ABSTRACT
Although it is known that the spatial coordination of Rac and Rho activity is essential for cell migration, the molecular mechanisms regulating these GTPases during migration are unknown. We found that the expression of constitutively activated R-Ras (38V) blocked membrane protrusion and random migration. In contrast, expression of dominant negative R-Ras (41A) enhanced migrational persistence and membrane protrusion. Endogenous R-Ras is necessary for cell migration, as cells that were transfected with siRNA for R-Ras did not migrate. Expression of R-Ras (38V) decreased Rac activity and increased Rho activity around the entire cell periphery, whereas expression of dominant negative R-Ras (41A) showed the converse, suggesting that R-Ras can spatially activate Rho and inactivate Rac. Consistent with this role, endogenous R-Ras localized and was preferentially activated at the leading edge of migratory cells in response to adhesion. The effects of R-Ras on cell migration are mediated by PI3-Kinase, as an effector mutant that uncouples PI3-Kinase binding from R-Ras (38V) rescued migration. From these data, we hypothesize that R-Ras plays a key role in cell migration by locally regulating the switch from Rac to Rho activity after membrane protrusion and adhesion.
Subject(s)
Cell Membrane/metabolism , GTP Phosphohydrolases/physiology , rac GTP-Binding Proteins/metabolism , ras Proteins/physiology , rho GTP-Binding Proteins/metabolism , Cell Adhesion , Cell Line, Tumor , Cell Movement , Collagen/metabolism , Dose-Response Relationship, Drug , Genes, Dominant , Humans , Microscopy, Fluorescence , Microscopy, Video , Models, Biological , Mutation , Phosphatidylinositol 3-Kinases/metabolism , RNA, Small Interfering/metabolism , Time Factors , TransfectionABSTRACT
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.
Subject(s)
Integrins/physiology , Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Cell Movement , Cell Survival , Crk-Associated Substrate Protein , Fluorescence Resonance Energy Transfer , Humans , Integrins/genetics , Phosphorylation , Protein Conformation , Protein Structure, Tertiary , Proteins/chemistry , Proteins/genetics , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-abl/metabolism , Proto-Oncogene Proteins c-crk , Retinoblastoma-Like Protein p130ABSTRACT
Recently we demonstrated the existence of a phosphatidylinositol 3-kinase (PI3K)-independent F-actin polymerization during neutrophil pseudopod extension. Here we examine the use of the PI3K-dependent and PI3K-independent pathways of activation by the N-formyl peptide receptor and the chemokine receptors, and the priming of the 2 pathways by granulocyte-macrophage colony-stimulating factor (GM-CSF) and insulin. The inhibition of PI3K activity with wortmannin showed that rate of pseudopod extension stimulated with N-formyl-Met-Leu-Phe (fMLP was mostly dependent on PI3K, while the rate of interleukin-8 (IL-8)-stimulated pseudopod extension was less dependent on PI3K. The incubation of cells with either GM-CSF or insulin increased the rate of pseudopod extension by 50% when the cells were stimulated with IL-8 but not with fMLP. The stimulation with IL-8 phosphorylated the PI3K regulatory subunit. This phosphorylation was enhanced by GM-CSF, which increased PI3K activity and total phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) production. The effect of GM-CSF was blocked with wortmannin. In contrast, insulin did not increase p85 phosphorylation and did not enhance PI3K activity or PtdIns(3,4,5)P3 production. The effect of insulin was insensitive to wortmannin; however, it was blocked by an Src homology 2 (SH2)-binding peptide. These data indicate that priming of IL-8 activation with GM-CSF was mediated via the PI3Ks of class IA, while priming with insulin used a PI3K-independent pathway.
Subject(s)
Actins/metabolism , Neutrophils/metabolism , Adult , Androstadienes/pharmacology , Carrier Proteins/metabolism , Chemotactic Factors/pharmacology , Chemotaxis/drug effects , Chemotaxis/immunology , Enzyme Inhibitors/pharmacology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Hypoglycemic Agents/pharmacology , Insulin/pharmacology , Neutrophils/drug effects , Neutrophils/ultrastructure , Phosphatidylinositol 3-Kinases/pharmacology , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphatidylinositol Phosphates/metabolism , Polymers/metabolism , Pseudopodia/drug effects , Pseudopodia/metabolism , WortmanninABSTRACT
Cell migration requires establishment of a single pseudopodium in the direction of movement. Here we highlight recent advances in our understanding of the molecular signaling mechanisms that regulate formation of pseudopodia. We discuss how signal transduction processes are spatially and temporally organized to establish cell polarity through directed extension and stabilization of dominant pseudopodia. We also highlight recent advances in technology that will further the understanding of signaling dynamics specific to pseudopodia extension and cell migration.
Subject(s)
Cell Movement , Pseudopodia/physiology , Actin Cytoskeleton/physiology , Animals , Cell Polarity , Humans , Integrins/physiology , Signal TransductionABSTRACT
We characterized the overall rate of F-actin polymerization in the pseudopod region by measuring the rate of extension of single pseudopods stimulated by f-Met-Leu-Phe. The rate of pseudopod extension was measured in the presence of inhibitors for signaling molecules that are known to be involved in motility. Our data show the existence of 2 distinct signaling pathways of actin polymerization in the pseudopod region: a phosphoinositide 3-kinase gamma (PI3Kgamma)-dependent and -independent pathway. The PI3Kgamma dependent signaling of F-actin polymerization also depends on protein kinase C zeta and protein kinase B (Akt/PKB). The PI3Kgamma-independent pathway depends on GTPase RhoA, the RhoA ROCK kinase, Src family tyrosine kinases, and NADPH, and is modulated by cAMP.
Subject(s)
Actins/metabolism , Neutrophils/metabolism , Receptors, Immunologic/physiology , Receptors, Peptide/physiology , Class Ib Phosphatidylinositol 3-Kinase , Cyclic AMP/physiology , Humans , Intracellular Signaling Peptides and Proteins , Isoenzymes/metabolism , Kinetics , Microscopy, Video , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Pseudopodia/drug effects , Pseudopodia/metabolism , Pseudopodia/ultrastructure , Receptors, Formyl Peptide , Signal Transduction/physiology , rho-Associated Kinases , rhoA GTP-Binding Protein/metabolism , src-Family Kinases/metabolismABSTRACT
The formation of pseudopods and lamellae after ligation of chemoattractant sensitive G-protein coupled receptors (GPCRs) is essential for chemotaxis. Here, pseudopod extension was stimulated with chemoattractant delivered from a micropipet. The chemoattractant diffusion and convection mass transport were considered, and it is shown that when the delivery of chemoattractant was limited by diffusion there was a strong chemoattractant gradient along the cell surface. The diffusion-limited delivery of chemoattractant from a micropipet allowed for maintaining an almost constant chemoattractant concentration at the leading edge of single pseudopods during their growth. In these conditions, the rate of pseudopod extension was dependent on the concentration of chemoattractant in the pipet delivering chemoattractant. The pseudopod extension induced using micropipets was oscillatory even in the presence of a constant delivery of chemoattractant. This oscillatory pseudopod extension was controlled by activated RhoA and its downstream effector kinase ROCK and was abolished after the inhibition of RhoA activation with Clostridium botulinium C3 exoenzyme (C3) or the blocking of ROCK activation with Y-27632. The ability of the micropipet assay to establish a well-defined chemoattractant distribution around the activated cell over a wide range of molecular weights of the used chemoattractants allowed for comparison of the effect of chemoattractant stimulation on the dynamics of pseudopod growth. Pseudopod growth was stimulated using N-formylated peptide (N-formyl-methionyl-leucyl-phenylalanine (fMLP)), platelet activating factor (PAF), leukotriene B4 (LTB(4)), C5a anaphylotoxin (C5a), and interleukin-8 (IL-8), which represent the typical ligands for G-protein coupled chemotactic receptors. The dependence of the rate of pseudopod extension on the concentration of these chemoattractants and their equimolar mixture was measured and shown to be similar for all chemoattractants. The inhibition of the activity of phosphoinositide-3 kinase (PI3K) with wortmannin showed that 72%-80% of the rate of pseudopod extension induced with N-formyl-methionyl-leucyl-phenylalanine, platelet activating factor, and leukotriene B4 was phosphoinositide-3 kinase-dependent, in contrast to 55% of the rate of pseudopod extension induced with interleukin-8. The dependence of the rate of pseudopod extension on the concentration of individual chemoattractants and their equimolar mixture suggests that there is a common rate-limiting mechanism for the polymerization of cytoskeletal F-actin in the pseudopod region induced by G-protein coupled chemoattractant receptors.
Subject(s)
Actins/metabolism , Chemotaxis/physiology , Neutrophils/metabolism , Pseudopodia/metabolism , rhoA GTP-Binding Protein/metabolism , ADP Ribose Transferases/pharmacology , Actins/drug effects , Amides/pharmacology , Anaphylatoxins/pharmacology , Androstadienes/pharmacology , Botulinum Toxins/pharmacology , Chemotactic Factors/chemistry , Chemotaxis/drug effects , Humans , Interleukin-8/pharmacology , Intracellular Signaling Peptides and Proteins , Leukotriene B4/pharmacology , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Platelet Activating Factor/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Pseudopodia/drug effects , Pyridines/pharmacology , Wortmannin , rho-Associated Kinases , rhoA GTP-Binding Protein/antagonists & inhibitorsABSTRACT
Chemoattractant-stimulated pseudopod growth in human neutrophils was used as a model system to study the rate-limiting mechanism of cytoskeleton rearrangement induced by activated G-protein-coupled receptors. Cells were activated with N-formyl-Met-Leu-Phe, and the temperature dependence of the rate of pseudopod extension was measured in the presence of pharmacological inhibitors with known mechanisms of action. Three groups of inhibitors were used: (i) inhibitors sequestering substrates involved in F-actin polymerization (latrunculin A for G-actin and cytochalasin D for actin filament-free barbed ends) or sequestering secondary messengers (PIP-binding peptide for phosphoinositide lipids); (ii) competitively binding inhibitors (Akt-inhibitor for Akt/protein kinase B); and (iii) inhibitors that reduce enzyme activity (wortmannin for phosphoinositide 3-kinase and chelerythrine for protein kinase C). The experimental data are consistent with a model in which the relative involvement of a given pathway of F-actin polymerization to the measured rate of pseudopod extension is limited by a slowest (bottleneck) reaction in the cascade of reactions involved in the overall signaling pathway. The approach we developed was used to demonstrate that chemoattractant-induced pseudopod growth and mechanically stimulated cytoskeleton rearrangement are controlled by distinct pathways of F-actin polymerization.