulate signaling pathways and processes such as cell migration, polarization and proliferation. Regulation of raft/caveolae trafficking involves multiple steps regulated by different proteins to ensure coordination of signaling cascades. The best studied raft-mediated endocytic route is controlled by caveolins. Recent data suggest integrin-mediated cell adhesion is a key regulator of caveolar endocytosis. In this review we examine the regulation of caveolar trafficking and the interplay between integrins, cell adhesion and caveolae internalization.     

The Galpha13-Rho signaling axis is required for SDF-1-induced migration through CXCR4

The CXC chemokine stromal cell-derived factor-1alpha (SDF-1) binds to CXCR4, a seven-transmembrane G protein-coupled receptor that plays a critical role in many physiological processes that involve cell migration and cell fate decisions, ranging from stem cell homing, angiogenesis, and neuronal development to immune cell trafficking. CXCR4 is also implicated in various pathological conditions, including metastatic spread and human immunodeficiency virus infection. Although SDF-1-induced cell migration in CXCR4-expressing cells is sensitive to pertussis toxin treatment, hence involving heterotrimeric G proteins of the G(i) family, whether other G proteins participate in the chemotactic response to SDF-1 is still unknown. In this study, we took advantage of the potent chemotactic activity of SDF-1 in Jurkat T-cells to examine the nature of the heterotrimeric G protein subunits contributing to CXCR4-mediated cell migration. We observed that whereas G(i) and Gbetagamma subunits are involved in SDF-1-induced Rac activation and cell migration, CXCR4 can also stimulate Rho potently leading to the phosphorylation of myosin light chain through the Rho effector, Rho kinase, but independently of G(i). Furthermore, we found that Galpha(13) mediates the activation of Rho by CXCR4 and that the functional activity of both Galpha(13) and Rho is required for directional cell migration in response to SDF-1. Collectively, our data indicate that signaling by CXCR4 to Rho through Galpha(13) contributes to cell migration when stimulated by SDF-1, thus identifying the Galpha(13)-Rho signaling axis as a potential pharmacological target in many human diseases that involve the aberrant function of CXCR4.