共查询到20条相似文献,搜索用时 46 毫秒
1.
C B Thien D D Bowtell W Y Langdon 《Journal of immunology (Baltimore, Md. : 1950)》1999,162(12):7133-7139
Recent studies indicate that c-Cbl and its oncogenic variants can modulate the activity of protein tyrosine kinases. This finding is supported by studies showing that c-Cbl interacts directly with a negative regulatory tyrosine in ZAP-70, and that the levels of tyrosine-phosphorylated ZAP-70 and numerous other proteins are increased in TCR-stimulated thymocytes from c-Cbl-deficient mice. Here, we demonstrate that this enhanced phosphorylation of ZAP-70 and that of two substrates, LAT and SLP-76, is not due to altered protein levels but is the consequence of two separate events. First, we find increased expression of tyrosine-phosphorylated TCRzeta chain in c-Cbl-deficient thymocytes, which results in a higher level of zeta-chain-associated ZAP-70 that is initially accessible for activation. Thus, more ZAP-70 is activated and more of its substrates (LAT and SLP-76) become tyrosine-phosphorylated after TCR stimulation. However, an additional mechanism of ZAP-70 regulation is evident at a later time poststimulation. At this time, ZAP-70 from both normal and c-Cbl-/- thymocytes becomes hyperphosphorylated; however, only in normal thymocytes does this correlate with ZAP-70 down-regulation and a diminished ability to phosphorylate LAT and SLP-76. In contrast, c-Cbl-deficient thymocytes display altered phosphorylation kinetics, for which LAT phosphorylation is increased and SLP-76 phosphorylation is sustained. Thus, the ability to down-regulate the phosphorylation of two ZAP-70 substrates is impaired in c-Cbl-/- thymocytes. These findings provide evidence that c-Cbl is involved in the negative regulation of the phosphorylation of LAT and SLP-76 by ZAP-70. 相似文献
2.
3.
Transforming growth factor-beta (TGF-beta) is thought to regulate ductal and lobuloalveolar development as well as involution in the mammary gland. In an attempt to understand the role TGF-beta plays during normal mammary gland development, and ultimately cancer, we previously generated transgenic mice that express a dominant-negative TGF-beta type II receptor under control of the metallothionine promoter (MT-DNIIR). Upon stimulation with zinc sulfate, the transgene was expressed in the mammary stroma and resulted in an increase in ductal side branching. In this study, mammary gland transplantation experiments confirm that the increase in side branching observed was due to DNIIR activity in the stroma. Development during puberty through the end buds was also accelerated. Cbl is a multifunctional intracellular adaptor protein that regulates receptor tyrosine kinase ubiquitination and downregulation. Mice with a targeted disruption of the c-Cbl gene displayed increased side branching similar to that observed in MT-DNIIR mice; however, end bud development during puberty was normal. Transplantation experiments showed that the mammary stroma was responsible for the increased side branching observed in Cbl-null mice. Cbl expression was reduced in mammary glands from DNIIR mice compared to controls and TGF-beta stimulated expression of Cbl in cultures of primary mammary fibroblasts. In addition, both TGF-beta and Cbl regulated platelet-derived growth factor receptor-alpha (PDGFR alpha) expression in vivo and in isolated mammary fibroblasts. The hypothesis that TGF-beta mediates the levels of PDGFR alpha protein via regulation of c-Cbl was tested. We conclude that TGF-beta regulates PDGFR alpha in the mammary stroma via a c-Cbl-independent mechanism. Finally, the effects of PDGF-AA on branching were determined. Treatment in vivo with PDGF-AA did not affect branching making a functional interaction between TGF-beta and PDGF unlikely. 相似文献
4.
Zhang J Chiang YJ Hodes RJ Siraganian RP 《Journal of immunology (Baltimore, Md. : 1950)》2004,173(3):1811-1818
The Cbl family of proteins negatively regulate signaling from tyrosine kinase-coupled receptors. Among the three members of this family, only c-Cbl and Cbl-b are expressed in hemopoietic cells. To examine the role of c-Cbl and Cbl-b in Fc epsilon RI signaling, mast cell cultures from wild-type, c-Cbl(-/-), and Cbl-b(-/-) mice were generated. Cell growth rates and cell surface expression of Fc epsilon RI were similar in the different cell populations. Compared with control cells, Cbl-b inactivation resulted in increases in Fc epsilon RI-induced Ca(2+) response and histamine release. Fc epsilon RI-induced tyrosine phosphorylation of total cellular proteins, Syk, and phospholipase C-gamma was also enhanced by Cbl-b deficiency, whereas receptor-initiated phosphorylation of Vav, JNK, and p38 kinases was not changed in these cells. In contrast to Cbl-b, c-Cbl deficiency had no detectable effect on Fc epsilon RI-induced histamine release or on the phosphorylation of total cellular proteins or Syk. The absence of c-Cbl increased the phosphorylation of ERK after receptor stimulation, but resulted in slightly reduced p38 phosphorylation and Ca(2+) response. These results suggest that Cbl-b and c-Cbl have divergent effects on Fc epsilon RI signal transduction and that Cbl-b, but not c-Cbl, functions as a negative regulator of Fc epsilon RI-induced degranulation. 相似文献
5.
The linker phosphorylation site Tyr292 mediates the negative regulatory effect of Cbl on ZAP-70 in T cells 总被引:2,自引:0,他引:2
Rao N Lupher ML Ota S Reedquist KA Druker BJ Band H 《Journal of immunology (Baltimore, Md. : 1950)》2000,164(9):4616-4626
The protooncogene product Cbl has emerged as a negative regulator of tyrosine kinases. We have shown previously that Cbl binds to ZAP-70 through its N-terminal tyrosine kinase binding (TKB) domain. In this study, we demonstrate that overexpression of Cbl in Jurkat T cells decreases the TCR-induced phosphorylation of ZAP-70 and other cellular phosphoproteins. Coexpression of Cbl with ZAP-70 in COS cells reproduced the Cbl-induced reduction in the level of phosphorylated ZAP-70. The effect of Cbl was eliminated by the TKB-inactivating G306E mutation in Cbl as well as by a phenylalanine mutation of Tyr292 within the TKB domain binding site on ZAP-70. Notably, the oncogenic Cbl-70Z/3 mutant associated with ZAP-70, but did not reduce the levels of phosphorylated ZAP-70. Overexpression of Cbl, but not Cbl-G306E, in Jurkat T cells led to a decrease in the TCR-induced NF-AT luciferase reporter activity. Overexpression of the TKB domain itself, but not its G306E mutant, functioned in a dominant-negative manner and led to an increase in NF-AT reporter activity. Cbl-70Z/3-overexpressing cells exhibited an increase in both basal and TCR-induced NF-AT luciferase reporter activity, and this trend was reversed by the G306E mutation. Finally, by reconstituting a ZAP-70-deficient Jurkat T cell line, p116, we demonstrate that wild-type ZAP-70 is susceptible to the negative regulatory effect of Cbl, whereas the ZAP-70-Y292F mutant is resistant. Together, our results establish that the linker phosphorylation site Tyr292 mediates the negative regulatory effect of Cbl on ZAP-70 in T cells. 相似文献
6.
The 120-kDa proto-oncogenic protein c-Cbl is a multidomain adaptor protein that is phosphorylated in response to the stimulation of a broad range of cell surface receptors and participates in the assembly of signaling complexes that are formed as a result of the activation of various signal transduction pathways. Several structural features of c-Cbl, including the phosphotyrosine-binding domain, proline-rich domain, and motifs containing phosphotyrosine and phosphoserine residues, mediate the association of c-Cbl with other components of these complexes. In addition to those domains that have been demonstrated to play a role in the binding of c-Cbl to other signaling molecules, c-Cbl also contains a RING finger motif and a putative leucine zipper. In this study, we demonstrate that the previously identified putative leucine zipper mediates the formation of Cbl homodimers. Using the yeast two-hybrid system, we show that deletion of the leucine zipper domain is sufficient to abolish Cbl homodimerization, while Cbl mutants carrying extensive N-terminal truncations retain the ability to dimerize with the full-length Cbl. The requirement of the leucine zipper for the homodimerization of Cbl was confirmed by in vitro binding assays, using deletion variants of the C-terminal half of Cbl with and without the leucine zipper domain, and in cells using Myc and green fluorescent protein (GFP) N-terminal-tagged Cbl variants. In cells, the deletion of the leucine zipper caused a decrease in both the tyrosine phosphorylation of Cbl and its association with the epidermal growth factor receptor following stimulation with epidermal growth factor, thus demonstrating a role for the leucine zipper in c-Cbl's signaling functions. Thus, the leucine zipper domain enables c-Cbl to homodimerize, and homodimerization influences Cbl's signaling function, modulating the activity of Cbl itself and/or affecting Cbl's associations with other signaling proteins in the cell. 相似文献
7.
Takahashi Y Shimokawa N Esmaeili-Mahani S Morita A Masuda H Iwasaki T Tamura J Haglund K Koibuchi N 《FEBS letters》2011,585(12):1741-1747
Nerve growth factor (NGF) binding to its receptor TrkA, which belongs to the family of receptor tyrosine kinases (RTKs), is known to induce its internalization, endosomal trafficking and subsequent lysosomal degradation. The Cbl family of ubiquitin ligases plays a major role in mediating ubiquitination and degradation of RTKs. However, it is not known whether Cbl participates in mediating ubiquitination of TrkA. Here we report that c-Cbl mediates ligand-induced ubiquitination and degradation of TrkA. TrkA ubiquitination and degradation required direct interactions between c-Cbl and phosphorylated TrkA. c-Cbl and ubiquitinated TrkA are found in a complex after NGF stimulation and are degraded in lysosomes. Taken together, our data demonstrate that c-Cbl can induce downregulation of NGF-TrkA complexes through ubiquitination and degradation of TrkA. 相似文献
8.
c-Cbl is involved in Met signaling in B cells and mediates hepatocyte growth factor-induced receptor ubiquitination 总被引:9,自引:0,他引:9
Taher TE Tjin EP Beuling EA Borst J Spaargaren M Pals ST 《Journal of immunology (Baltimore, Md. : 1950)》2002,169(7):3793-3800
Hepatocyte growth factor/scatter factor (HGF) and its receptor tyrosine kinase Met are key regulators of epithelial motility and morphogenesis. Recent studies indicate that the HGF/Met pathway also plays a role in B cell differentiation, whereas uncontrolled Met signaling may lead to B cell neoplasia. These observations prompted us to explore HGF/Met signaling in B cells. In this study, we demonstrate that HGF induces strong tyrosine phosphorylation of the proto-oncogene product c-Cbl in B cells and increases Cbl association with the Src family tyrosine kinases Fyn and Lyn, as well as with phosphatidylinositol-3 kinase and CrkL. In addition, we demonstrate that c-Cbl mediates HGF-induced ubiquitination of Met. This requires the juxtamembrane tyrosine Y1001 (Y2) of Met, but not the multifunctional docking site (Y14/15) or any additional C-terminal tyrosine residues (Y13-16). In contrast to wild-type c-Cbl, the transforming mutants v-Cbl and 70Z/3 Cbl, which lack the ubiquitin ligase RING finger domain, suppress Met ubiquitination. Our findings identify c-Cbl as a negative regulator of HGF/Met signaling in B cells, mediating ubiquitination and, consequently, proteosomal degradation of Met, and suggest a role for Cbl in Met-mediated tumorigenesis. 相似文献
9.
Sprouty fine-tunes EGF signaling through interlinked positive and negative feedback loops 总被引:8,自引:0,他引:8
BACKGROUND: Growth factors and their receptor tyrosine kinases play pivotal roles in development, normal physiology, and pathology. Signal transduction is regulated primarily by receptor endocytosis and degradation in lysosomes ("receptor downregulation"). c-Cbl is an adaptor that modulates this process by recruiting binding partners, such as ubiquitin-conjugating enzymes. The role of another group of adaptors, Sprouty proteins, is less understood; although, studies in insects implicated the founder protein in the negative regulation of several receptor tyrosine kinases. RESULTS: By utilizing transfection of living cells, as well as reconstituted in vitro systems, we identified a dual regulatory mechanism that combines human Sprouty2 and c-Cbl. Upon activation of the receptor for the epidermal growth factor (EGFR), Sprouty2 undergoes phosphorylation at a conserved tyrosine that recruits the Src homology 2 domain of c-Cbl. Subsequently, the flanking RING finger of c-Cbl mediates poly-ubiquitination of Sprouty2, which is followed by proteasomal degradation. Because phosphorylated Sprouty2 sequesters active c-Cbl molecules, it impedes receptor ubiquitination, downregulation, and degradation in lysosomes. This competitive interplay occurs in endosomes, and it regulates the amplitude and longevity of intracellular signals. CONCLUSIONS: Sprouty2 emerges as an inducible antagonist of c-Cbl, and together they set a time window for receptor activation. When incorporated in signaling networks, the coupling of positive (Sprouty) to negative (Cbl) feedback loops can greatly enhance output diversification. 相似文献
10.
Cbl functions as an adaptor protein by interacting with other signalling molecules to form multimolecular complexes. Previous studies have proposed that Cbl is also a positive regulator of CrkL–C3G signalling, which leads to Rap1 activation. However, there is a lack of genetic evidence for a physiological function of Cbl in regulating this pathway. Here, we show that Cbl deficiency results in enhanced activation of Rap1. Cbl was shown to promote the ubiquitylation of CrkL without any apparent effect on its stability. Remarkably, the membrane translocation of C3G, its association with CrkL, and the guanine-nucleotide exchange activity of C3G were all increased in Cbl−/− thymocytes. Consistent with a function of Rap1 in integrin activation, enhanced integrin-mediated cell adhesion was also seen in Cbl−/− thymocytes. Thus, Cbl negatively regulates Rap1 activation, probably through a proteolysis-independent E3-ubiquitin-ligase activity of Cbl that modulates protein–protein interactions. 相似文献
11.
c-Cbl and Cbl-b E3 ubiquitin ligases are abundantly expressed in hemopoietic cells where they negatively regulate the activity and levels of many cell surface receptors and associated signaling molecules. By comparing bone marrow-derived mast cells from c-Cbl and Cbl-b-deficient mice it has recently been shown that Cbl-b is the dominant family member for negatively regulating signaling responses from high-affinity IgE receptors. In this study, we suggest that a possible reason for the greater enhancement of IgE receptor signaling in Cbl-b-deficient mice is the relatively higher levels of Cbl-b protein over c-Cbl in mast cells compared with other hemopoietic cells. We also directly compare mast cells from c-Cbl and Cbl-b-deficient mice and find that loss of Cbl-b, but not c-Cbl, increases cell growth, retards receptor internalization, and causes the sustained tyrosine phosphorylation of Syk and its substrates. However, loss of Cbl-b does not enhance the activation of ERK or Akt, nor does it promote a greater calcium response. Furthermore, loss of Cbl-b or c-Cbl does not increase levels of the Syk or Lyn protein tyrosine kinases. Most notable, however, is the extremely large increase in the production of proinflammatory cytokines TNF-alpha, IL-6, and MCP-1 by Cbl-b(-/-) mast cells compared with levels produced by c-Cbl(-/-) or wild-type cells. This marked induction, which appears to be restricted to these three cytokines, is dependent on IgE receptor activation and correlates with enhanced IkappaB kinase phosphorylation. Thus, Cbl-b functions as a potent negative regulator of cytokines that promote allergic and inflammatory reactions. 相似文献
12.
Binding of ZAP-70 to phosphorylated T-cell receptor zeta and eta enhances its autophosphorylation and generates specific binding sites for SH2 domain-containing proteins. 总被引:10,自引:3,他引:7
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
E N Neumeister Y Zhu S Richard C Terhorst A C Chan A S Shaw 《Molecular and cellular biology》1995,15(6):3171-3178
ZAP-70 is a protein tyrosine kinase thought to play a critical role in T-cell receptor (TCR) signal transduction. During T-cell activation, ZAP-70 binds to a conserved signalling motif known as the immune receptor tyrosine activating motif (ITAM) and becomes tyrosine phosphorylated. To determine whether binding of ZAP-70 to the phosphorylated ITAM was able to activate its kinase activity, we measured the kinase activity of ZAP-70 both when it was bound and when it was unbound to phosphorylated TCR subunits. The ability of ZAP-70 to phosphorylate itself, but not exogenous substrates, was enhanced when it was bound to the tyrosine-phosphorylated TCR zeta and eta chains or to a construct that contained duplicated epsilon ITAMs. No enhanced ZAP-70 autophosphorylation was noted when it was bound to tyrosine-phosphorylated CD3 gamma or epsilon. In addition, autophosphorylation of ZAP-70 when bound to zeta or eta resulted in the generation of multiple distinct ZAP-70 phosphorylated tyrosine residues which had the capacity to bind the SH2 domains of fyn, lck, GAP, and abl. As the effect was noted only when ZAP-70 was bound to TCR subunits containing multiple ITAMs, we propose that one of the roles of the tandem ITAMs is to facilitate the autophosphorylation of ZAP-70. Tyrosine-phosphorylated ZAP-70 then mediates downstream signalling by recruiting SH2 domain-containing signalling proteins. 相似文献
13.
Kozlov G Peschard P Zimmerman B Lin T Moldoveanu T Mansur-Azzam N Gehring K Park M 《The Journal of biological chemistry》2007,282(37):27547-27555
Ligand-induced down-regulation by the ubiquitin-protein ligases, c-Cbl and Cbl-b, controls signaling downstream from many receptor-tyrosine kinases (RTK). Cbl proteins bind to phosphotyrosine residues on activated RTKs to affect ligand-dependent ubiquitylation of these receptors targeting them for degradation in the lysosome. Both c-Cbl and Cbl-b contain a ubiquitin-associated (UBA) domain, which is important for Cbl dimerization and tyrosine phosphorylation; however, the mechanism of UBA-mediated dimerization and its requirement for Cbl biological activity is unclear. Here, we report the crystal structure of the UBA domain of c-Cbl refined to 2.1-A resolution. The structure reveals the protein is a symmetric dimer tightly packed along a large hydrophobic surface formed by helices 2 and 3. NMR chemical shift mapping reveals heterodimerization can occur with the related Cbl-b UBA domain via the same surface employed for homodimerization. Disruption of c-Cbl dimerization by site-directed mutagenesis impairs c-Cbl phosphorylation following activation of the Met/hepatocyte growth factor RTK and c-Cbl-dependent ubiquitination of Met. This provides direct evidence for a role of Cbl dimerization in terminating signaling following activation of RTKs. 相似文献
14.
The product of the cbl oncogene forms stable complexes in vivo with endogenous Crk in a tyrosine phosphorylation-dependent manner. 总被引:5,自引:1,他引:4
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The cellular homologs of the v-Crk oncogene product are composed exclusively of Src homology region 2 (SH2) and SH3 domains. v-Crk overexpression in fibroblasts causes cell transformation and elevated tyrosine phosphorylation of specific cellular proteins. Among these proteins is a 130-kDa protein, identified as p130cas, that forms a stable complex in vivo with v-Crk. We have explored the role of endogenous Crk proteins in Bcr-Abl-transformed cells. In the K562 human chronic myelogenous leukemia cell line, p130cas is not tyrosine phosphorylated or bound to Crk. Instead, Crk proteins predominantly associate with the tyrosine-phosphorylated proto-oncogene product of Cbl. In vitro analysis showed that this interaction is mediated by the SH2 domain of Crk and can be inhibited with a phosphopeptide containing the Crk-SH2 binding motif. In NIH 3T3 cells transformed by Bcr-Abl, c-Cbl becomes strongly tyrosine phosphorylated and associates with c-Crk. The complex between c-Crk and c-Cbl is also seen upon T-cell receptor cross-linking or with the transforming, tyrosine-phosphorylated c-Cbl. These results indicate that Crk binds to c-Cbl in a tyrosine phosphorylation-dependent manner, suggesting a physiological role for the Crk-c-Cbl complex in Bcr-Abl tyrosine phosphorylation-mediated transformation. 相似文献
15.
Ota S Hazeki K Rao N Lupher ML Andoniou CE Druker B Band H 《The Journal of biological chemistry》2000,275(1):414-422
The proto-oncogene product Cbl has emerged as a negative regulator of a number of protein-tyrosine kinases, including the ZAP-70/Syk tyrosine kinases that are critical for signaling in hematopoietic cells. The evolutionarily conserved N-terminal tyrosine kinase-binding domain is required for Cbl to associate with ZAP-70/Syk and for their subsequent negative regulation. However, the role of the remaining C-terminal regions of Cbl remains unclear. Here, we used a COS-7 cell reconstitution system to address this question. Analysis of a series of C-terminally truncated Cbl mutants revealed that the N-terminal half of the protein, including the TKB and RING finger domains, was sufficient to mediate negative regulation of Syk. Further truncations, which delete the RING finger domain, abrogated the negative regulatory effects of Cbl on Syk. Point mutations of conserved cysteine residues or a histidine in the RING finger domain, which are required for zinc binding, abrogated the ability of Cbl to negatively regulate Syk in COS-7 cells and Ramos B lymphocytic cells. In addition, Syk-dependent transactivation of a serum response element-luciferase reporter in transfected 293T cells was reduced by wild type Cbl; mutations of the RING finger domain or its deletion abrogated this effect. These results establish the RING finger domain as an essential element in Cbl-mediated negative regulation of a tyrosine kinase and reveal that the evolutionarily conserved N-terminal half of the protein is sufficient for this function. 相似文献
16.
The proto-oncogenic protein c-Cbl was discovered as the cellular form of v-Cbl, a retroviral transforming protein. This was followed over the years by important discoveries, which identified c-Cbl and other Cbl-family proteins as key players in several signaling pathways. c-Cbl has donned the role of a multivalent adaptor protein, capable of interacting with a plethora of proteins, and has been shown to positively influence certain biological processes. The identity of c-Cbl as an E3 ubiquitin ligase unveiled the existence of an important negative regulatory pathway involved in maintaining homeostasis in protein tyrosine kinase (PTK) signaling. Recent years have also seen the emergence of novel regulators of Cbl, which have provided further insights into the complexity of Cbl-influenced pathways. This review will endeavor to provide a summary of current studies focused on the effects of Cbl proteins on various biological processes and the mechanism of these effects. The major sections of the review are as follows: Structure and genomic organization of Cbl proteins; Phosphorylation of Cbl; Interactions of Cbl; Localization of Cbl; Mechanism of effects of Cbl: (a) Ubiquitylation-dependent events: This section elucidates the mechanism of Cbl-mediated downregulation of EGFR and details the PTK and non-PTKs targeted by Cbl. In addition, it addresses the functional requirements for E3 Ubiquitin ligase activity of Cbl and negative regulation of Cbl-mediated downregulation of PTKs, (b) Adaptor functions: This section discusses the mechanisms of adaptor functions of Cbl in mitogen-activated protein kinase (MAPK) activation, insulin signaling, regulation of Ras-related protein 1 (Rap1), PI-3' kinase signaling, and regulation of Rho-family GTPases and cytoskeleton; Biological functions: This section gives an account of the diverse biological functions of Cbl and includes the role of Cbl in transformation, T-cell signaling and thymus development, B-cell signaling, mast-cell degranulation, macrophage functions, bone development, neurite growth, platelet activation, muscle degeneration, and bacterial invasion; Conclusions and perspectives. 相似文献
17.
Y. Jeffrey Chiang Martha S. Jordan Reiko Horai Pamela L. Schwartzberg Gary A. Koretzky Richard J. Hodes 《The Journal of biological chemistry》2009,284(7):4429-4438
A signaling pathway involving ZAP-70, LAT, and SLP76 has been regarded as
essential for receptor-driven T cell development and activation. Consistent
with this model, mice deficient in SLP76 have a complete block at the double
negative 3 stage of T cell development. Recently, however, it has been
reported that inactivation of Cbl, a ubiquitin-protein isopeptide ligase,
partially rescues T cell development in SLP76-deficient mice. To probe the
influence of Cbl on domain-specific SLP76 functions, we reconstituted
SLP76-/- Cbl-/- mice with Slp76 transgenes
bearing mutations in each of three functional domains of SLP76 as follows:
Y3F, in which the amino-terminal tyrosine residues of SLP76 were mutated,
eliminating sites of SLP76 interaction with Vav, Nck, and Itk; Δ20, in
which 20 amino acids in the proline-rich region of SLP76 were deleted,
removing a binding site for Gads; and RK, in which arginine 448 of SLP76 was
replaced by lysine, abolishing function of the Src homology 2 domain. Although
each of these transgenes has been shown to partially rescue T cell development
in SLP76-/- mice, we report here that Cbl inactivation completely
reverses the severe double negative 3 developmental block that occurs in
SLP76-deficient mice expressing the Y3F transgene (Y3F mice) and
partially rescues the defect in positive selection in T cell receptor
transgenic Y3F mice, but in contrast fails to rescue thymic development of
SLP76-deficient mice expressing the Δ20 or RK transgene. Rescue in
SLP76-/-Cbl-/-Y3F double-positive thymocytes is
associated with enhanced tyrosine phosphorylation of signaling molecules,
including Lck, Vav, PLC-γ1, and ERKs, but not Itk, in response to T cell
receptor stimulation. Thus, our data demonstrate that Cbl suppresses
activation of a bypass signaling pathway and thereby enforces SLP76 dependence
of early T cell development.T cell development proceeds through multiple stages that regulate the
generation and selection of T cells whose T cell receptors
(TCR)2 have an
appropriate range of affinity for peptides presented by major
histocompatibility complex (MHC) molecules
(1). Precursors give rise to
immature CD4-CD8- double negative (DN) cells that can be
further divided into DN1, DN2, DN3, and DN4 stages, distinguished by cell
surface phenotype as well as by critical events, including expansion of DN3
cells that have successfully rearranged TCRβ and have expressed and
signaled through the pre-TCR complex
(2). DN3 cells differentiate to
the DN4 and then CD4+CD8+ double-positive (DP) stage
following pre-TCR signaling. DP thymocytes rearrange TCRα, express a
mature TCRαβ receptor, and develop into mature
CD4+CD8- or CD4-CD8+
single-positive (SP) cells through a process of positive and negative
selection that is based on signaling through this mature TCR and selection of
a T cell repertoire that is tolerant to self but capable of responding to
foreign-peptide-MHC (pMHC) complexes
(1,
3,
4). Finally, SP cells exit from
the thymus as mature T cells capable of recognizing and responding to foreign
antigens.The signals from pre-TCR and TCR, which determine the fate of developing
thymocytes, have been intensely studied. Ligation of the TCR by pMHC complexes
results in activation of a signaling cascade initiated by phosphorylation and
activation of TCR-ζ, Lck, and ZAP-70, which in turn phosphorylate
downstream targets, including LAT and SLP76. ZAP-70, LAT and SLP76 proteins
(3) have been shown to be
essential for thymocyte development by studies, including genetic manipulation
in mice
(5–8).
There are essentially no detectable DP or SP thymocytes or peripheral T cells
in LAT-/- or SLP76-/- mice, in which thymocyte
development is blocked at the DN3 stage
(5,
7). ZAP70-/-
thymocytes are blocked at the DP stage of T cell development, and
ZAP70-/- mice have very few SP thymocytes or peripheral T cells
(6). These studies suggest that
signal transduction required for early T cell development proceeds through a
pathway that involves critical roles of multiple molecules, including ZAP-70,
LAT, and SLP76.SLP76 consists of three functional domains as follows: an amino-terminal
domain containing targets for tyrosine phosphorylation, a proline-rich region,
and a carboxyl-terminal SH2 domain
(9). The amino-terminal
tyrosine residues (Tyr-112, Tyr-128, and Tyr-145) are phosphorylated by
tyrosine kinases following TCR engagement, enabling SLP76 to interact with
Vav, a Rho guanine nucleotide exchange factor, Nck, an adaptor protein, and
Itk, a member of Tec family PTK. The proline-rich region of SLP76 has the
capacity to bind Gads, a Grb2 homolog, which results in the recruitment of
SLP76 to cell surface membrane lipid rafts through binding to LAT following
TCR engagement. The carboxyl-terminal SH2 domain of SLP76 interacts with ADAP
(adhesion and degranulation-promoting protein)
(10) an adaptor protein, and
HPK-1, a serine kinase (9).
Reconstitution of SLP76-deficient mice with transgenes containing mutations in
each of these domains has demonstrated that each region is required for normal
thymocyte development (5,
8). Two groups have
reconstituted SLP76-deficient mice with T cell-specific expression of
wild-type and mutant SLP76 transgenes, including a mutant in which three
tyrosine residues (Tyr-112, Tyr-128, and Tyr-145) in the amino-terminal domain
of SLP76 were substituted by phenylalanines (Y3F); a mutant in which 20 amino
acids (amino acids 224–244) in the proline-rich region of SLP76 were
deleted (Δ20); and a mutant in which arginine 448 of SLP76 was replaced
by lysine (RK) (11,
12). The profound defects in T
cell development and activation that are observed in SLP76 knock-out mice are
completely reversed by reconstitution with a wild-type SLP76 transgene. In
contrast, however, reconstitution with SLP76 that has been mutated in any of
its three functional domains only partially rescues T cell development in
SLP76 knock-out mice.c-Cbl (Cbl) is a ubiquitin ligase and adaptor protein (regulator) with
multiple domains that associate with multiple molecules involved in signal
transduction (13). Thymocytes
from Cbl knock-out mice have enhanced cell surface expression of TCR and CD3
in comparison with control mice
(14,
15). In addition, it has been
observed that phosphorylation of ZAP-70, LAT, and SLP76 is increased in
Cbl-/- mouse thymocytes
(14,
15). Recently, we reported
that inactivation of Cbl partially rescues T cell development in LAT and
SLP76-deficient mice (16), and
Myers et al. (17)
reported that inactivation of Cbl partially rescues T cell development in
ZAP-70-deficient mice. These observations indicate that Cbl mediates
requirements for LAT, SLP76, and ZAP-70 by preventing signaling that is
capable of supporting T cell differentiation independent of LAT, SLP76, or
ZAP-70. However, the rescue of T cell development in these model systems is
strikingly incomplete, failing to substantially reconstitute development
through the pre-TCR-dependent DN3-DN4 transition and thus failing to generate
normal numbers of DP or functionally mature SP thymocytes. These findings
suggest that Cbl inactivation functions to enable pathways that are capable of
bypassing some but not all of the requirements for ZAP-70, LAT, and SLP76
during T cell development. To define these signaling pathways, normally
suppressed by Cbl, that can support T cell development, we assessed the
ability of Cbl inactivation to rescue T cell development in the presence of
Y3F, Δ20, or RK SLP76 mutant transgenes. In this study, we report that
Cbl inactivation completely reverses the DN3-DN4 developmental defect and
partially reverses alterations in positive selection in thymocytes of SLP76
knock-out mice reconstituted with the SLP76 mutant Y3F, which lacks
amino-terminal phosphotyrosine residues. In contrast, Cbl inactivation has no
effect on the thymic developmental defects observed in SLP76 knock-out mice
reconstituted with Slp76 transgenes mutated in the proline-rich
Gads-binding region (Δ20) or the carboxyl-terminal SH2 domain (RK).
Biochemical studies revealed that rescue of development in
SLP76-/-Y3F thymocytes by inactivation of Cbl was marked by
reversal of defects in tyrosine phosphorylation of multiple molecules,
including Lck, Vav, PLC-γ1, and ERKs in response to TCR stimulation of
DP thymocytes. Thus, Cbl normally enforces SLP76 dependence of T cell
development by inhibiting an alternative pathway that may be independent of
SLP76 association with Vav, Nck, and Itk
(18). 相似文献
18.
The Cbl protein functions both as a multivalent adaptor and a negative regulator of receptor tyrosine kinases (RTKs), the latter by directing polyubiquitination of RTKs. To study the function of Cbl in endothelial cell signalling and angiogenesis, wild-type Cbl and tyrosine kinase binding (TKB) domain mutated Cbl (G306E) were overexpressed in murine immortalised brain endothelial (IBE) cells. Wild-type Cbl cells exhibited enhanced proliferation in low serum compared with the control and G306E Cbl cells. Furthermore, up-regulated phosphorylation of fibroblast growth factor receptor 1 (FGFR-1) and Akt were observed in wild-type Cbl cells upon FGF-2 stimulation. A Cbl TKB domain mutant, G306E, disrupted the phosphorylation of the FGFR-1 but not that of FRS2. In the tubular morphogenesis assay, cells expressing wild-type Cbl initially formed tubular structures. These showed decreased stability and converted into cell aggregates, possibly due to a failure to cease proliferating. Our data support the idea that the wild-type Cbl cells exhibit enhanced proliferation, and thus lose their ability to differentiate appropriately. The present study reveals a role of the Cbl protein in FGF-2 dependent signalling in endothelial cells by its destabilisation of tubular structures. 相似文献
19.
Liu J DeYoung SM Hwang JB O'Leary EE Saltiel AR 《The Journal of biological chemistry》2003,278(38):36754-36762
Previous studies suggest that the stimulation of glucose transport by insulin involves the tyrosine phosphorylation of c-Cbl and the translocation of the c-Cbl/CAP complex to lipid raft subdomains of the plasma membrane. We now demonstrate that Cbl-b also undergoes tyrosine phosphorylation and membrane translocation in response to insulin in 3T3-L1 adipocytes. Ectopic expression of APS facilitated insulin-stimulated phosphorylation of tyrosines 665 and 709 in Cbl-b. The phosphorylation of APS produced by insulin drove the translocation of both c-Cbl and Cbl-b to the plasma membrane. Like c-Cbl, Cbl-b associates constitutively with CAP and interacts with Crk upon insulin stimulation. Cbl proteins formed homo- and heterodimers in vivo, which required the participation of a conserved leucine zipper domain. A Cbl mutant incapable of dimerization failed to interact with APS and to undergo tyrosine phosphorylation in response to insulin, indicating an essential role of Cbl dimerization in these processes. Thus, both c-Cbl and Cbl-b can initiate a phosphatidylinositol 3-kinase/protein kinase B-independent signaling pathway critical to insulin-stimulated GLUT4 translocation. 相似文献
20.
The c-Cbl protooncogene can function as a negative regulator of receptor protein tyrosine kinases (RPTKs) by targeting activated receptors for polyubiquitination and downregulation. This function requires its tyrosine kinase binding (TKB) domain for targeting RPTKs and RING finger domain to recruit E2 ubiquitin-conjugating enzymes. It has therefore been proposed that oncogenic Cbl proteins act in a dominant-negative manner to block this c-Cbl activity. In testing this hypothesis, we found that although mutations spanning the RING finger abolish c-Cbl-directed polyubiquitination and downregulation of RPTKs, they do not induce transformation. In contrast, it is mutations within a highly conserved alpha-helical structure linking the SH2 and RING finger domains that render Cbl proteins oncogenic. Thus, Cbl transformation involves effects additional to polyubiquitination of RPTKs that are independent of the RING finger and its ability to recruit E2-conjugating enzymes. 相似文献