RNF11 is a GGA protein cargo and acts as a molecular adaptor for GGA3 ubiquitination mediated by Itch.

Ring finger protein 11 (RNF11) is a RING (really interesting new gene)-H2 E3 ligase that is overexpressed in several human tumor tissues. The mature protein, which is anchored to membranes via a double acylation, localizes to early endosome and recycling compartments. Apart from its subcellular localization, additional lines of evidence implicate RNF11 in the mechanisms underlying vesicle traffic. Here we identify two acidic-cluster dileucine (Ac-LL) motifs, which are recognized by the VHS domains of Golgi-localized, gamma adaptin era-containing, ADP-ribosylation factor-binding protein (GGA) adaptors, as the molecular determinants governing RNF11 sorting at the trans-Golgi network and its internalization from the plasma membrane. We also show that RNF11 recruits itch to drive the ubiquitination of GGA3. This function is experimentally detectable only in cells overexpressing an RNF11 variant that is inactivated in the RING domain, indicating that RNF11 recruits GGA3 and controls its ubiquitination by regulating itch activity. Accordingly, our data demonstrate the involvement of itch in regulating GGA3 stability. Indeed, we observe that the endogenous levels of GGA3 are increased in cells knocked down for itch and endogenous GGA3 is hyperubiquitinated in an itch-dependent manner in a cell line expressing catalytically inactive RNF11. Our data are consistent with a model whereby the RING E3 ligase RNF11 is a novel GGA cargo actively participating in regulating the ubiquitination of the GGA protein family. The results that we are presenting put RNF11 at the center of a finally regulated system where it acts both as an adaptor and a modulator of itch-mediated control of ubiquitination events underlying membrane traffic.

HPV16 E5 affects the KGFR/FGFR2b-mediated epithelial growth through alteration of the receptor expression, signaling and endocytic traffic.

The E5 oncoprotein of the human papillomavirus type 16 (HPV16 E5) cooperates in cervical carcinogenesis and in epithelial transformation deregulating cell growth, survival and differentiation through the modulation of growth factor receptors. Among the epithelial receptor tyrosine kinases, the keratinocyte growth factor receptor/fibroblast growth factor receptor 2b (KGFR/FGFR2b) is a major paracrine mediator of epithelial homeostasis and appears to have an unique and unusual role in epithelial tissues, exerting a tumor-suppressive function in vitro and in vivo. With the aim to better elucidate the molecular events involved in the pathological activity of 16E5, we investigated if the viral protein would be able to affect the KGFR expression, signaling and turnover by interference with its degradative and recycling endocytic pathways. Quantitative reverse transcriptase-PCR and biochemical approaches on human keratinocytes transfected with 16E5-HA showed that E5 protein is able to induce KGFR down-modulation at both transcript and protein levels. Immunofluorescence microscopy in double-transfected cells expressing both E5 and KGFR revealed that the viral protein alters the receptor endocytic trafficking and triggers its endosomal sorting to the indirect juxtanuclear recycling pathway. The shift from lysosomal degradation to recycling at the plasma membrane correlates with a reduced phosphorylation of the fibroblast growth factor receptor substrate-2α tyrosine 196, the major docking site for Grb2-Cbl complexes responsible for receptor ubiquitination and degradation. 5'-Bromo-deoxyuridine incorporation assay demonstrated that expression of 16E5 induces a decrease in the growth response to the receptor ligands as a consequence of KGFR down-modulation, suggesting that 16E5 might have a role on HPV infection in perturbing the KGFR-mediated physiological behavior of confluent keratinocytes committed to differentiation.

Multiple modification and protein interaction signals drive the Ring finger protein 11 (RNF11) E3 ligase to the endosomal compartment.

Ring finger protein 11 (RNF11) is a small RING E3-ligase overexpressed in numerous human prostate, colon and invasive breast cancers. Although functional studies have implicated RNF11 in a variety of biological processes, including signal transduction and apoptosis, the molecular mechanisms underlying its function are still poorly understood. In this study we show that RNF11 is a membrane-associated E3 ligase co-localizing with markers of both the early and the recycling endosomes. Several modification and protein interaction signals in the RNF11 sequence are shown to affect its compartmentalization. Membrane binding requires two acylation motifs driving the myristoylation of Gly2 and the S-palmitoylation of Cys4. Accordingly, genetic removal of the myristoylating signal results in diffuse staining, whereas an RNF11 protein mutated in the palmitoylation signal is retained in compartments of the early secretory pathway. However, amino-terminal fusion to green fluorescent protein of a 10-residue peptide containing both acylation signals re-localizes the chimera to the plasma membrane, but it is not sufficient to direct it to the recycling compartment suggesting that additional signals contribute to the correct localization. In addition, we show that membrane anchoring through acylation is necessary for RNF11 to be post-translationally modified by the addition of several ubiquitin moieties and that loss of acylation severely impairs the in vivo ubiquitination mediated by the HECT E3-ligases Itch and Nedd4. Finally, in cells transfected with RNF11 we observe a correlation between high RNF11 expression, as in tumor cells, and a swelling of the endosomal compartment suggesting a possible role of the dysregulation of the endosome compartment in tumorigenesis.

Endosomal trafficking of the Menkes copper ATPase ATP7A is mediated by vesicles containing the Rab7 and Rab5 GTPase proteins.

The Cu-ATPase ATP7A (MNK) is localized in the trans-Golgi network (TGN) and relocalizes in the plasma membrane via vesicle-mediated traffic following exposure of the cells to high concentrations of copper. Rab proteins are organelle-specific GTPases, markers of different endosomal compartments; their role has been recently reviewed (Trends Cell Biol. 11(2001) 487). In this article we analyze the endosomal pathway of trafficking of the MNK protein in stably transfected clones of CHO cells, expressing chimeric Rab5-myc or Rab7-myc proteins, markers of early or late endosome compartments, respectively. We demonstrate by immunofluorescence and confocal and electron microscopy techniques that the increase in the concentration of copper in the medium (189 microM) rapidly induces a redistribution of the MNK protein from early sorting endosomes, positive for Rab5-myc protein, to late endosomes, containing the Rab7-myc protein. Cell fractionation experiments confirm these results; i.e., the MNK protein is recruited to the endosomal fraction on copper stimulation and colocalizes with Rab5 and Rab7 proteins. These findings allow the first characterization of the vesicles involved in the intracellular routing of the MNK protein from the TGN to the plasma membrane, a key mechanism allowing appropriate efflux of copper in cells grown in high concentrations of the metal.

Up-modulation of the expression of functional keratinocyte growth factor receptors induced by high cell density in the human keratinocyte HaCaT cell line.

Keratinocyte growth factor (KGF) is involved in the control of proliferation and differentiation of human keratinocytes. It binds to, and activates, the tyrosine kinase KGF receptor (KGFR), a splicing transcript variant of the fibroblast growth factor receptor 2. We have previously shown (C. Marchese et al., Cell Growth Differ., 8: 989-997, 1997) that differentiation of primary cultured keratinocytes triggered by high Ca2+ concentrations in the growing medium induced up-regulation of KGFR expression, which suggested that KGFR may play a crucial role in the control of the proliferative/differentiative program during transition from basal to suprabasal cells. Here we analyzed the process of modulation of the expression of KGFRs in the human keratinocyte cell line HaCaT, widely used as a model to study keratinocyte differentiation. Western blot and double immunofluorescence for KGFR and the K1 differentiation marker showed that cell differentiation and stratification induced by confluence and high cell density correlated with an increase in KGFR expression. KGFRs, present on suprabasal differentiated cells, appeared to be efficiently tyrosine-phosphorylated by KGF, which indicated that the receptors up-regulated by differentiation can be functionally activated by ligand binding. Bromodeoxyuridine incorporation assay revealed that a significant portion of suprabasal differentiated cells expressing KGFR seemed to be still able to synthesize DNA and to proliferate in response to KGF, which suggested that increased KGFR expression may be required for retention of the proliferative activity.

Endocytosis of a chimera between human pro-urokinase and the plant toxin saporin: an unusual internalization mechanism.

A fluorescent derivative of a chimeric toxin between human pro-urokinase and the plant ribosome-inactivating protein saporin (p-uPA-Sap(TRITC)), has been prepared in order to study the endocytosis of this potentially antimetastatic conjugate in the murine model cell line LB6 clone19 (Cl19) transfected with the human urokinase receptor gene. The physiological internalization of urokinase-inhibitor complexes is triggered by the interaction of plasminogen inhibitors (PAIs) with receptors belonging to the low density lipoprotein-related receptor protein (LRP) family, and involves a macro-quaternary structure including uPAR, LRP, and PAIs. However, in contrast to this mechanism, we observed a two-step process: first, the urokinase receptor (uPAR) acts as the anchoring factor on the plasma membrane; subsequently, LRP acts as the endocytic trigger. Once the chimera is bound to the plasma membrane by interaction with uPAR, we suggest that a possible exchange may occur to transfer the toxin to LRP via the saporin moiety and begin the internalization. So an unusual endocytic process is described, where the toxin enters the cell via a receptor different from that used to bind the plasma membrane.

Eps15 is recruited to the plasma membrane upon epidermal growth factor receptor activation and localizes to components of the endocytic pathway during receptor internalization.

Eps15 is a substrate for the tyrosine kinase of the epidermal growth factor receptor (EGFR) and is characterized by the presence of a novel protein:protein interaction domain, the EH domain. Eps15 also stably binds the clathrin adaptor protein complex AP-2. Previous work demonstrated an essential role for eps15 in receptor-mediated endocytosis. In this study we show that, upon activation of the EGFR kinase, eps15 undergoes dramatic relocalization consisting of 1) initial relocalization to the plasma membrane and 2) subsequent colocalization with the EGFR in various intracellular compartments of the endocytic pathway, with the notable exclusion of coated vesicles. Relocalization of eps15 is independent of its binding to the EGFR or of binding of the receptor to AP-2. Furthermore, eps15 appears to undergo tyrosine phosphorylation both at the plasma membrane and in a nocodazole-sensitive compartment, suggesting sustained phosphorylation in endocytic compartments. Our results are consistent with a model in which eps15 undergoes cycles of association:dissociation with membranes and suggest multiple roles for this protein in the endocytic pathway.

Receptor-mediated endocytosis of keratinocyte growth factor.

Keratinocyte growth factor (KGF) is a fibroblast growth factor which acts specifically on epithelial cells, regulating their proliferation and differentiation. KGF elicits its activity through binding to and activation of KGF receptor, a splicing transcript variant of fibroblast growth factor receptor 2 (FGFR2). Here we analyzed the pathway of internalization of KGF and its receptor using several approaches, including the utilization in immunofluorescence and in immunoelectron microscopy of a functional KGF-HFc chimeric protein as a specific tool to follow the endocytosis of the growth factor and of its receptor. Western blot analysis with anti-FGFR2 and anti-phosphotyrosine antibodies, as well as parallel double immunofluorescence and confocal analysis of NIH3T3 KGFR transfectants treated with KGF at 4 degrees C, followed by incubations at 37 degrees C for different time points, showed that KGF induced endocytosis of tyrosine activated KGFRs. The use of KGF-HFc in immunofluorescence and in immunogold electron microscopy on KGFR transfectants, A253 epithelial tumor cells and human cultured keratinocytes allowed us to follow the early steps of KGF internalization and revealed that this process occurred through clathrin-coated pits. A quantitative ELISA assay confirmed that KGF-HFc binding on the cell surface rapidly decreased because of internalization. Our results demonstrate that KGF is internalized by receptor-mediated endocytosis and illustrate the involvement of clathrin-coated pits in this process.

eps15 and eps15R are essential components of the endocytic pathway.

eps15 and eps1SR are substrates of the epidermal growth factor (EGF) receptor kinase that are characterized by the presence of a protein:protein interaction domain, the EH domain, and by their ability to bind to the clathrin adaptor protein complex adaptor protein 2. Indirect evidence suggests that eps15 and eps15R are involved in endocytosis. Here we show that microinjection of antibodies against eps15 and eps15R inhibits internalization of EGF and transferrin. In addition, fragments of eps15 (encompassing its EH domains or the COOH-terminal region that binds to adaptor protein 2) inhibit EGF internalization or endocytosis of Sindbis virus. These results demonstrate that eps15 and eps15R are essential components of the endocytic machinery.