ABSTRACT
Upon starvation, individual Dictyostelium discoideum cells enter a developmental program that leads to collective migration and the formation of a multicellular organism. The process is mediated by extracellular cAMP binding to the G protein-coupled cAMP receptor 1, which initiates a signaling cascade leading to the activation of adenylyl cyclase A (ACA), the synthesis and secretion of additional cAMP, and an autocrine and paracrine activation loop. The release of cAMP allows neighboring cells to polarize and migrate directionally and form characteristic chains of cells called streams. We now report that cAMP relay can be measured biochemically by assessing ACA, ERK2, and TORC2 activities at successive time points in development after stimulating cells with subsaturating concentrations of cAMP. We also find that the activation profiles of ACA, ERK2, and TORC2 change in the course of development, with later developed cells showing a loss of sensitivity to the relayed signal. We examined mutants in PKA activity that have been associated with precocious development and find that this loss in responsiveness occurs earlier in these mutants. Remarkably, we show that this loss in sensitivity correlates with a switch in migration patterns as cells transition from streams to aggregates. We propose that as cells proceed through development, the cAMP-induced desensitization and down-regulation of cAMP receptor 1 impacts the sensitivities of chemotactic signaling cascades leading to changes in migration patterns.
Subject(s)
Chemotaxis/physiology , Dictyostelium/growth & development , Adenylyl Cyclases/metabolism , Biochemistry/methods , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Fluorescence Resonance Energy Transfer/methods , MAP Kinase Signaling System , Models, Biological , Mutation , Phosphorylation , Receptors, Cyclic AMP/metabolism , Signal TransductionABSTRACT
Cyclic AMP has a crucial role during the entire developmental program of the social amoebae Dictyostelium, acting both as an intracellular second messenger and, when secreted, as a directional cue that is relayed to neighboring cells during chemotaxis. Although significant knowledge about cAMP production in chemotaxing cells has been derived from studies performed on cell populations, cAMP dynamics at the single cell level have not been investigated. To examine this, we used a FRET-based cAMP sensor that possesses high cAMP sensitivity and great temporal resolution. We show the transient profile of cAMP accumulation in live Dictyostelium cells and establish that chemoattractants control intracellular cAMP dynamics by regulating synthesis via the adenylyl cyclase ACA. aca(-) cells show no significant change in FRET response following chemoattractant addition. Furthermore, cells lacking ACB, the other adenylyl cyclase expressed in chemotaxing cells, behave similarly to wild-type cells. We also establish that the RegA is the major phosphodiesterase that degrades intracellular cAMP in chemotaxis-competent cells. Interestingly, we failed to measure intracellular cAMP compartmentalization in actively chemotaxing cells. We conclude that cytosolic cAMP, which is destined to activate PKA, is regulated by ACA and RegA and does not compartmentalize during chemotaxis.
Subject(s)
Cyclic AMP/metabolism , Dictyostelium/cytology , Dictyostelium/metabolism , Adenylyl Cyclases/genetics , Adenylyl Cyclases/metabolism , Chemotaxis , Cytosol/metabolism , Dictyostelium/enzymology , Dictyostelium/genetics , Fluorescence Resonance Energy Transfer , Protozoan Proteins/genetics , Protozoan Proteins/metabolismABSTRACT
The ability of cells to sense external chemical cues and respond by directionally migrating towards them is a fundamental process called chemotaxis. This phenomenon is essential for many biological responses in the human body, including the invasion of neutrophils to sites of inflammation. Remarkably, many of the molecular mechanisms involved in controlling neutrophils chemotaxis arose millions of years ago in the simple eukaryotic organism Dictyostelium discoideum. Both neutrophils and Dictyostelium use G protein-coupled signaling cascades to mediate chemotactic responses, which are responsible for transducing external cues into highly organized cytoskeletal rearrangements that ultimately lead to directed migration. By using the genetically and biochemically tractable organism Dictyostelium as a model system, it has been possible to decipher many of the signal transduction events that are involved in chemotaxis.
Subject(s)
Chemotaxis, Leukocyte , Models, Biological , Actins/metabolism , Animals , Chemotactic Factors/metabolism , Chemotaxis , Cytoskeleton/metabolism , Dictyostelium/enzymology , Humans , Myosin Type II/metabolism , Neutrophils/enzymology , PTEN Phosphohydrolase/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Receptors, G-Protein-Coupled/metabolism , Signal TransductionABSTRACT
Metastasis results from a sequence of selective events often involving interactions with elements of the tumor-specific physiological microenvironment. The low-serum component of this microenvironment confers increased motility and invasion in breast cancer cells by activating the Na+/H+ exchanger isoform 1 (NHE1). The present study was undertaken to characterize the signal transduction mechanisms underlying this serum deprivation-dependent activation of both the NHE1 and the concomitant invasive characteristics such as leading edge pseudopodia development and penetration of matrigel in breast cancer cell lines representing different stages of metastatic progression. Using pharmacological and genetic manipulation together with transport and kinase activity assays, we observe that the activation of the NHE1 and subsequent invasion by serum deprivation in metastatic human breast cells is coordinated by a sequential RhoA/p160ROCK/p38MAPK signaling pathway gated by direct protein kinase A phosphorylation and inhibition of RhoA. Fluorescence resonance energy transfer imaging of RhoA activity and immunofluorescence analysis of phospho-RhoA and NHE1 show that serum deprivation dynamically remodels the cell, forming long, leading edge pseudopodia and that this signal module is preferentially compartmentalized in these leading edge pseudopodia, suggesting a tight topographic relation of the signaling module to an invasion-specific cell structure.
Subject(s)
Breast Neoplasms/pathology , Cation Transport Proteins/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Gene Expression Regulation, Neoplastic , Membrane Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Pseudopodia/metabolism , Sodium-Hydrogen Exchangers/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , rhoA GTP-Binding Protein/metabolism , Cell Line, Tumor , Collagen/metabolism , Culture Media, Serum-Free/pharmacology , Disease Progression , Down-Regulation , Drug Combinations , Enzyme Activation , Fluorescence Resonance Energy Transfer , Genetic Vectors , Humans , Hydrogen-Ion Concentration , Intracellular Signaling Peptides and Proteins , Laminin/metabolism , Microscopy, Fluorescence , Models, Biological , Neoplasm Invasiveness , Neoplasm Metastasis , Phosphorylation , Proteoglycans/metabolism , Serine/chemistry , Signal Transduction , Sodium-Hydrogen Exchanger 1 , Subcellular Fractions , Time Factors , Up-Regulation , rho-Associated KinasesABSTRACT
INTRODUCTION: An increasing body of evidence shows that the tumour microenvironment is essential in driving neoplastic progression. The low serum component of this microenvironment stimulates motility/invasion in human breast cancer cells via activation of the Na+-H+ exchanger (NHE) isoform 1, but the signal transduction systems that underlie this process are still poorly understood. We undertook the present study to elucidate the role and pattern of regulation by the Rho GTPases of this serum deprivation-dependent activation of both NHE1 and subsequent invasive characteristics, such as pseudopodia and invadiopodia protrusion, directed cell motility and penetration of normal tissues. METHODS: The present study was performed in a well characterized human mammary epithelial cell line representing late stage metastatic progression, MDA-MB-435. The activity of RhoA and Rac1 was modified using their dominant negative and constitutively active mutants and the activity of NHE1, cell motility/invasion, F-actin content and cell shape were measured. RESULTS: We show for the first time that serum deprivation induces NHE1-dependent morphological and cytoskeletal changes in metastatic cells via a reciprocal interaction of RhoA and Rac1, resulting in increased chemotaxis and invasion. Deprivation changed cell shape by reducing the amount of F-actin and inducing the formation of leading edge pseudopodia. Serum deprivation inhibited RhoA activity and stimulated Rac1 activity. Rac1 and RhoA were antagonistic regulators of both basal and stimulated tumour cell NHE1 activity. The regulation of NHE1 activity by RhoA and Rac1 in both conditions was mediated by an alteration in intracellular proton affinity of the exchanger. Interestingly, the role of each of these G-proteins was reversed during serum deprivation; basal NHE1 activity was regulated positively by RhoA and negatively by Rac1, whereas RhoA negatively and Rac1 positively directed the stimulation of NHE1 during serum deprivation. Importantly, the same pattern of RhoA and Rac1 regulation found for NHE1 activity was observed in both basal and serum deprivation dependent increases in motility, invasion and actin cytoskeletal organization. CONCLUSION: Our findings suggest that the reported antagonistic roles of RhoA and Rac1 in cell motility/invasion and cytoskeletal organization may be due, in part, to their concerted action on NHE1 activity as a convergence point.
Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cation Transport Proteins/metabolism , Cell Movement/physiology , Cytoskeleton/metabolism , Membrane Proteins/metabolism , Sodium-Hydrogen Exchangers/metabolism , rac1 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/metabolism , Actins/metabolism , Breast Neoplasms/enzymology , Cell Line, Tumor , Culture Media, Serum-Free , Cytoskeleton/pathology , Enzyme Activation , Humans , Neoplasm Invasiveness , Signal Transduction/physiology , Sodium-Hydrogen Exchanger 1ABSTRACT
Although Cystic fibrosis transmembrane conductance regulator (CFTR) has been shown to regulate the activity of NHE3, the potential reciprocal interaction of NHE3 to modulate the protein kinase A (PKA)-dependent regulation of CFTR in epithelial cells is still unknown. In the present work, we describe experiments to define the interactions between CFTR and NHE3 with the regulatory, scaffolding protein, NHERF that organize their PKA-dependent regulation in a renal epithelial cell line that expresses endogenous CFTR. The expression of rat NHE3 significantly decreased PKA-dependent activation of CFTR without altering CFTR expression, and this decrease was prevented by mutation of either of the two rat NHE3 PKA target serines to alanine (S552A or S605A). Inhibition of CFTR expression by antisense treatment resulted in an acute decrease in PKA-dependent regulation of NHE3 activity. CFTR, NHE3, and ezrin were recognized by NHERF-2 but not NHERF-1 in glutathione S-transferase pull-down experiments. Ezrin may function as a protein kinase A anchoring protein (AKAP) in this signaling complex, because blocking the binding of PKA to an AKAP by incubation with the S-Ht31 peptide inhibited the PKA-dependent regulation of CFTR in the absence of NHE3. In the A6-NHE3 cells S-Ht31 blocked the PKA regulation of NHE3 whereas it now failed to affect the regulation of CFTR. We conclude that CFTR and NHE3 reciprocally interact via a shared regulatory complex comprised of NHERF-2, ezrin, and PKA.