Van Gogh-like 2 is essential for the architectural patterning of the mammalian biliary tree.

BACKGROUND & AIMS: In the developing liver, bipotent epithelial progenitor cells undergo lineage segregation to form hepatocytes, which constitute the bulk of the liver parenchyma, and biliary epithelial cells (cholangiocytes), which comprise the bile duct (a complex tubular network that is critical for normal liver function). Notch and TGFß signalling promote the formation of a sheet of biliary epithelial cells, the ductal plate, that organises into discontinuous tubular structures. How these structures elongate and connect to form a continuous duct remains undefined. We aimed to define the mechanisms by which the ductal plate transitions from a simple sheet of epithelial cells into a complex and connected bile duct. METHODS: By combining single-cell RNA sequencing of embryonic mouse livers with genetic tools and organoid models we functionally dissected the role of planar cell polarity in duct patterning. RESULTS: We show that the planar cell polarity protein VANGL2 is expressed late in intrahepatic bile duct development and patterns the formation of cell-cell contacts between biliary cells. The patterning of these cell contacts regulates the normal polarisation of the actin cytoskeleton within biliary cells and loss of Vangl2 function results in the abnormal distribution of cortical actin remodelling, leading to the failure of bile duct formation. CONCLUSIONS: Planar cell polarity is a critical step in the post-specification sculpture of the bile duct and is essential for establishing normal tissue architecture. IMPACT AND IMPLICATIONS: Like other branched tissues, such as the lung and kidney, the bile ducts use planar cell polarity signalling to coordinate cell movements; however, how these biochemical signals are linked to ductular patterning remains unclear. Here we show that the core planar cell polarity protein VANGL2 patterns how cell-cell contacts form in the mammalian bile duct and how ductular cells transmit confluent mechanical changes along the length of a duct. This work sheds light on how biological tubes are patterned across mammalian tissues (including within the liver) and will be important in how we promote ductular growth in patients where the duct is mis-patterned or poorly formed.

Macrophage fumarate hydratase restrains mtRNA-mediated interferon production.

Metabolic rewiring underlies the effector functions of macrophages1-3, but the mechanisms involved remain incompletely defined. Here, using unbiased metabolomics and stable isotope-assisted tracing, we show that an inflammatory aspartate-argininosuccinate shunt is induced following lipopolysaccharide stimulation. The shunt, supported by increased argininosuccinate synthase (ASS1) expression, also leads to increased cytosolic fumarate levels and fumarate-mediated protein succination. Pharmacological inhibition and genetic ablation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) further increases intracellular fumarate levels. Mitochondrial respiration is also suppressed and mitochondrial membrane potential increased. RNA sequencing and proteomics analyses demonstrate that there are strong inflammatory effects resulting from FH inhibition. Notably, acute FH inhibition suppresses interleukin-10 expression, which leads to increased tumour necrosis factor secretion, an effect recapitulated by fumarate esters. Moreover, FH inhibition, but not fumarate esters, increases interferon-ß production through mechanisms that are driven by mitochondrial RNA (mtRNA) release and activation of the RNA sensors TLR7, RIG-I and MDA5. This effect is recapitulated endogenously when FH is suppressed following prolonged lipopolysaccharide stimulation. Furthermore, cells from patients with systemic lupus erythematosus also exhibit FH suppression, which indicates a potential pathogenic role for this process in human disease. We therefore identify a protective role for FH in maintaining appropriate macrophage cytokine and interferon responses.

The RNA-bound proteome of MRSA reveals post-transcriptional roles for helix-turn-helix DNA-binding and Rossmann-fold proteins.

RNA-binding proteins play key roles in controlling gene expression in many organisms, but relatively few have been identified and characterised in detail in Gram-positive bacteria. Here, we globally analyse RNA-binding proteins in methicillin-resistant Staphylococcus aureus (MRSA) using two complementary biochemical approaches. We identify hundreds of putative RNA-binding proteins, many containing unconventional RNA-binding domains such as Rossmann-fold domains. Remarkably, more than half of the proteins containing helix-turn-helix (HTH) domains, which are frequently found in prokaryotic transcription factors, bind RNA in vivo. In particular, the CcpA transcription factor, a master regulator of carbon metabolism, uses its HTH domain to bind hundreds of RNAs near intrinsic transcription terminators in vivo. We propose that CcpA, besides acting as a transcription factor, post-transcriptionally regulates the stability of many RNAs.

IQGAP1 Is a Scaffold of the Core Proteins of the Hippo Pathway and Negatively Regulates the Pro-Apoptotic Signal Mediated by This Pathway.

The Hippo pathway regulates a complex signalling network which mediates several biological functions including cell proliferation, organ size and apoptosis. Several scaffold proteins regulate the crosstalk of the members of the pathway with other signalling pathways and play an important role in the diverse output controlled by this pathway. In this study we have identified the scaffold protein IQGAP1 as a novel interactor of the core kinases of the Hippo pathway, MST2 and LATS1. Our results indicate that IQGAP1 scaffolds MST2 and LATS1 supresses their kinase activity and YAP1-dependent transcription. Additionally, we show that IQGAP1 is a negative regulator of the non-canonical pro-apoptotic pathway and may enable the crosstalk between this pathway and the ERK and AKT signalling modules. Our data also show that bile acids regulate the IQGAP1-MST2-LATS1 signalling module in hepatocellular carcinoma cells, which could be necessary for the inhibition of MST2-dependent apoptosis and hepatocyte transformation.

The RhoA regulators Myo9b and GEF-H1 are targets of cyclic nucleotide-dependent kinases in platelets.

BACKGROUND: Circulating platelets are maintained in an inactive state by the endothelial lining of the vasculature. Endothelium-derived prostacyclin and nitric oxide stimulate cAMP- and cGMP-dependent kinases, PKA and PKG, to inhibit platelets. PKA and PKG effects include the inhibition of the GTPase RhoA, which has been suggested to involve the direct phosphorylation of RhoA on serine 188. OBJECTIVES: We wanted to confirm RhoA S188 phosphorylation by cyclic nucleotide-dependent kinases and to identify possible alternative mechanisms of RhoA regulation in platelets. METHODS: Phosphoproteomics data of human platelets were used to identify candidate PKA and PKG substrates. Phosphorylation of individual proteins was studied by Western blotting and Phos-tag gel electrophoresis in human platelets and transfected HEK293T cells. Pull-down assays were performed to analyze protein interaction and function. RESULTS: Our data indicate that RhoA is not phosphorylated by PKA in platelets. Instead, we provide evidence that cyclic nucleotide effects are mediated through the phosphorylation of the RhoA-specific GTPase-activating protein Myo9b and the guanine nucleotide exchange factor GEF-H1. We identify Myo9b S1354 and guanine nucleotide exchange factor-H1 (GEF-H1) S886 as PKA and PKG phosphorylation sites. Myo9b S1354 phosphorylation enhances its GTPase activating protein function leading to reduced RhoA-GTP levels. GEF-H1 S886 phosphorylation stimulates binding of 14-3-3ß and has been shown to inhibit GEF function by facilitating binding of GEF-H1 to microtubules. Microtubule disruption increases RhoA-GTP levels confirming the importance of GEF-H1 in platelets. CONCLUSION: Phosphorylation of RhoA regulatory proteins Myo9b and GEF-H1, but not RhoA itself, is involved in cyclic nucleotide-mediated control of RhoA in human platelets.

Nucleolar-nucleoplasmic shuttling of TARG1 and its control by DNA damage-induced poly-ADP-ribosylation and by nucleolar transcription.

Macrodomains are conserved protein folds associated with ADP-ribose binding and turnover. ADP-ribosylation is a posttranslational modification catalyzed primarily by ARTD (aka PARP) enzymes in cells. ARTDs transfer either single or multiple ADP-ribose units to substrates, resulting in mono- or poly-ADP-ribosylation. TARG1/C6orf130 is a macrodomain protein that hydrolyzes mono-ADP-ribosylation and interacts with poly-ADP-ribose chains. Interactome analyses revealed that TARG1 binds strongly to ribosomes and proteins associated with rRNA processing and ribosomal assembly factors. TARG1 localized to transcriptionally active nucleoli, which occurred independently of ADP-ribose binding. TARG1 shuttled continuously between nucleoli and nucleoplasm. In response to DNA damage, which activates ARTD1/2 (PARP1/2) and promotes synthesis of poly-ADP-ribose chains, TARG1 re-localized to the nucleoplasm. This was dependent on the ability of TARG1 to bind to poly-ADP-ribose. These findings are consistent with the observed ability of TARG1 to competitively interact with RNA and PAR chains. We propose a nucleolar role of TARG1 in ribosome assembly or quality control that is stalled when TARG1 is re-located to sites of DNA damage.

Transcriptionally inducible Pleckstrin homology-like domain, family A, member 1, attenuates ErbB receptor activity by inhibiting receptor oligomerization.

Feedback control is a key mechanism in signal transduction, intimately involved in regulating the outcome of the cellular response. Here, we report a novel mechanism by which PHLDA1, Pleckstrin homology-like domain, family A, member 1, negatively regulates ErbB receptor signaling by inhibition of receptor oligomerization. We have found that the ErbB3 ligand, heregulin, induces PHILDA1 expression in MCF-7 cells. Transcriptionally-induced PHLDA1 protein directly binds to ErbB3, whereas knockdown of PHLDA1 increases complex formation between ErbB3 and ErbB2. To provide insight into the mechanism for our time-course and single-cell experimental observations, we performed a systematic computational search of network topologies of the mathematical models based on receptor dimer-tetramer formation in the ErbB activation processes. Our results indicate that only a model in which PHLDA1 inhibits formation of both dimers and tetramer can explain the experimental data. Predictions made from this model were further validated by single-molecule imaging experiments. Our studies suggest a unique regulatory feature of PHLDA1 to inhibit the ErbB receptor oligomerization process and thereby control the activity of receptor signaling network.

Common and Distinctive Functions of the Hippo Effectors Taz and Yap in Skeletal Muscle Stem Cell Function.

Hippo pathway downstream effectors Yap and Taz play key roles in cell proliferation and regeneration, regulating gene expression especially via Tead transcription factors. To investigate their role in skeletal muscle stem cells, we analyzed Taz in vivo and ex vivo in comparison with Yap. Small interfering RNA knockdown or retroviral-mediated expression of wild-type human or constitutively active TAZ mutants in satellite cells showed that TAZ promoted proliferation, a function shared with YAP. However, at later stages of myogenesis, TAZ also enhanced myogenic differentiation of myoblasts, whereas YAP inhibits such differentiation. Functionally, while muscle growth was mildly affected in Taz (gene Wwtr1-/- ) knockout mice, there were no overt effects on regeneration. Conversely, conditional knockout of Yap in satellite cells of Pax7Cre-ERT2/+ : Yapfl °x/fl °x :Rosa26Lacz mice produced a regeneration deficit. To identify potential mechanisms, microarray analysis showed many common TAZ/YAP target genes, but TAZ also regulates some genes independently of YAP, including myogenic genes such as Pax7, Myf5, and Myod1 (ArrayExpress-E-MTAB-5395). Proteomic analysis revealed many novel binding partners of TAZ/YAP in myogenic cells, but TAZ also interacts with proteins distinct from YAP that are often involved in myogenesis and aspects of cytoskeleton organization (ProteomeXchange-PXD005751). Neither TAZ nor YAP bind members of the Wnt destruction complex but both regulated expression of Wnt and Wnt-cross talking genes with known roles in myogenesis. Finally, TAZ operates through Tead4 to enhance myogenic differentiation. In summary, Taz and Yap have overlapping functions in promoting myoblast proliferation but Taz then switches to enhance myogenic differentiation. Stem Cells 2017;35:1958-1972.

Carbon dioxide-dependent regulation of NF-κB family members RelB and p100 gives molecular insight into CO2-dependent immune regulation.

CO2 is a physiological gas normally produced in the body during aerobic respiration. Hypercapnia (elevated blood pCO2 >≈50 mm Hg) is a feature of several lung pathologies, e.g. chronic obstructive pulmonary disease. Hypercapnia is associated with increased susceptibility to bacterial infections and suppression of inflammatory signaling. The NF-κB pathway has been implicated in these effects; however, the molecular mechanisms underpinning cellular sensitivity of the NF-κB pathway to CO2 are not fully elucidated. Here, we identify several novel CO2-dependent changes in the NF-κB pathway. NF-κB family members p100 and RelB translocate to the nucleus in response to CO2 A cohort of RelB protein-protein interactions (e.g. with Raf-1 and IκBα) are altered by CO2 exposure, although others are maintained (e.g. with p100). RelB is processed by CO2 in a manner dependent on a key C-terminal domain located in its transactivation domain. Loss of the RelB transactivation domain alters NF-κB-dependent transcriptional activity, and loss of p100 alters sensitivity of RelB to CO2 Thus, we provide molecular insight into the CO2 sensitivity of the NF-κB pathway and implicate altered RelB/p100-dependent signaling in the CO2-dependent regulation of inflammatory signaling.

Hypoxia Reduces the Pathogenicity of Pseudomonas aeruginosa by Decreasing the Expression of Multiple Virulence Factors.

Our understanding of how the course of opportunistic bacterial infection is influenced by the microenvironment is limited. We demonstrate that the pathogenicity of Pseudomonas aeruginosa strains derived from acute clinical infections is higher than that of strains derived from chronic infections, where tissues are hypoxic. Exposure to hypoxia attenuated the pathogenicity of strains from acute (but not chronic) infections, implicating a role for hypoxia in regulating bacterial virulence. Mass spectrometric analysis of the secretome of P. aeruginosa derived from an acute infection revealed hypoxia-induced repression of multiple virulence factors independent of altered bacterial growth. Pseudomonas aeruginosa lacking the Pseudomonas prolyl-hydroxylase domain-containing protein, which has been implicated in bacterial oxygen sensing, displays reduced virulence factor expression. Furthermore, pharmacological hydroxylase inhibition reduces virulence factor expression and pathogenicity in a murine model of pneumonia. We hypothesize that hypoxia reduces P. aeruginosa virulence at least in part through the regulation of bacterial hydroxylases.

Protein-Protein Interaction Detection Via Mass Spectrometry-Based Proteomics.

Analysis of protein-protein interactions is one of the mainstays of mass spectrometry-based proteomics and recent developments, which have simplified the methodology, have permitted non-specialised laboratories to adopt the approach. We introduce and review three complimentary methods which allow for the targeted, global and site-specific analysis of protein complexes. Co-precipitation of endogenous or ectopically expressed proteins and their complexes followed by proteomic analysis allows for the discovery and accurate quantification of specific protein interactions. Whereas complimentary methods, such as co-purification of entire complexes based on physico-chemical attributes, can give a snap-shot of the composition and dynamics of protein complexes on a global scale. Cross-linking on the other hand can pinpoint the amino acids involved in protein-protein interactions to such a resolution that the likely complex can be reconstructed computationally.

Rapamycin regulates autophagy and cell adhesion in induced pluripotent stem cells.

BACKGROUND: Cellular reprogramming is a stressful process, which requires cells to engulf somatic features and produce and maintain stemness machineries. Autophagy is a process to degrade unwanted proteins and is required for the derivation of induced pluripotent stem cells (iPSCs). However, the role of autophagy during iPSC maintenance remains undefined. METHODS: Human iPSCs were investigated by microscopy, immunofluorescence, and immunoblotting to detect autophagy machinery. Cells were treated with rapamycin to activate autophagy and with bafilomycin to block autophagy during iPSC maintenance. High concentrations of rapamycin treatment unexpectedly resulted in spontaneous formation of round floating spheres of uniform size, which were analyzed for differentiation into three germ layers. Mass spectrometry was deployed to reveal altered protein expression and pathways associated with rapamycin treatment. RESULTS: We demonstrate that human iPSCs express high basal levels of autophagy, including key components of APMKα, ULK1/2, BECLIN-1, ATG13, ATG101, ATG12, ATG3, ATG5, and LC3B. Block of autophagy by bafilomycin induces iPSC death and rapamycin attenuates the bafilomycin effect. Rapamycin treatment upregulates autophagy in iPSCs in a dose/time-dependent manner. High concentration of rapamycin reduces NANOG expression and induces spontaneous formation of round and uniformly sized embryoid bodies (EBs) with accelerated differentiation into three germ layers. Mass spectrometry analysis identifies actin cytoskeleton and adherens junctions as the major targets of rapamycin in mediating iPSC detachment and differentiation. CONCLUSIONS: High levels of basal autophagy activity are present during iPSC derivation and maintenance. Rapamycin alters expression of actin cytoskeleton and adherens junctions, induces uniform EB formation, and accelerates differentiation. IPSCs are sensitive to enzyme dissociation and require a lengthy differentiation time. The shape and size of EBs also play a role in the heterogeneity of end cell products. This research therefore highlights the potential of rapamycin in producing uniform EBs and in shortening iPSC differentiation duration.

Cyclic Nucleotide-dependent Protein Kinases Target ARHGAP17 and ARHGEF6 Complexes in Platelets.

Endothelial cells release prostacyclin (PGI2) and nitric oxide (NO) to inhibit platelet functions. PGI2 and NO effects are mediated by cyclic nucleotides, cAMP- and cGMP-dependent protein kinases (PKA, PKG), and largely unknown PKA and PKG substrate proteins. The small G-protein Rac1 plays a key role in platelets and was suggested to be a target of cyclic nucleotide signaling. We confirm that PKA and PKG activation reduces Rac1-GTP levels. Screening for potential mediators of this effect resulted in the identification of the Rac1-specific GTPase-activating protein ARHGAP17 and the guanine nucleotide exchange factor ARHGEF6 as new PKA and PKG substrates in platelets. We mapped the PKA/PKG phosphorylation sites to serine 702 on ARHGAP17 using Phos-tag gels and to serine 684 on ARHGEF6. We show that ARHGAP17 binds to the actin-regulating CIP4 protein in platelets and that Ser-702 phosphorylation interferes with this interaction. Reduced CIP4 binding results in enhanced inhibition of cell migration by ARHGAP17. Furthermore, we show that ARHGEF6 is constitutively linked to GIT1, a GAP of Arf family small G proteins, and that ARHGEF6 phosphorylation enables binding of the 14-3-3 adaptor protein to the ARHGEF6/GIT1 complex. PKA and PKG induced rearrangement of ARHGAP17- and ARHGEF6-associated protein complexes might contribute to Rac1 regulation and platelet inhibition.

Nuclear FAK controls chemokine transcription, Tregs, and evasion of anti-tumor immunity.

Focal adhesion kinase (FAK) promotes anti-tumor immune evasion. Specifically, the kinase activity of nuclear-targeted FAK in squamous cell carcinoma (SCC) cells drives exhaustion of CD8(+) T cells and recruitment of regulatory T cells (Tregs) in the tumor microenvironment by regulating chemokine/cytokine and ligand-receptor networks, including via transcription of Ccl5, which is crucial. These changes inhibit antigen-primed cytotoxic CD8(+) T cell activity, permitting growth of FAK-expressing tumors. Mechanistically, nuclear FAK is associated with chromatin and exists in complex with transcription factors and their upstream regulators that control Ccl5 expression. Furthermore, FAK's immuno-modulatory nuclear activities may be specific to cancerous squamous epithelial cells, as normal keratinocytes do not have nuclear FAK. Finally, we show that a small-molecule FAK kinase inhibitor, VS-4718, which is currently in clinical development, also drives depletion of Tregs and promotes a CD8(+) T cell-mediated anti-tumor response. Therefore, FAK inhibitors may trigger immune-mediated tumor regression, providing previously unrecognized therapeutic opportunities.

Nonlinear signalling networks and cell-to-cell variability transform external signals into broadly distributed or bimodal responses.

We show theoretically and experimentally a mechanism behind the emergence of wide or bimodal protein distributions in biochemical networks with nonlinear input-output characteristics (the dose-response curve) and variability in protein abundance. Large cell-to-cell variation in the nonlinear dose-response characteristics can be beneficial to facilitate two distinct groups of response levels as opposed to a graded response. Under the circumstances that we quantify mathematically, the two distinct responses can coexist within a cellular population, leading to the emergence of a bimodal protein distribution. Using flow cytometry, we demonstrate the appearance of wide distributions in the hypoxia-inducible factor-mediated response network in HCT116 cells. With help of our theoretical framework, we perform a novel calculation of the magnitude of cell-to-cell heterogeneity in the dose-response obtained experimentally.

On-beads digestion in conjunction with data-dependent mass spectrometry: a shortcut to quantitative and dynamic interaction proteomics.

With the advent of the "-omics" era, biological research has shifted from functionally analyzing single proteins to understanding how entire protein networks connect and adapt to environmental cues. Frequently, pathological processes are initiated by a malfunctioning protein network rather than a single protein. It is therefore crucial to investigate the regulation of proteins in the context of a pathway first and signaling network second. In this study, we demonstrate that a quantitative interaction proteomic approach, combining immunoprecipitation, in-solution digestion and label-free quantification mass spectrometry, provides data of high accuracy and depth. This protocol is applicable, both to tagged, exogenous and untagged, endogenous proteins. Furthermore, it is fast, reliable and, due to a label-free quantitation approach, allows the comparison of multiple conditions. We further show that we are able to generate data in a medium throughput fashion and that we can quantify dynamic interaction changes in signaling pathways in response to mitogenic stimuli, making our approach a suitable method to generate data for system biology approaches.

RCP-driven α5ß1 recycling suppresses Rac and promotes RhoA activity via the RacGAP1-IQGAP1 complex.

Inhibition of αvß3 or expression of mutant p53 promotes invasion into fibronectin (FN)-containing extracellular matrix (ECM) by enhancing Rab-coupling protein (RCP)-dependent recycling of α5ß1 integrin. RCP and α5ß1 cooperatively recruit receptor tyrosine kinases, including EGFR1, to regulate their trafficking and downstream signaling via protein kinase B (PKB)/Akt, which, in turn, promotes invasive migration. In this paper, we identify a novel PKB/Akt substrate, RacGAP1, which is phosphorylated as a consequence of RCP-dependent α5ß1 trafficking. Phosphorylation of RacGAP1 promotes its recruitment to IQGAP1 at the tips of invasive pseudopods, and RacGAP1 then locally suppresses the activity of the cytoskeletal regulator Rac and promotes the activity of RhoA in this subcellular region. This Rac to RhoA switch promotes the extension of pseudopodial processes and invasive migration into FN-containing matrices, in a RhoA-dependent manner. Thus, the localized endocytic trafficking of α5ß1 within the tips of invasive pseudopods elicits signals that promote the reorganization of the actin cytoskeleton, protrusion, and invasion into FN-rich ECM.

Signalling by protein phosphatases and drug development: a systems-centred view.

Protein modification cycles catalysed by opposing enzymes, such as kinases and phosphatases, form the backbone of signalling networks. Although, historically, kinases have been at the research forefront, a systems-centred approach reveals predominant roles for phosphatases in controlling the network response times and spatio-temporal profiles of signalling activities. Emerging evidence suggests that phosphatase kinetics are critical for network function and cell-fate decisions. Protein phosphatases operate as both immediate and delayed regulators of signal transduction, capable of attenuating or amplifying signalling. This versatility of phosphatase action emphasizes the need for systems biology approaches to understand cellular signalling networks and predict the cellular outcomes of combinatorial drug interventions.

Mammalian protein expression noise: scaling principles and the implications for knockdown experiments.

The abundance of a particular protein varies both over time within a single mammalian cell and between cells of a genetically identical population. Here, we investigate the properties of such noisy protein expression in mammalian cells by combining theoretical and experimental approaches. The gamma distribution model is well-known to describe cell-to-cell variability in protein expression in a variety of common scenarios. This model predicts, and experiments show, that when protein levels are manipulated by altering transcription rates or mRNA half-life, protein expression noise, defined as the squared coefficient of variation, is constant. In contrast, we also demonstrate that when protein levels are manipulated by changing protein half-life, as mean levels increase, noise decreases. Thus, in mammalian cells, the scaling relationship between mean protein levels and expression noise depends on how mean levels are perturbed. Therefore it may be important to consider how common experimental manipulations of protein expression affect not only mean levels, but also noise levels. In the context of knockdown experiments, natural cell-to-cell variability in protein expression implies that a particular cell from the knockdown population may have higher protein levels than a cell from the control population. Simulations and experimental data suggest that approximately three-fold knockdown in mean expression levels can reduce such so-called "overlap probability" to less than ~10%. This has implications for the interpretation of knockdown experiments when the readout is a single cell measure.

ERK2 drives tumour cell migration in three-dimensional microenvironments by suppressing expression of Rab17 and liprin-ß2.

Upregulation of the extracellular signal-regulated kinase (ERK) pathway has been shown to contribute to tumour invasion and progression. Because the two predominant ERK isoforms (ERK1 and ERK2, also known as MAPK3 and MAPK1, respectively) are highly homologous and have indistinguishable kinase activities in vitro, both enzymes were believed to be redundant and interchangeable. To challenge this view, we show that ERK2 silencing inhibits invasive migration of MDA-MB-231 cells, and re-expression of ERK2 but not ERK1 restores the normal invasive phenotype. A detailed quantitative analysis of cell movement on 3D matrices indicates that ERK2 knockdown impairs cellular motility by decreasing the migration velocity as well as increasing the time that cells spend not moving. Using gene expression arrays we found that the expression of the genes for Rab17 and liprin-ß2 was increased by knockdown of ERK2 and restored to normal levels following re-expression of ERK2, but not ERK1. Both play inhibitory roles in the invasive behaviour of three independent cancer cell lines. Importantly, knockdown of either Rab17 or liprin-ß2 restores invasiveness of ERK2-depleted cells, indicating that ERK2 drives invasion of MDA-MB-231 cells by suppressing expression of these genes.