Comprehensive interrogation of gene lists from genome-scale cancer screens with oncoEnrichR.

Genome-scale screening experiments in cancer produce long lists of candidate genes that require extensive interpretation for biological insight and prioritization for follow-up studies. Interrogation of gene lists frequently represents a significant and time-consuming undertaking, in which experimental biologists typically combine results from a variety of bioinformatics resources in an attempt to portray and understand cancer relevance. As a means to simplify and strengthen the support for this endeavor, we have developed oncoEnrichR, a flexible bioinformatics tool that allows cancer researchers to comprehensively interrogate a given gene list along multiple facets of cancer relevance. oncoEnrichR differs from general gene set analysis frameworks through the integration of an extensive set of prior knowledge specifically relevant for cancer, including ranked gene-tumor type associations, literature-supported proto-oncogene and tumor suppressor gene annotations, target druggability data, regulatory interactions, synthetic lethality predictions, as well as prognostic associations, gene aberrations and co-expression patterns across tumor types. The software produces a structured and user-friendly analysis report as its main output, where versions of all underlying data resources are explicitly logged, the latter being a critical component for reproducible science. We demonstrate the usefulness of oncoEnrichR through interrogation of two candidate lists from proteomic and CRISPR screens. oncoEnrichR is freely available as a web-based service hosted by the Galaxy platform (https://oncotools.elixir.no), and can also be accessed as a stand-alone R package (https://github.com/sigven/oncoEnrichR).

A cell competition-based small molecule screen identifies a novel compound that induces dual c-Myc depletion and p53 activation.

Breakpoint Cluster Region-Abelson kinase (BCR-Abl) is a driver oncogene that causes chronic myeloid leukemia and a subset of acute lymphoid leukemias. Although tyrosine kinase inhibitors provide an effective treatment for these diseases, they generally do not kill leukemic stem cells (LSCs), the cancer-initiating cells that compete with normal hematopoietic stem cells for the bone marrow niche. New strategies to target cancers driven by BCR-Abl are therefore urgently needed. We performed a small molecule screen based on competition between isogenic untransformed cells and BCR-Abl-transformed cells and identified several compounds that selectively impair the fitness of BCR-Abl-transformed cells. Interestingly, systems-level analysis of one of these novel compounds, DJ34, revealed that it induced depletion of c-Myc and activation of p53. DJ34-mediated c-Myc depletion occurred in a wide range of tumor cell types, including lymphoma, lung, glioblastoma, breast cancer, and several forms of leukemia, with primary LSCs being particularly sensitive to DJ34. Further analyses revealed that DJ34 interferes with c-Myc synthesis at the level of transcription, and we provide data showing that DJ34 is a DNA intercalator and topoisomerase II inhibitor. Physiologically, DJ34 induced apoptosis, cell cycle arrest, and cell differentiation. Taken together, we have identified a novel compound that dually targets c-Myc and p53 in a wide variety of cancers, and with particularly strong activity against LSCs.

Strategies to inhibit FGFR4 V550L-driven rhabdomyosarcoma.

BACKGROUND: Rhabdomyosarcoma (RMS) is a paediatric cancer driven either by fusion proteins (e.g., PAX3-FOXO1) or by mutations in key signalling molecules (e.g., RAS or FGFR4). Despite the latter providing opportunities for precision medicine approaches in RMS, there are currently no such treatments implemented in the clinic. METHODS: We evaluated biologic properties and targeting strategies for the FGFR4 V550L activating mutation in RMS559 cells, which have a high allelic fraction of this mutation and are oncogenically dependent on FGFR4 signalling. Signalling and trafficking of FGFR4 V550L were characterised by confocal microscopy and proteomics. Drug effects were determined by live-cell imaging, MTS assay, and in a mouse model. RESULTS: Among recently developed FGFR4-specific inhibitors, FGF401 inhibited FGFR4 V550L-dependent signalling and cell proliferation at low nanomolar concentrations. Two other FGFR4 inhibitors, BLU9931 and H3B6527, lacked potent activity against FGFR4 V550L. Alternate targeting strategies were identified by RMS559 phosphoproteomic analyses, demonstrating that RAS/MAPK and PI3K/AKT are essential druggable pathways downstream of FGFR4 V550L. Furthermore, we found that FGFR4 V550L is HSP90-dependent, and HSP90 inhibitors efficiently impeded RMS559 proliferation. In a RMS559 mouse xenograft model, the pan-FGFR inhibitor, LY2874455, did not efficiently inhibit growth, whereas FGF401 potently abrogated growth. CONCLUSIONS: Our results pave the way for precision medicine approaches against FGFR4 V550L-driven RMS.

Protein Tyrosine Phosphatase Receptor Type G (PTPRG) Controls Fibroblast Growth Factor Receptor (FGFR) 1 Activity and Influences Sensitivity to FGFR Kinase Inhibitors.

Recently, FGFR1 was found to be overexpressed in osteosarcoma and represents an important target for precision medicine. However, because targeted cancer therapy based on FGFR inhibitors has so far been less efficient than expected, a detailed understanding of the target is important. We have here applied proximity-dependent biotin labeling combined with label-free quantitative mass spectrometry to identify determinants of FGFR1 activity in an osteosarcoma cell line. Many known FGFR interactors were identified (e.g. FRS2, PLCG1, RSK2, SRC), but the data also suggested novel determinants. A strong hit in our screen was the tyrosine phosphatase PTPRG. We show that PTPRG and FGFR1 interact and colocalize at the plasma membrane where PTPRG directly dephosphorylates activated FGFR1. We further show that osteosarcoma cell lines depleted for PTPRG display increased FGFR activity and are hypersensitive to stimulation by FGF1. In addition, PTPRG depletion elevated cell growth and negatively affected the efficacy of FGFR kinase inhibitors. Thus, PTPRG may have future clinical relevance by being a predictor of outcome after FGFR inhibitor treatment.

Proximity Labeling by a Recombinant APEX2-FGF1 Fusion Protein Reveals Interaction of FGF1 with the Proteoglycans CD44 and CSPG4.

Fibroblast growth factor 1 (FGF1) binds to specific FGF receptors (FGFRs) at the surface of target cells to initiate intracellular signaling. While heparan sulfate proteoglycans (HSPGs) are well-described coreceptors, it is uncertain whether there are additional binding sites for FGF1 at the cell surface. To address this, we devised and tested a method to identify novel binding sites for FGF1 at the cell surface, which may also be applicable for other protein ligands. We constructed an APEX2-FGF1 fusion protein to perform proximal biotin labeling of proteins following binding of the fusion protein to the cell surface. After functional validation of the fusion protein by a signaling assay, we used this method to identify binding sites for FGF1 on cell surfaces of living cells. We confirmed the feasibility of our approach by detection of FGFR4, a well-known and specific receptor for FGF1. We subsequently screened for novel interactors using RPE1 cells and identified the proteoglycans CSPG4 (NG2) and CD44. We found that FGF1 binds CD44 through its heparin-binding moiety. Moreover, we found that FGF1 was colocalized with both CSPG4 and CD44 at the cell surface, suggesting that these receptors act as storage molecules that create a reservoir of FGF1. Importantly, our data demonstrate that recombinant ligand-APEX2 fusion proteins can be used to identify novel receptor interactions on the cell surface.

Photoactivation approaches reveal a role for Rab11 in FGFR4 recycling and signalling.

Fibroblast growth factor receptor 4 (FGFR4) plays important roles during development and in the adult to maintain tissue homeostasis. Moreover, overexpression of FGFR4 or activating mutations in FGFR4 has been identified as tumour-promoting events in several forms of cancer. Endocytosis is important for regulation of signalling receptors and we have previously shown that FGFR4 is mainly localized to transferrin-positive structures after ligand-induced endocytosis. Here, using a cell line with a defined pericentriolar endocytic recycling compartment, we show that FGFR4 accumulates in this compartment after endocytosis. Furthermore, using classical recycling assays and a new, photoactivatable FGFR4-PA-GFP fusion protein combined with live-cell imaging, we demonstrate that recycling of FGFR4 is dependent on Rab11. Upon Rab11b depletion, FGFR4 is trapped in the pericentriolar recycling compartment and the total levels of FGFR4 in cells are increased. Moreover, fibroblast growth factor 1 (FGF1)-induced autophosphorylation of FGFR4 as well as phosphorylation of phospholipase C (PLC)-γ is prolonged in cells depleted of Rab11. Interestingly, the activation of mitogen-activated protein kinase and AKT pathways were not prolonged but rather reduced in Rab11-depleted cells, indicating that recycling of FGFR4 is important for the nature of its signalling output. Thus, Rab11-dependent recycling of FGFR4 maintains proper levels of FGFR4 in cells and regulates FGF1-induced FGFR4 signalling.

Proximity Labeling Reveals Molecular Determinants of FGFR4 Endosomal Transport.

The fibroblast growth factor receptors (FGFRs) are important oncogenes promoting tumor progression in many types of cancer, such as breast, bladder, and lung cancer as well as multiple myeloma and rhabdomyosarcoma. However, little is known about how these receptors are internalized and down-regulated in cells. We have here applied proximity biotin labeling to identify proteins involved in FGFR4 signaling and trafficking. For this purpose we fused a mutated biotin ligase, BirA*, to the C-terminal tail of FGFR4 (FGFR4-BirA*) and the fusion protein was stably expressed in U2OS cells. Upon addition of biotin to these cells, proteins in proximity to the FGFR4-BirA* fusion protein became biotinylated and could be isolated and identified by quantitative mass spectrometry. We identified in total 291 proteins, including 80 proteins that were enriched in samples where the receptor was activated by the ligand (FGF1), among them several proteins previously found to be involved in FGFR signaling (e.g., FRS2, PLCγ, RSK2 and NCK2). Interestingly, many of the identified proteins were implicated in endosomal transport, and by precise annotation we were able to trace the intracellular pathways of activated FGFR4. Validating the data by confocal and three-dimensional structured illumination microscopy analysis, we concluded that FGFR4 uses clathrin-mediated endocytosis for internalization and is further sorted from early endosomes to the recycling compartment and the trans-Golgi network. Depletion of cells for clathrin heavy chain led to accumulation of FGFR4 at the cell surface and increased levels of active FGFR4 and PLCγ, while AKT and ERK signaling was diminished, demonstrating that functional clathrin-mediated endocytosis is required for proper FGFR4 signaling. Thus, this study reveals proteins and pathways involved in FGFR4 transport and signaling that provide possible targets and opportunities for therapeutic intervention in FGFR4 aberrant cancer.

Production of phosphatidylinositol 5-phosphate via PIKfyve and MTMR3 regulates cell migration.

Although phosphatidylinositol 5-phosphate (PtdIns5P) is present in many cell types and its biogenesis is increased by diverse stimuli, its precise cellular function remains elusive. Here we show that PtdIns5P levels increase when cells are stimulated to move and we find PtdIns5P to promote cell migration in tissue culture and in a Drosophila in vivo model. First, class III phosphatidylinositol 3-kinase, which produces PtdIns3P, was shown to be involved in migration of fibroblasts. In a cell migration screen for proteins containing PtdIns3P-binding motifs, we identified the phosphoinositide 5-kinase PIKfyve and the phosphoinositide 3-phosphatase MTMR3, which together constitute a phosphoinositide loop that produces PtdIns5P via PtdIns(3,5)P(2). The ability of PtdIns5P to stimulate cell migration was demonstrated directly with exogenous PtdIns5P and a PtdIns5P-producing bacterial enzyme. Thus, the identified phosphoinositide loop defines a new role for PtdIns5P in cell migration.

PIKfyve, MTMR3 and their product PtdIns5P regulate cancer cell migration and invasion through activation of Rac1.

Previously, we have shown that the phosphoinositide metabolizing enzymes PIKfyve (phosphoinositide 5-kinase, FYVE finger containing) and MTMR3 (myotubularin-related protein 3), together with their lipid product PtdIns5P, are important for migration of normal human fibroblasts. As these proteins are a kinase and a phosphatase respectively, and thereby considered druggable, we wanted to test their involvement in cancer cell migration and invasion. First, we showed that PIKfyve and MTMR3 are expressed in most cancer cells. Next, we demonstrated that depletion of PIKfyve or MTMR3 resulted in decreased velocity in three different cancer cell lines by using new software for cell tracking. Inhibition of the enzymatic activity of PIKfyve by the inhibitor YM201636 also led to a strong reduction in cell velocity. Mechanistically, we show that PIKfyve and MTMR3 regulate the activation of the Rho family GTPase Rac1. Further experiments also implicated PtdIns5P in the activation of Rac1. The results suggest a model for the activation of Rac1 in cell migration where PIKfyve and MTMR3 produce PtdIns5P on cellular membranes which may then serve to recruit effectors to activate Rac1. Finally, in an invasion assay, we demonstrate that both PIKfyve and MTMR3 are implicated in invasive behaviour of cancer cells. Thus PIKfyve and MTMR3 could represent novel therapeutic targets in metastatic cancer.

Automated tracking of cell migration in phase contrast images with CellTraxx.

The ability of cells to move and migrate is required during development, but also in the adult in processes such as wound healing and immune responses. In addition, cancer cells exploit the cells' ability to migrate and invade to spread into nearby tissue and eventually metastasize. The majority of cancer deaths are caused by metastasis and the process of cell migration is therefore intensively studied. A common way to study cell migration is to observe cells through an optical microscope and record their movements over time. However, segmenting and tracking moving cells in phase contrast time-lapse video sequences is a challenging task. Several tools to track the velocity of migrating cells have been developed. Unfortunately, most of the automated tools are made for fluorescence images even though unlabelled cells are often preferred to avoid phototoxicity. Consequently, researchers are constrained with laborious manual tracking tools using ImageJ or similar software. We have therefore developed a freely available, user-friendly, automated tracking tool called CellTraxx. This software makes it easy to measure the velocity and directness of migrating cells in phase contrast images. Here, we demonstrate that our tool efficiently recognizes and tracks unlabelled cells of different morphologies and sizes (HeLa, RPE1, MDA-MB-231, HT1080, U2OS, PC-3) in several types of cell migration assays (random migration, wound healing and cells embedded in collagen). We also provide a detailed protocol and download instructions for CellTraxx.

Fibroblast growth factors and their receptors in cancer.

FGFs (fibroblast growth factors) and their receptors (FGFRs) play essential roles in tightly regulating cell proliferation, survival, migration and differentiation during development and adult life. Deregulation of FGFR signalling, on the other hand, has been associated with many developmental syndromes, and with human cancer. In cancer, FGFRs have been found to become overactivated by several mechanisms, including gene amplification, chromosomal translocation and mutations. FGFR alterations are detected in a variety of human cancers, such as breast, bladder, prostate, endometrial and lung cancers, as well as haematological malignancies. Accumulating evidence indicates that FGFs and FGFRs may act in an oncogenic fashion to promote multiple steps of cancer progression by inducing mitogenic and survival signals, as well as promoting epithelial-mesenchymal transition, invasion and tumour angiogenesis. Therapeutic strategies targeting FGFs and FGFRs in human cancer are therefore currently being explored. In the present review we will give an overview of FGF signalling, the main FGFR alterations found in human cancer to date, how they may contribute to specific cancer types and strategies for therapeutic intervention.

Negative Regulation of FGFR (Fibroblast Growth Factor Receptor) Signaling.

FGFR (fibroblast growth factor receptor) signaling controls fundamental processes in embryonic, fetal and adult human life. The magnitude, duration, and location of FGFR signaling must be strictly controlled in order to induce the correct biological response. Uncontrolled receptor signaling has been shown to lead to a variety of diseases, such as skeletal disorders and cancer. Here we review the numerous cellular mechanisms that regulate and turn off FGFR signaling, once the receptor is activated. These mechanisms include endocytosis and endocytic sorting, phosphatase activity, negative regulatory proteins and negative feedback phosphorylation events. The mechanisms act together simultaneously or sequentially, controlling the same or different steps in FGFR signaling. Although more work is needed to fully understand the regulation of FGFR signaling, it is clear that the cells in our body have evolved an extensive repertoire of mechanisms that together keep FGFR signaling tightly controlled and prevent excess FGFR signaling.

Efficacy and Selectivity of FGF2-Saporin Cytosolically Delivered by PCI in Cells Overexpressing FGFR1.

Fibroblast growth factor receptors (FGFRs) have become an attractive target in cancer research and therapy due to their implication in several cancers. Limitations of current treatment options require a need for additional, more specific and potent strategies to overcome cancers driven by FGFRs. Photochemical internalization (PCI) is a light-controlled method for cytosolic delivery of drugs that are entrapped in endosomes and lysosomes. We here evaluated the efficacy and selectivity of PCI of FGF2-saporin (FGF-SAP) in cells overexpressing FGFR1. FGF-SAP is a conjugate of FGF2 and the highly cytotoxic ribosome-inactivating protein (RIP) saporin, which is used as payload to eliminate cancer cells. Evaluation of the targeting effect of PCI of FGF-SAP was done by comparing the cytotoxic response in osteosarcoma cells with very low levels of FGFR1 (U2OS) to cells overexpressing FGFR1 (U2OS-R1). We demonstrate that PCI greatly enhances cytotoxicity of the drug showing efficient cell killing at pM concentrations of the drug in U2OS-R1 cells. However, U2OS cells were also sensitive to the toxin after PCI. Binding experiments using confocal microscopy and Western blotting techniques indicate that FGF-SAP is taken up by cells through heparan sulfate proteoglycans (HSPGs) in U2OS cells. We further show that the cytotoxicity of FGF-SAP in U2OS cells was reduced when cells were co-treated with heparin to compete out binding to HSPG, demonstrating that the cytotoxic effect was due to internalization by HSPGs. We conclude that to prevent off-target effects of FGF-based toxins, it will be necessary to circumvent binding to HSPGs, for example by mutating the binding site of FGF2 to HSPGs.

GAK and PRKCD are positive regulators of PRKN-independent mitophagy.

The mechanisms involved in programmed or damage-induced removal of mitochondria by mitophagy remains elusive. Here, we have screened for regulators of PRKN-independent mitophagy using an siRNA library targeting 197 proteins containing lipid interacting domains. We identify Cyclin G-associated kinase (GAK) and Protein Kinase C Delta (PRKCD) as regulators of PRKN-independent mitophagy, with both being dispensable for PRKN-dependent mitophagy and starvation-induced autophagy. We demonstrate that the kinase activity of both GAK and PRKCD are required for efficient mitophagy in vitro, that PRKCD is present on mitochondria, and that PRKCD facilitates recruitment of ULK1/ATG13 to early autophagic structures. Importantly, we demonstrate in vivo relevance for both kinases in the regulation of basal mitophagy. Knockdown of GAK homologue (gakh-1) in C. elegans or knockout of PRKCD homologues in zebrafish led to significant inhibition of basal mitophagy, highlighting the evolutionary relevance of these kinases in mitophagy regulation.

Size limitation in translocation of fibroblast growth factor 1 fusion proteins across the endosomal membrane.

After binding to its receptor on the surface of mammalian cells and subsequent endocytosis, FGF1 is translocated across the membrane into the cytosol. The growth factor is then further transported into the nucleus. In order to characterize more closely the translocation mechanism utilized by FGF1, we introduced additional amino acids into FGF1 to test the size dependence of the translocated substrate. We constructed mutants containing an increasing number of copies of the myc tag (1-13 copies) in a surface loop of the FGF1 molecule. All of the constructs bound to specific FGF receptors and to heparin and were taken up by endocytosis. However, only FGF1 mutants harboring up to three myc tags (53 amino acids) were translocated while mutants with five myc tags (77 amino acids) or more were not translocated through the membrane. We further showed that insertion of other, unrelated polypeptides into FGF1, i.e., 3xFLAG tag (22 amino acids) and streptavidin binding peptide (50 amino acids), was also translocated. Larger insertions into FGF1, like the CBP-SBP tag (82 amino acids) or ricin A-chain (272 amino acids), resulted in fusion proteins that failed to translocate. The presented data imply that it is possible to employ FGF1 to import various polypeptides into the cytosol and nucleus of cells. Furthermore, the strict size dependence of FGF1 fusion proteins in membrane translocation argues against simple leakage of FGF1 from ruptured endosomal membranes but rather points to a specific translocation apparatus involving a proteinaceous pore.

Cancer Mutations in FGFR2 Prevent a Negative Feedback Loop Mediated by the ERK1/2 Pathway.

Tight regulation of signaling from receptor tyrosine kinases is required for normal cellular functions and uncontrolled signaling can lead to cancer. Fibroblast growth factor receptor 2 (FGFR2) is a receptor tyrosine kinase that induces proliferation and migration. Deregulation of FGFR2 contributes to tumor progression and activating mutations in FGFR2 are found in several types of cancer. Here, we identified a negative feedback loop regulating FGFR2 signaling. FGFR2 stimulates the Ras/MAPK signaling pathway consisting of Ras-Raf-MEK1/2-ERK1/2. Inhibition of this pathway using a MEK1/2 inhibitor increased FGFR2 signaling. The putative ERK1/2 phosphorylation site at serine 780 (S780) in FGFR2 corresponds to serine 777 in FGFR1 which is directly phosphorylated by ERK1/2. Substitution of S780 in FGFR2 to an alanine also increased signaling. Truncated forms of FGFR2 lacking the C-terminal tail, including S780, have been identified in cancer and S780 has been found mutated to leucine in bladder cancer. Substituting S780 in FGFR2 with leucine increased FGFR2 signaling. Importantly, cells expressing these mutated versions of S780 migrated faster than cells expressing wild-type FGFR2. Thus, ERK1/2-mediated phosphorylation of S780 in FGFR2 constitutes a negative feedback loop and inactivation of this feedback loop in cancer cells causes hyperactivation of FGFR2 signaling, which may result in increased invasive properties.

Phosphorylation-regulated nucleocytoplasmic trafficking of internalized fibroblast growth factor-1.

Fibroblast growth factor-1 (FGF-1), which stimulates cell growth, differentiation, and migration, is capable of crossing cellular membranes to reach the cytosol and the nucleus in cells containing specific FGF receptors. The cell entry process can be monitored by phosphorylation of the translocated FGF-1. We present evidence that phosphorylation of FGF-1 occurs in the nucleus by protein kinase C (PKC)delta. The phosphorylated FGF-1 is subsequently exported to the cytosol. A mutant growth factor where serine at the phosphorylation site is exchanged with glutamic acid, to mimic phosphorylated FGF-1, is constitutively transported to the cytosol, whereas a mutant containing alanine at this site remains in the nucleus. The export can be blocked by leptomycin B, indicating active and receptor-mediated nuclear export of FGF-1. Thapsigargin, but not leptomycin B, prevents the appearance of active PKCdelta in the nucleus, and FGF-1 is in this case phosphorylated in the cytosol. Leptomycin B increases the amount of phosphorylated FGF-1 in the cells by preventing dephosphorylation of the growth factor, which seems to occur more rapidly in the cytoplasm than in the nucleus. The nucleocytoplasmic trafficking of the phosphorylated growth factor is likely to play a role in the activity of internalized FGF-1.

The inositol phosphatase SHIP2 enables sustained ERK activation downstream of FGF receptors by recruiting Src kinases.

Sustained activation of extracellular signal-regulated kinase (ERK) drives pathologies caused by mutations in fibroblast growth factor receptors (FGFRs). We previously identified the inositol phosphatase SHIP2 (also known as INPPL1) as an FGFR-interacting protein and a target of the tyrosine kinase activities of FGFR1, FGFR3, and FGFR4. We report that loss of SHIP2 converted FGF-mediated sustained ERK activation into a transient signal and rescued cell phenotypes triggered by pathologic FGFR-ERK signaling. Mutant forms of SHIP2 lacking phosphoinositide phosphatase activity still associated with FGFRs and did not prevent FGF-induced sustained ERK activation, demonstrating that the adaptor rather than the catalytic activity of SHIP2 was required. SHIP2 recruited Src family kinases to the FGFRs, which promoted FGFR-mediated phosphorylation and assembly of protein complexes that relayed signaling to ERK. SHIP2 interacted with FGFRs, was phosphorylated by active FGFRs, and promoted FGFR-ERK signaling at the level of phosphorylation of the adaptor FRS2 and recruitment of the tyrosine phosphatase PTPN11. Thus, SHIP2 is an essential component of canonical FGF-FGFR signal transduction and a potential therapeutic target in FGFR-related disorders.

Translocation of FGF-1 and FGF-2 across vesicular membranes occurs during G1-phase by a common mechanism.

The entry of exogenous fibroblast growth factor 2 (FGF-2) to the cytosolic/nuclear compartment was studied and compared with the translocation mechanism used by FGF-1. To differentiate between external and endogenous growth factor, we used FGF-2 modified to contain a farnesylation signal, a CaaX-box. Because farnesylation occurs only in the cytosol and nucleoplasm, farnesylation of exogenous FGF-2-CaaX was taken as evidence that the growth factor had translocated across cellular membranes. We found that FGF-2 translocation occurred in endothelial cells and fibroblasts, which express FGF receptors, and that the efficiency of translocation was increased in the presence of heparin. Concomitantly with translocation, the 18-kDa FGF-2 was N-terminally cleaved to yield a 16-kDa form. Translocation of FGF-2 required PI3-kinase activity but not transport through the Golgi apparatus. Inhibition of endosomal acidification did not prevent translocation, whereas dissipation of the vesicular membrane potential completely blocked it. The data indicate that translocation occurs from intracellular vesicles containing proton pumps and that an electrical potential across the vesicle membrane is required. Translocation of both FGF-1 and FGF-2 occurred during most of G(1) but decreased shortly before the G(1)-->S transition. A common mechanism for FGF-1 and FGF-2 translocation into cells is postulated.

Phosphatidylinositol 5-phosphate is a second messenger important for cell migration.

We recently showed that production of phosphatidylinositol 5-phosphate (PtdIns5P or PI5P) upon growth factor stimulation is important for cell migration. However, it was not entirely clear if PI5P itself could be a second messenger in cell migration, or, if it was rather an intermediate for the production of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2 or PI(4,5)P2). Indeed, PI5P can be converted to PI(4,5)P2 by type II PIP4 kinases (PIP4K2s). We therefore decided to knock down PIP4K2α by siRNA to test if further conversion of PI5P to PI(4,5)P2 is important for cell migration. Even though we obtained an efficient knockdown of PIP4K2α in BJ human fibroblasts, we did not observe any change in cell velocity. Conversely, ectopic overexpression of PIP4K2α would consume PI5P to produce PI(4,5)P2 and we found that overexpressing PIP4K2α decreased cell migration speed. Taken together, the data clearly indicate that it is PI5P, and not PI(4,5)P2 produced from PI5P, that is the crucial signaling molecule in cell migration. We conclude, therefore, that PI5P is a true second messenger important for cell migration.