Regulation of Rab5 function during phagocytosis of live Pseudomonas aeruginosa in macrophages.

Pseudomonas aeruginosa, a Gram-negative opportunistic human pathogen, is a frequent cause of severe hospital-acquired infections. Effectors produced by the type III secretion system disrupt mammalian cell membrane trafficking and signaling and are integral to the establishment of P. aeruginosa infection. One of these effectors, ExoS, ADP-ribosylates several host cell proteins, including Ras and Rab GTPases. In this study, we demonstrated that Rab5 plays a critical role during early stages of P. aeruginosa invasion of J774-Eclone macrophages. We showed that live, but not heat-inactivated, P. aeruginosa inhibited phagocytosis and that this occurred in conjunction with downregulation of Rab5 activity. Inactivation of Rab5 was dependent on ExoS ADP-ribosyltransferase activity, and in J744-Eclone cells, ExoS ADP-ribosyltransferase activity caused a more severe inhibition of phagocytosis than ExoS Rho GTPase activity. Furthermore, we found that expression of Rin1, a Rab5 guanine exchange factor, but not Rabex5 and Rap6, partially reversed the inactivation of Rab5 during invasion of live P. aeruginosa. These studies provide evidence that live P. aeruginosa cells are able to influence their rate of phagocytosis in macrophages by directly regulating activation of Rab5.

Saw palmetto ethanol extract inhibits adipocyte differentiation.

The fruits of saw palmetto have been used for the treatment of a variety of urinary and reproductive system problems. In this study we investigated whether the fruit extracts affect in vitro adipogenesis. Saw palmetto ethanol extract inhibited the lipid droplet accumulation by induction media in a dose-dependent manner, and it also attenuated the protein expressions of C-EBPα and PPARγ. Phosphorylation of Erk1/2 and Akt1 were also decreased by saw palmetto ethanol extract. This report suggests that saw palmetto extracts selectively affect the adipocyte differentiation through the modulation of several key factors that play a critical role during adipogenesis.

Effect of EGF-receptor tyrosine kinase inhibitor on Rab5 function during endocytosis.

Tyrosine autophosphorylation within the cytoplasmic tail of EGF-receptor is a key event, which in turn recruits several factors including Shc, Grb2 and Rin1 that are essential activities for receptor-mediated endocytosis and signaling. In this study, we demonstrated that treatment with AG1478, an EGF-receptor kinase inhibitor, blocked the formation of Rab5-positive endosomes as well as the activation of Rab5 upon addition of EGF. We also found that EGF-receptor catalytically inactive mutant failed to activate Rab5 upon EGF stimulation. Additionally, endosomal co-localization of Rab5 and EGF-receptor was inhibited by AG1478. Interestingly, AG1478 inhibitor did not block the formation of enlarged Rab5-positive endosomes in cells expressing Rab5 GTP hydrolysis defective mutant (Rab5:Q79L). AG1478 inhibitor also blocked the in vitro endosome fusion in a concentration-dependent manner, and more importantly, Rab5:Q79L mutant rescued it. Furthermore, addition of Rin1, a Rab5 guanine nucleotide exchange factor, partially restored endosome fusion in the presence of AG1478 inhibitor. Consistent with these observations, we also observed that Rin1 was unable to localize to membranes upon EGF-stimulation in the presence of AG1478 inhibitor. These results constitute first evidence that the enzymatic activity of a tyrosine kinase receptor is required endosome fusion via the activation of Rab5.

Inhibition of Rab5 Activation During Insulin Receptor-Mediated Endocytosis.

Activation of receptor tyrosine kinases is a key feature in receptor signaling and membrane trafficking processes. In this study, we found that the insulin receptor tyrosine kinase activity is required for fusion between early endosomes. AG1024, a receptor tyrosine kinase inhibitor, blocked the in vitro endosome fusion in a concentration-dependent manner. We observed that Rab5: wild type partially rescued the fusion reaction, whereas Rab5: Q79L mutant fully rescued it. We also observed that treatment of cells with insulin receptor kinase inhibitor HNMPA-(AM)3 blocked the formation of Rab5-positive endosomes as well as the activation of Rab5 upon addition of insulin in intact cells. HNMPA-(AM)3 inhibitor also affected the endosomal co-localization of Rab5 and insulin receptor. However, the formation of Rab5: Q79L mutant-positive endosomes were not affected by the HNMPA-(AM)3 inhibitor. In addition, HNMPA-(AM)3 inhibitor affected the association of Rin1 to membrane upon insulin stimulation. Furthermore, Rin1 did not fully support endosome fusion in the presence of the AG1024 inhibitor. These results constitute the first evidence that, at least in part, the enzymatic activity of insulin receptor is required for the fusion events via the activation of Rab5.

Functional determinants of ras interference 1 mutants required for their inhbitory activity on endocytosis.

In this study, we initiated experiments to address the structure-function relationship of Rin1. A total of ten substitute mutations were created, and their effects on Rin1 function were examined. Of the ten mutants, four of them (P541A, E574A, Y577F, T580A) were defective in Rab5 binding, while two other Rin1 mutants (D537A, Y561F) partially interacted with Rab5. Mutations in several other residues (Y506F, Y523F, T572A, Y578F) resulted in partial loss of Rab5 function. Biochemical studies showed that six of them (D537A, P541A, Y561F, E574A, Y577F, T580A) were unable to activate Rab5 in an in vitro assay. In addition, Rin1: D537A and Rin1: Y561F mutants showed dominant inhibition of Rab5 function. Consistent with the biochemical studies, we observed that these two Rin1 mutants have lost their ability to stimulate the endocytosis of EGF, form enlarged Rab5-positive endosomes, or support in vitro endosome fusion. Based on these data, our results showed that mutations in the Vps9 domain of Rin1 lead to a loss-of-function phenotype, indicating a specific structure-function relationship between Rab5 and Rin1.

Inhibition of early endosome fusion by Rab5-binding defective Ras interference 1 mutants.

Rin1 has been shown to play an important role in endocytosis. In this study we demonstrated that depletion of Rin1 from the cytosol blocked the fusion reaction. More importantly, endosome fusion was rescued by the addition of Rin1 proteins depending on the presence of Rab5, and its effector EEA1. Furthermore, we found that Syntaxin 13, but not Syntaxin 7, was required by Rin1 to support endosome fusion. We also identified six mutations on the Vps9 domain of Rin1 that failed to rescue the fusion reaction. Two of them, Rin1: D537A and Rin1: Y561F mutants showed dramatic inhibitory effect on the fusion reaction, which correlate with their inability to properly activate Rab5 or to bind endosomal membranes. Taken together, our results suggest that specific residues on the Vsp9 domain of Rin1 are required for its interaction with Rab5, binding to the endosomal membranes and subsequent regulation of the fusion reaction.

The hominoid-specific oncogene TBC1D3 activates Ras and modulates epidermal growth factor receptor signaling and trafficking.

Hominoid- and human-specific genes may have evolved to modulate signaling pathways of a higher order of complexity. TBC1D3 is a hominoid-specific oncogene encoded by a cluster of eight paralogs on chromosome 17. Initial work indicates that TBC1D3 is widely expressed in human tissues ( Hodzic, D., Kong, C., Wainszelbaum, M. J., Charron, A. J., Su, X., and Stahl, P. D. (2006) Genomics 88, 731-736 ). In this study, we show that TBC1D3 expression has a powerful effect on cell proliferation that is further enhanced by epidermal growth factor (EGF) in both human and mouse cell lines. EGF activation of the Erk and protein kinase B/Akt pathways is enhanced, both in amplitude and duration, by TBC1D3 expression, whereas RNA interference silencing of TBC1D3 suppresses the activation. Light microscopy and Western blot experiments demonstrate that increased signaling in response to EGF is coupled with a significant delay in EGF receptor (EGFR) trafficking and degradation, which significantly extends the life span of EGFR. Moreover, TBC1D3 suppresses polyubiquitination of the EGFR and the recruitment of c-Cbl. Using the Ras binding domain of Raf1 to monitor GTP-Ras we show that TBC1D3 expression enhances Ras activation in quiescent cells, which is further increased by EGF treatment. We speculate that TBC1D3 may alter Ras GTP loading. We conclude that the expression of TBC1D3 generates a delay in EGFR degradation, a decrease in ubiquitination, and a failure to recruit adapter proteins that ultimately dysregulate EGFR signal transduction and enhance cell proliferation. Altered growth factor receptor trafficking and GTP-Ras turnover may be sites where recently evolved genes such as TBC1D3 selectively modulate signaling in hominoids and humans.

IL4/PGE2 induction of an enlarged early endosomal compartment in mouse macrophages is Rab5-dependent.

The endosomal compartment and the plasma membrane form a complex partnership that controls signal transduction and trafficking of different molecules. The specificity and functionality of the early endocytic pathway are regulated by a growing number of Rab GTPases, particularly Rab5. In this study, we demonstrate that IL4 (a Th-2 cytokine) and prostaglandin E2 (PGE2) synergistically induce Rab5 and several Rab effector proteins, including Rin1 and EEA1, and promote the formation of an enlarged early endocytic (EEE) compartment. Endosome enlargement is linked to a substantial induction of the mannose receptor (MR), a well-characterized macrophage endocytic receptor. Both MR levels and MR-mediated endocytosis are enhanced approximately 7-fold. Fluid-phase endocytosis is also elevated in treated cells. Light microscopy and fractionation studies reveal that MR colocalizes predominantly with Rab5a and partially with Rab11, an endosomal recycling pathway marker. Using retroviral expression of Rab5a:S34N, a dominant negative mutant, and siRNA Rab5a silencing, we demonstrate that Rab5a is essential for the large endosome phenotype and for localization of MR in these structures. We speculate that the EEE is maintained by activated Rab5, and that the EEE phenotype is part of some macrophage developmental program such as cell fusion, a characteristic of IL4-stimulated cells.

Rab22a regulates the sorting of transferrin to recycling endosomes.

Rab22a is a member of the Rab family of small GTPases that localizes in the endocytic pathway. In CHO cells, expression of canine Rab22a (cRab22a) causes a dramatic enlargement of early endocytic compartments. We wondered whether transferrin recycling is altered in these cells. Expression of the wild-type protein and a GTP hydrolysis-deficient mutant led to the redistribution of transferrin receptor to large cRab22a-positive structures in the periphery of the cell and to a significant decrease in the plasma membrane receptor. Kinetic analysis of transferrin uptake indicates that internalization and early recycling were not affected by cRab22a expression. However, recycling from large cRab22a-positive compartments was strongly inhibited. A similar effect on transferrin transport was observed when human but not canine Rab22a was expressed in HeLa cells. After internalization for short periods of time (5 to 8 min) or at a reduced temperature (16 degrees C), transferrin localized with endogenous Rab22a in small vesicles that did not tubulate with brefeldin A, suggesting that the endogenous protein is present in early/sorting endosomes. Rab22a depletion by small interfering RNA disorganized the perinuclear recycling center and strongly inhibited transferrin recycling. We speculate that Rab22a controls the transport of the transferrin receptor from sorting to recycling endosomes.

Role of rab5 in EGF receptor-mediated signal transduction.

Activated epidermal growth factor receptor (EGFR) recruits intracellular proteins that mediate receptor trafficking and signaling. Rab5 and Rin1, a multifunctional protein with a Rab5 guanine nucleotide exchange factor domain, have been shown to regulate EGFR endocytosis (Barbieri et al., 2000; Tall et al., 2001). In this study, we demonstrate that overexpression of both dominant negative Rab5 (Rab5:S34N) and full-length Rin1 selectively block EGF activation of the Raf-Erk1/2 kinase pathway and EGF-stimulated incorporation of [3H]thymidine into DNA without affecting the activity of JN and p38 kinase pathways. Expression of Rab5:S34N and Rin1 also block EGF induction of cyclin D1 transcription. In contrast, expression of Rin1:delta, a natural splice variant of Rin1 lacking 47 amino acids in the Vps9p domain or Rab5, increase both activation of Raf-Erk1/2- and cyclin D1 transcription in response to EGF. These results indicate that Rab5 and the Raf/Erk signal transduction pathway play essential and selective roles in EGF-induced cell proliferation, and highlight a new function for Rab5 in EGF signaling.

The SRC homology 2 domain of Rin1 mediates its binding to the epidermal growth factor receptor and regulates receptor endocytosis.

Activated epidermal growth factor receptors (EGFRs) recruit intracellular proteins that mediate receptor signaling and endocytic trafficking. Rin1, a multifunctional protein, has been shown to regulate EGFR internalization (1). Here we show that EGF stimulation induces a specific, rapid, and transient membrane recruitment of Rin1 and that recruitment is dependent on the Src homology 2 (SH2) domain of Rin1. Immunoprecipitation of EGFR is accompanied by co-immunoprecipitation of Rin1 in a time- and ligand-dependent manner. Association of Rin1 and specifically the SH2 domain of Rin1 with the EGFR was dependent on tyrosine phosphorylation of the intracellular domain of the EGFR. The recruitment of Rin1, observed by light microscopy, indicated that although initially cytosolic, Rin1 was recruited to both plasma membrane and endosomes following EGF addition. Moreover, the expression of the SH2 domain of Rin1 substantially impaired the internalization of EGF without affecting internalization of transferrin. Finally, we found that Rin1 co-immunoprecipitated with a number of tyrosine kinase receptors but not with cargo endocytic receptors. These results indicate that Rin1 provides a link via its SH2 domain between activated tyrosine kinase receptors and the endocytic pathway through the recruitment and activation of Rab5a.

1,25-Dihydroxyvitamin D down-regulates cell membrane growth- and nuclear growth-promoting signals by the epidermal growth factor receptor.

1,25(OH)(2)D(3) antiproliferative properties are widely known. However, the molecular bases of these properties are only partially elucidated. Since 1,25(OH)(2)D(3) effectively arrests growth in many tumors and hyperplastic tissues whose growth is driven by co-expression of EGFR and its ligand TGF-alpha, it was hypothesized that 1,25(OH)(2)D(3) could affect the TGF-alpha/EGFR-autocrine growth loop. This study examined 1,25(OH)(2)D(3) regulation of EGFR-growth signals, using human epidermoid A431 cells, in which the overexpression of EGFR and TGF-alpha constitute the major autocrine mitogenic signal. 1,25(OH)(2)D(3) inhibited autocrine and EGF-induced A431 cell proliferation. Furthermore, 1,25(OH)(2)D(3) changed the cellular localization of both TGF-alpha and EGFR and inhibited ligand-dependent phosphorylation of EGFR and ERK1/2. In addition, 1,25(OH)(2)D(3) impaired autocrine and EGF-induced nuclear translocation of activated EGFR and, consequently, its binding to AT-rich DNA sequences and transcriptional activation of the cyclin D1 promoter. These results demonstrate that 1,25(OH)(2)D(3) alters EGFR membrane trafficking and down-regulates EGFR growth signaling.

Multivesicular bodies and multivesicular endosomes: the "ins and outs" of endosomal traffic.

Multivesicular endosomes (MVEs) are complex intracellular organelles that function in endocytosis. A major function of the endocytic pathway is to sort internalized macromolecules and membrane proteins. Appropriately sorted proteins, such as epidermal growth factor (EGF) receptor (EGFR), are incorporated into MVEs before transport to the lysosomal compartment, where degradation occurs. Thus, MVEs operate in the endosome-to-lysosome portion of the pathway. In yeast cells, where MVE formation has been extensively studied, the pathway terminates in the yeast vacuole, which is equivalent to the vertebrate lysosome. MVEs arise by invagination of the limiting membrane of an endosomal vesicle such that many small internal vesicles are formed, hence the term "multivesicular endosome." In part, the internalization and targeting of membrane proteins to the MVE involves ubiquitin, a small protein associated with protein degradation. In reticulocytes and certain antigen-presenting cells, MVEs are routed to the plasma membrane rather than the lysosome, releasing small vesicles called "exosomes" back into the extracellular space.