On the molecular, cellular and tissue origin of cancer.

The EMBO Journal highlights the multifaceted aspects of tumour biology in a series of complementary review articles published over the course of 2021.

Profilin binding couples chloride intracellular channel protein CLIC4 to RhoA-mDia2 signaling and filopodium formation.

Chloride intracellular channel 4 (CLIC4) is a cytosolic protein implicated in diverse actin-based processes, including integrin trafficking, cell adhesion, and tubulogenesis. CLIC4 is rapidly recruited to the plasma membrane by RhoA-activating agonists and then partly colocalizes with ß1 integrins. Agonist-induced CLIC4 translocation depends on actin polymerization and requires conserved residues that make up a putative binding groove. However, the mechanism and significance of CLIC4 trafficking have been elusive. Here, we show that RhoA activation by either lysophosphatidic acid (LPA) or epidermal growth factor is necessary and sufficient for CLIC4 translocation to the plasma membrane and involves regulation by the RhoA effector mDia2, a driver of actin polymerization and filopodium formation. We found that CLIC4 binds the G-actin-binding protein profilin-1 via the same residues that are required for CLIC4 trafficking. Consistently, shRNA-induced profilin-1 silencing impaired agonist-induced CLIC4 trafficking and the formation of mDia2-dependent filopodia. Conversely, CLIC4 knockdown increased filopodium formation in an integrin-dependent manner, a phenotype rescued by wild-type CLIC4 but not by the trafficking-incompetent mutant CLIC4(C35A). Furthermore, CLIC4 accelerated LPA-induced filopodium retraction. We conclude that through profilin-1 binding, CLIC4 functions in a RhoA-mDia2-regulated signaling network to integrate cortical actin assembly and membrane protrusion. We propose that agonist-induced CLIC4 translocation provides a feedback mechanism that counteracts formin-driven filopodium formation.

Emerging biological roles of Cl- intracellular channel proteins.

Cl- intracellular channels (CLICs) are a family of six evolutionary conserved cytosolic proteins that exist in both soluble and membrane-associated forms; however, their functions have long been elusive. Soluble CLICs adopt a glutathione S-transferase (GST)-fold, can induce ion currents in artificial membranes and show oxidoreductase activity in vitro, but there is no convincing evidence of CLICs having such activities in vivo. Recent studies have revealed a role for CLIC proteins in Rho-regulated cortical actin dynamics as well as vesicular trafficking and integrin recycling, the latter of which are under the control of Rab GTPases. In this Commentary, we discuss the emerging roles of CLIC proteins in these processes and the lessons learned from gene-targeting studies. We also highlight outstanding questions regarding the molecular function(s) of these important but still poorly understood proteins.

Rapid Remodeling of Invadosomes by Gi-coupled Receptors: DISSECTING THE ROLE OF Rho GTPases.

Invadosomes are actin-rich membrane protrusions that degrade the extracellular matrix to drive tumor cell invasion. Key players in invadosome formation are c-Src and Rho family GTPases. Invadosomes can reassemble into circular rosette-like superstructures, but the underlying signaling mechanisms remain obscure. Here we show that Src-induced invadosomes in human melanoma cells (A375M and MDA-MB-435) undergo rapid remodeling into dynamic extracellular matrix-degrading rosettes by distinct G protein-coupled receptor agonists, notably lysophosphatidic acid (LPA; acting through the LPA1 receptor) and endothelin. Agonist-induced rosette formation is blocked by pertussis toxin, dependent on PI3K activity and accompanied by localized production of phosphatidylinositol 3,4,5-trisphosphate, whereas MAPK and Ca(2+) signaling are dispensable. Using FRET-based biosensors, we show that LPA and endothelin transiently activate Cdc42 through Gi, concurrent with a biphasic decrease in Rac activity and differential effects on RhoA. Cdc42 activity is essential for rosette formation, whereas G12/13-mediated RhoA-ROCK signaling suppresses the remodeling process. Our results reveal a Gi-mediated Cdc42 signaling axis by which G protein-coupled receptors trigger invadosome remodeling, the degree of which is dictated by the Cdc42-RhoA activity balance.

Threshold-controlled ubiquitination of the EGFR directs receptor fate.

How the cell converts graded signals into threshold-activated responses is a question of great biological relevance. Here, we uncover a nonlinear modality of epidermal growth factor receptor (EGFR)-activated signal transduction, by demonstrating that the ubiquitination of the EGFR at the PM is threshold controlled. The ubiquitination threshold is mechanistically determined by the cooperative recruitment of the E3 ligase Cbl, in complex with Grb2, to the EGFR. This, in turn, is dependent on the simultaneous presence of two phosphotyrosines, pY1045 and either one of pY1068 or pY1086, on the same EGFR moiety. The dose-response curve of EGFR ubiquitination correlate precisely with the non-clathrin endocytosis (NCE) mode of EGFR internalization. Finally, EGFR-NCE mechanistically depends on EGFR ubiquitination, as the two events can be simultaneously re-engineered on a phosphorylation/ubiquitination-incompetent EGFR backbone. Since NCE controls the degradation of the EGFR, our findings have implications for how the cell responds to increasing levels of EGFR signalling, by varying the balance of receptor signalling and degradation/attenuation.

Quantitative Proteomics Illuminates a Functional Interaction between mDia2 and the Proteasome.

Formin mDia2 is a cytoskeleton-regulatory protein that switches reversibly between a closed, autoinhibited and an open, active conformation. Although the open conformation of mDia2 induces actin assembly thereby controlling many cellular processes, mDia2 possesses also actin-independent and conformation-insensitive scaffolding roles related to microtubules and p53, respectively. Thus, we hypothesize that mDia2 may have other unappreciated functions and regulatory modes. Here we identify and validate proteasome and Ubiquitin as mDia2-interacting partners using stable isotope labeling with amino acids in cell culture-based quantitative proteomics and biochemistry, respectively. Although mDia2 is ubiquitinated, binds ubiquitinated proteins and free Ubiquitin, it is not a proteasome substrate. Surprisingly, knockdown of mDia2 increases the activity of the proteasome in vitro, whereas mDia2 overexpression has opposite effects only when it adopts the open conformation and cannot induce actin assembly. Consistently, a combination of candidate and unbiased proteome-wide analyses indicates that mDia2 regulates the cellular levels of proteasome substrate ß-catenin and a number of ubiquitinated actin-regulatory proteins. Hence, these findings add more complexity to the mDia2 activity cycle by showing that the open conformation may control actin dynamics also through actin-independent regulation of the proteasome.

CLIC4 regulates cell adhesion and ß1 integrin trafficking.

Chloride intracellular channel protein 4 (CLIC4) exists in both soluble and membrane-associated forms, and is implicated in diverse cellular processes, ranging from ion channel formation to intracellular membrane remodeling. CLIC4 is rapidly recruited to the plasma membrane by lysophosphatidic acid (LPA) and serum, suggesting a possible role for CLIC4 in exocytic-endocytic trafficking. However, the function and subcellular target(s) of CLIC4 remain elusive. Here, we show that in HeLa and MDA-MB-231 cells, CLIC4 knockdown decreases cell-matrix adhesion, cell spreading and integrin signaling, whereas it increases cell motility. LPA stimulates the recruitment of CLIC4 to ß1 integrin at the plasma membrane and in Rab35-positive endosomes. CLIC4 is required for both the internalization and the serum- or LPA-induced recycling of ß1 integrin, but not for EGF receptor trafficking. Furthermore, we show that CLIC4 suppresses Rab35 activity and antagonizes Rab35-dependent regulation of ß1 integrin trafficking. Our results define CLIC4 as a regulator of Rab35 activity and serum- and LPA-dependent integrin trafficking.

Proteomic snapshot of the EGF-induced ubiquitin network.

The activity, localization and fate of many cellular proteins are regulated through ubiquitination, a process whereby one or more ubiquitin (Ub) monomers or chains are covalently attached to target proteins. While Ub-conjugated and Ub-associated proteomes have been described, we lack a high-resolution picture of the dynamics of ubiquitination in response to signaling. In this study, we describe the epidermal growth factor (EGF)-regulated Ubiproteome, as obtained by two complementary purification strategies coupled to quantitative proteomics. Our results unveil the complex impact of growth factor signaling on Ub-based intracellular networks to levels that extend well beyond what might have been expected. In addition to endocytic proteins, the EGF-regulated Ubiproteome includes a large number of signaling proteins, ubiquitinating and deubiquitinating enzymes, transporters and proteins involved in translation and transcription. The Ub-based signaling network appears to intersect both housekeeping and regulatory circuitries of cellular physiology. Finally, as proof of principle of the biological relevance of the EGF-Ubiproteome, we demonstrated that EphA2 is a novel, downstream ubiquitinated target of epidermal growth factor receptor (EGFR), critically involved in EGFR biological responses.

The chloride intracellular channel protein CLIC4 inhibits filopodium formation induced by constitutively active mutants of formin mDia2.

Chloride intracellular channel 4 (CLIC4) functions in diverse actin-dependent processes. Upon Rho activation, CLIC4 reversibly translocates from the cytosol to the plasma membrane to regulate cell adhesion and migration. At the plasma membrane, CLIC4 counters the formation of filopodia, which requires actin assembly by the formin mammalian Diaphanous (mDia)2. To this end, mDia2 must be activated through conversion from the closed to the open conformation. Thus, CLIC4 could harness the activation or the open conformation of mDia2 to inhibit filopodium formation. Here, we find that CLIC4 silencing enhances the filopodia induced by two constitutively active mDia2 mutants. Furthermore, we report that CLIC4 binds the actin-regulatory region of mDia2 in vitro. These results suggest that CLIC4 modulates the activity of the open conformation of mDia2, shedding new light into how cells may control filopodia.

Flat clathrin lattices are dynamic actin-controlled hubs for clathrin-mediated endocytosis and signalling of specific receptors.

Clathrin lattices at the plasma membrane coat both invaginated and flat regions forming clathrin-coated pits and clathrin plaques, respectively. The function and regulation of clathrin-coated pits in endocytosis are well understood but clathrin plaques remain enigmatic nanodomains. Here we use super-resolution microscopy, molecular genetics and cell biology to show that clathrin plaques contain the machinery for clathrin-mediated endocytosis and cell adhesion, and associate with both clathrin-coated pits and filamentous actin. We also find that actin polymerization promoted by N-WASP through the Arp2/3 complex is crucial for the regulation of plaques but not pits. Clathrin plaques oppose cell migration and undergo actin- and N-WASP-dependent disassembly upon activation of LPA receptor 1, but not EGF receptor. Most importantly, plaque disassembly correlates with the endocytosis of LPA receptor 1 and down-modulation of AKT activity. Thus, clathrin plaques serve as dynamic actin-controlled hubs for clathrin-mediated endocytosis and signalling that exhibit receptor specificity.

Quantitative analysis reveals how EGFR activation and downregulation are coupled in normal but not in cancer cells.

Ubiquitination of the epidermal growth factor receptor (EGFR) that occurs when Cbl and Grb2 bind to three phosphotyrosine residues (pY1045, pY1068 and pY1086) on the receptor displays a sharp threshold effect as a function of EGF concentration. Here we use a simple modelling approach together with experiments to show that the establishment of the threshold requires both the multiplicity of binding sites and cooperative binding of Cbl and Grb2 to the EGFR. While the threshold is remarkably robust, a more sophisticated model predicted that it could be modulated as a function of EGFR levels on the cell surface. We confirmed experimentally that the system has evolved to perform optimally at physiological levels of EGFR. As a consequence, this system displays an intrinsic weakness that causes--at the supraphysiological levels of receptor and/or ligand associated with cancer--uncoupling of the mechanisms leading to signalling through phosphorylation and attenuation through ubiquitination.

Clathrin-mediated internalization is essential for sustained EGFR signaling but dispensable for degradation.

Clathrin-mediated endocytosis (CME) is the major pathway of epidermal growth factor receptor (EGFR) internalization. It is commonly believed that CME mediates long-term attenuation of EGFR signaling by targeting the receptor for degradation. However, the EGFR can also be internalized through (a) clathrin-independent pathway(s), and it remains unclear why distinct mechanisms of internalization have evolved. Here, we report that EGFRs internalized via CME are not targeted for degradation, but instead are recycled to the cell surface. By contrast, clathrin-independent internalization preferentially commits the receptor to degradation. This finding has profound implications for signaling, as by skewing EGFR fate toward recycling rather than degradation, CME prolongs the duration of signaling. Our data show that CME determines the longevity of some EGFR-activated signaling pathways and that EGF-dependent biological responses, such as DNA synthesis, absolutely require CME. Thus, CME of the EGFR unexpectedly has a greater impact on receptor signaling than on receptor degradation.

Peptide identification using vectors of small fragment ions.

Traditionally, peptide identification using fragmentation spectra relies on extracting the maximum amount of information from spectra. Using different combinations of small ion masses, we show that identifying a small number of fragment ions in a spectrum is sufficient for peptide identification. We consider y2-, y3-, b2-, and b3-ions and find the combination of b2-y2 to be sufficient for many peptides. Adding either the y3- or the b3-ion increases specificity and allows reliable peptide identification in the human proteome. Fragmentation spectra and peptides are represented as n-dimensional vectors, where n is given by the number of fragment ions considered, and the peptide mass. The identification score is given by the Euclidian distance between the spectra and the matching peptide in n-dimensional space. We show that this approach, using minimal information, allows for precise and fast peptide identification.

Melanogenic response of the Kupffer cells of Rana esculenta L to melanocyte stimulating hormone.

We have previously shown that Kupffer cells (KCs) of Rana esculenta L. possess melanogenic ability. The melanogenic enzyme activities in these cells are different from those described in skin melanocytes, and very little is known about their regulation by extracellular signalling molecules. In order to study this regulation, we analysed the effects of NDP-MSH on the levels of expression of the tyrosinase gene and on dopa-oxidase activity, using primary cultures of KCs. Incubation of the cells with NDP-MSH increases tyrosinase gene transcription, within the first 24 h of stimulation. To gain insight into the signalling mechanism involved in the cell response to the hormone, KCs in culture were incubated with IBMX or forskolin. These agents mimic the effects of alpha-MSH on melanocytes by increasing the intracellular level of cAMP. The experimental results showed that while the hormonal treatment always activated the KC tyrosinase system, treatment with IBMX or forskolin never did. Therefore, in KCs the tyrosinase-stimulating action of NDP-MSH was not mimicked by cAMP elevating agents. Assays of cAMP levels in cells stimulated with NDP-MSH demonstrated that the hormone does not produce significant increases in intracellular cAMP. On the contrary, forskolin produced significant increases in cAMP starting from 30 min of incubation. These results suggest that tyrosinase induction by melanocortins in KCs is not mediated by the cAMP pathway, and highlight the existence of substantial differences in the hormone signal transduction mechanisms between amphibian KCs and melanocytes or melanoma cells.