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RabGEF1, a negative regulator of Ras signalling, mast cell activation and skin inflammation
Abstract
Mast cell activation induced by the aggregation of FcepsilonRI with IgE and antigen is mediated through the activation of multiple protein kinase cascades. This process induces mast cells to undergo degranulation, to synthesize and release lipid mediators, and to secrete multiple cytokines, chemokines and growth factors. We found that RabGEF1 (Rabex-5) binds to Ras and negatively regulates Ras activation and downstream effector pathways during FcepsilonRI-dependent mouse mast cell activation. Mast cells derived from RabGEF1-deficient mice exhibit significantly enhanced levels of degranulation, release of lipid mediators and secretion of cytokines in response to FcepsilonRI aggregation. RabGEF1 knockout mice have increased perinatal mortality and the mice that do survive develop severe skin inflammation and increased numbers of mast cells in the dermis, some of which exhibit morphological evidence of degranulation. These mice also show elevated concentrations of serum histamine and IgE. Thus, RabGEF1 is a negative regulator of Ras signalling and FcepsilonRI-dependent mast cell activation in vitro, and a lack of RabGEF1 results in the development of elevated numbers of mast cells in the skin and severe skin inflammation in vivo.
... Additionally, the Rab5 GEF Rabex-5 was shown to promote Ras ubiquitination in Drosophila and the resultant E3 ligase activity of Rabex-5 decreased the activation of downstream effectors and thus suppressed oncogenic Ras transformation. 42,43 More recently, Rabex-5 has been identified in mammalian cells and has a similar role: Rabex-5 ubiquitinates H-Ras leading to its internalization into EEA1 positive early endosomes resulting in the quenching of downstream signaling. 44 As Rabex-5 activity would promote Rab5 mediated endocytosis at the plasma membrane, 45 the internalization of H-Ras was perhaps not surprising. ...
... In addition, as mentioned above, Rabex-5 can promote H-Ras ubiquitination and blunt its activity through endosomal sequestering. [42][43][44] The role Rab5 plays in this is as yet unclear, but it is interesting to note that another Rab5 GEF, Rin1, which can interact with activated Ras, is required for the endosomal localization of H-Ras suggesting this in some way involves Rab5. 44 The yeast GEF Vps9p acts upon the yeast homolog of Rab5 (Ypt21p) and has also been demonstrated to be mono-ubiquitinated. ...
The regulation of the small GTPases leading to their membrane localization has long been attributed to processing of their C-terminal CAAX box. As deregulation of many of these GTPases have been implicated in cancer and other disorders, prenylation and methylation of this CAAX box has been studied in depth as a possibility for drug targeting, but unfortunately, to date no drug has proved clinically beneficial. However, these GTPases also undergo other modifications that may be important for their regulation. Ubiquitination has long been demonstrated to regulate the fate of numerous cellular proteins and recently it has become apparent that many GTPases, along with their GAPs, GeFs and GDis, undergo ubiquitination leading to a variety of fates such as re-localization or degradation. in this review we focus on the recent literature demonstrating that the regulation of small GTPases by ubiquitination, either directly or indirectly, plays a considerable role in controlling their function and that targeting these modifications could be important for disease treatment.
... Rabex-5 is a guanine nucleotide exchange factor (GEF) for activation of Rab5 [1], a small GTPase that is associated with early endosomal membrane and regulates early endosome fusion and endocytosis2345. Rabex-5 knockout mice die early and develop severe skin inflammation [6], suggesting a non-redundant function in vivo. Mast cells isolated from Rabex-5 knockout mice show enhanced IgE receptor-mediated degranulation and cytokine release and these effects are due to the loss of Rabex-5 GEF activity for Rab5 [7]. ...
... Rabex-5 functions as a GEF for Rab5 activation in the cell and plays a critical role in regulation of early endosome fusion and endocytosis [1,10]. Rabex-5 knockout mice develop severe skin inflammation and die early, indicating a non-redundant and essential function in vivo [6]. Although there are other Vps9 domain-containing Rab5 GEFs, such as the RIN proteins, they are subject to temporal regulation and become active only upon growth factor-mediated activation and recruitment to early endosomes during signal transduction processes [18]. ...
Rabex-5 is a guanine nucleotide exchange factor (GEF) that specifically activates Rab5, i.e., converting Rab5-GDP to Rab5-GTP, through two distinct pathways to promote endosome fusion and endocytosis. The direct pathway involves a pool of membrane-associated Rabex-5 that targets to the membrane via an early endosomal targeting (EET) domain. The indirect pathway, on the other hand, involves a cytosolic pool of Rabex-5/Rabaptin-5 complex. The complex is recruited to the membrane via Rabaptin-5 binding to Rab5-GTP, suggesting a positive feedback mechanism. The relationship of these two pathways for Rab5 activation in the cell is unclear.
We dissect the relative contribution of each pathway to Rab5 activation via mathematical modeling and kinetic analysis in the cell. These studies show that the indirect pathway constitutes a positive feedback loop for converting Rab5-GDP to Rab5-GTP on the endosomal membrane and allows sensitive regulation of endosome fusion activity by the levels of Rab5 and Rabex-5 in the cell. The onset of this positive feedback effect, however, contains a threshold, which requires above endogenous levels of Rab5 or Rabex-5 in the cell. We term this novel phenomenon "delayed response". The presence of the direct pathway reduces the delay by increasing the basal level of Rab5-GTP, thus facilitates the function of the Rabex-5/Rabaptin-5-mediated positive feedback loop.
Our data support the mathematical model. With the model's guidance, the data reveal the affinity of Rabex-5/Rabaptin-5/Rab5-GTP interaction in the cell, which is quantitatively related to the Rabex-5 concentration for the onset of the indirect positive feedback pathway. The presence of the direct pathway and increased Rab5 concentration can reduce the Rabex-5 concentration required for the onset of the positive feedback loop. Thus the direct and indirect pathways cooperate in the regulation of early endosome fusion.
... Gray arrows indicate catalysis, black arrows indicate transformation and red blunt arrows indicate inhibition. finger (ZnF) domain similar to that of A20 with E3 ligase activity [49][50][51], and that Rabex-5 interacts with Ras [52,53]. Using in vivo and in vitro ubiquitination assays along with RNAi technology, the authors showed that Rabex-5 is necessary and sufficient to catalyse H/N-Ras ubiquitination, promoting their endosomal localisation and resulting in suppressed ERK activation ( Figure 2) [48]. ...
Ubiquitination, the covalent attachment of ubiquitin to target proteins, has emerged as a ubiquitous post-translational modification (PTM) whose function extends far beyond its original role as a tag for protein degradation identified three decades ago. Although sharing parallel properties with phosphorylation, ubiquitination distinguishes itself in important ways. Nevertheless, the interplay and crosstalk between ubiquitination and phosphorylation events have become a recurrent theme in cell signalling regulation. Understanding how these two major PTMs intersect to regulate signal transduction is an important research question. In this review, we first discuss the involvement of ubiquitination in the regulation of the EGF-mediated ERK signalling pathway via the EGF receptor, highlighting the interplay between ubiquitination and phosphorylation in this cancer-implicated system and addressing open questions. The roles of ubiquitination in pathways crosstalking to EGFR/MAPK signalling will then be discussed. In the final part of the review, we demonstrate the rich and versatile dynamics of crosstalk between ubiquitination and phosphorylation by using quantitative modelling and analysis of network motifs commonly observed in cellular processes. We argue that given the overwhelming complexity arising from inter-connected PTMs, a quantitative framework based on systems biology and mathematical modelling is needed to efficiently understand their roles in cell signalling.
... PTEN, phosphatase and tensin homolog (Di Cristofano et al., 1999) pyrin (French FMF Consortium, 1997;International Mediterranean Fever Consortium, 1997) RabGEF1, guanine nucleotide exchange factor (Tam et al., 2005) RelB ( Barton et al., 2000;Burkly et al., 1995;Weih et al., 1995) RUNX3 (Fainaru et al., 2005) RXRα/β, retinoid X receptor alpha/beta (double deletion) (Li et al., 2005) SAP, serum amyloid P component (Bickerstaff et al., 1999) SH3BP2, src homology 3 domain binding protein 2 (Ueki et al., 2007) SHIP, src homology-2 domain-containing inositol 5-phosphatase (Helgason et al., 1998) 5 SHP-1, src homology-2 domain-containing protein tyrosine phosphatase (Shultz et al., 1993;Tsui et al., 1993) SOCS1, suppressor of cytokine singaling 1 (Alexander et al., 1999) surfactant protein D (Wert et al., 2000) TAK1, transforming growth factor beta-activated kinase 1 (Kajino-Sakamoto et al., 2008;Omori et al., 2006) T-bet (Finotto et al., 2002) TcR-α, T cell receptor alpha (Mombaerts et al., 1993) TcR-β, T cell receptor beta (Mombaerts et al., 1993) TGF-β1, transforming growth factor beta 1 (Kulkarni et al., 1993;Shull et al., 1992) TIA-1 (named for a monoclonal antibody) (Phillips et al., 2004) TIPE2, TNF-α-induced protein-2 (Sun et al., 2008) TNFR1, TNF receptor 1 (McDermott et al., 1999) TRAF6, TNF receptor associated factor 6 ( King et al., 2006) tristetraproline (Taylor et al., 1996) TSAd, T cell specific adaptor protein (Drappa et al., 2003) STAMP2, six-transmembrane protein of prostate 2 (Wellen et al., 2007) WASP, Wiskott-Aldrich syndrome protein (Leverrier et al., 2001;Snapper et al., 1998;Sullivan et al., 1994) XBP1, X box binding protein 1 (Kaser et al., 2008) ZAP70, T cell receptor zeta chain associated protein (Sakaguchi et al., 2003) ...
Nonresolving inflammation is a major driver of disease. Perpetuation of inflammation is an inherent risk because inflammation can damage tissue and necrosis can provoke inflammation. Nonetheless, multiple mechanisms normally ensure resolution. Cells like macrophages switch phenotypes, secreted molecules like reactive oxygen intermediates switch impact from pro- to anti-inflammatory, and additional mediators of resolution arise, including proteins, lipids, and gasses. Aside from persistence of initiating stimuli, nonresolution may result from deficiencies in these mechanisms when an inflammatory response begins either excessively or subnormally. This greatly complicates the development of anti-inflammatory therapies. The problem calls for conceptual, organizational, and statistical innovations.
Supplemental references.
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The receptor tyrosine kinase, c-kit, and its ligand, stem cell factor (SCF), function in a diverse range of biological functions. The role of c-kit in the maintenance and survival of hematopoietic stem cells and of mast cells is well recognized. c-kit also plays an important role in melanogenesis, erythropoiesis and spermatogenesis. Recent work from our laboratory highlights an important role of c-kit in the regulation of expression of two molecules in dendritic cells (DCs), interleukin-6 (IL-6) and Jagged-2 (a ligand of Notch), which are known to regulate T helper cell differentiation. Our study shows that induction of c-kit expression and its signaling in DCs promotes Th2 and Th17 responses but not Th1 response. c-kit inhibition by imatinib mesylate (Gleevec) in DCs was previously shown to promote natural killer cell activation which may be due to dampening of IL-6 production by the DCs. Since dysregulation of c-kit function has been associated with various disease states including cancer, in this perspective we have focused on known and novel functions of c-kit to include molecules such as IL-6 and Notch that were not previously recognized to be within the purview of c-kit biology. We have also reviewed the differential expression pattern of SCF and c-kit on various cell types and its variation during development or pathology. The recognition of previously unappreciated roles for c-kit will provide better insights into its function within and beyond the immune system and pave the way for developing better therapeutic strategies.
Mast cells are tissue-resident cells with important functions in allergy and inflammation. Pluripotential hematopoietic stem cells in the bone marrow give rise to committed mast cell progenitors that transit via the blood to tissues throughout the body, where they mature. Knowledge is limited about the factors that release mast cell progenitors from the bone marrow or recruit them to remote tissues. Mouse femoral bone marrow cells were cultured with IL-3 for 2 wk and a range of chemotactic agents were tested on the c-kit(+) population. Cells were remarkably refractory and no chemotaxis was induced by any chemokines tested. However, supernatants from activated mature mast cells induced pronounced chemotaxis, with the active principle identified as leukotriene (LT) B(4). Other activation products were inactive. LTB(4) was highly chemotactic for 2-wk-old cells, but not mature cells, correlating with a loss of mRNA for the LTB(4) receptor, BLT1. Immature cells also accumulated in vivo in response to intradermally injected LTB(4). Furthermore, LTB(4) was highly potent in attracting mast cell progenitors from freshly isolated bone marrow cell suspensions. Finally, LTB(4) was a potent chemoattractant for human cord blood-derived immature, but not mature, mast cells. These results suggest an autocrine role for LTB(4) in regulating tissue mast cell numbers.
Psychological conditions, including stress, compromise immune defenses. Although this concept is not novel, the molecular mechanism behind it remains unclear. Neuropeptide Y (NPY) in the central nervous system is a major regulator of numerous physiological functions, including stress. Postganglionic sympathetic nerves innervating lymphoid organs release NPY, which together with other peptides activate five Y receptors (Y1, Y2, Y4, Y5, and y(6)). Using Y1-deficient (Y1(-/-)) mice, we showed that Y1(-/-) T cells are hyperresponsive to activation and trigger severe colitis after transfer into lymphopenic mice. Thus, signaling through Y1 receptor on T cells inhibits T cell activation and controls the magnitude of T cell responses. Paradoxically, Y1(-/-) mice were resistant to T helper type 1 (Th1) cell-mediated inflammatory responses and showed reduced levels of the Th1 cell-promoting cytokine interleukin 12 and reduced interferon gamma production. This defect was due to functionally impaired antigen-presenting cells (APCs), and consequently, Y1(-/-) mice had reduced numbers of effector T cells. These results demonstrate a fundamental bimodal role for the Y1 receptor in the immune system, serving as a strong negative regulator on T cells as well as a key activator of APC function. Our findings uncover a sophisticated molecular mechanism regulating immune cell functions that can lead to stress-induced immunosuppression.
The high-affinity Fc receptor for IgE (FcepsilonRI), a multimeric immune receptor, is a crucial structure for IgE-mediated allergic reactions. In recent years, advances have been made in several important areas of the study of FcepsilonRI. The first area relates to FcepsilonRI-mediated biological responses that are antigen independent. The second area encompasses the biological relevance of the distinct signalling pathways that are activated by FcepsilonRI; and the third area relates to the accumulated evidence for the tight control of FcepsilonRI signalling through a broad array of inhibitory mechanisms, which are being developed into promising therapeutic approaches.
The significant contributions this past year to our understanding of IgE receptor (Fc epsilon RI) signaling in mast cells include studies with truncated Syk in a vaccinia expression system and Syk-negative variants of rat basophilic (RBL-2H3) cells. These studies demonstrate an essential role for Syk in initiating signals for secretion and release of arachidonic acid via phospholipase A2 and mitogen-activated protein kinase. A newly recognized addition to the repertoire of Fc epsilon RI-mediated signaling systems is the activation of sphingosine kinase, which contributes to calcium mobilization in mast cells. Advances have been made in our understanding of other receptors that regulate proliferation and differentiation of mast cells, and in our understanding of the ability of mast cells to mount acquired and acute responses to antigenic and bacterial challenge.