REVIEWS
d
c b
Clathrin-coated pit
a T K P B
P T K B
Clathrin
RF E2 P P P Y
Ub
Recycling vesicle
B
Y
P
P
P
K
RF E2 P P P Y P
T K P B
T K P B
RF E2 P P P Y P
RF E2 P P P Y P
e
T K P B
Endosome (low pH)
Coated vesicle
RF E2 P P P Y P
T K P B
Uncoated vesicle
RF E2 P P P Y P
h g
T
RF
T K P B Lysosome
i
RF E2 P P P Y P
T K P B
f
RF E2 P P P Y P
Proteasome
Figure 4 | Proposed model for the role of c-Cbl in directing multi-ubiquitylation and downregulation of the epidermal growth factor receptor. a | Ligand binding induces tyrosine phosphorylation (P) of the epidermal growth factor (EGFR), and initiates movement of the activated EGFR to clathrin-coated membrane areas. b | Cbl is recruited to the tyrosine-phosphorylated receptor (by adaptor proteins, not shown) and the tyrosine-kinase-binding (TKB) domain binds to phosphorylated Y1045 on the EGFR18. At this time, c-Cbl also becomes tyrosine phosphorylated. c | The E3 (ubiquitin ligase) function of Cbl catalyses transfer of a ubiquitin molecule (red circle) from the RING-finger-bound E2 (ubiquitin-conjugating enzyme) to the EGFR. d | Continued addition of ubiquitin moieties leads to multi-ubiquitylation of the EGFR. e | Multi-ubiquitylated receptor–Cbl complex is internalized by clathrin-coated pits. f | Internalized vesicles are rapidly uncoated and fuse into early endosomes (g). In endosomes, ligand dissociation normally occurs and the receptor is recycled back to the cell surface (h). In cells overexpressing c-Cbl, however, the EGFR is prevented from recycling and is directed instead towards proteasomal/lysosomal degradation (i). Cbl has been reported to be transiently ubiquitylated in macrophages, and has been detected in endosomal compartments, but does not itself become degraded, while the polyubiquitinated CSF-1 receptor is lysosomally degraded43,46. Adapted from REF. 45.
ANERGY
A state in which T cells cannot respond to antigen.
the constitutive activation of NF-AT, which is further enhanced following TCR stimulation80. Importantly, in the same experiment, c-Cbl overexpression did not promote the constitutive activation of NF-AT but rather produced a marked suppression following TCR stimulation. So, under identical experimental conditions, the targeting of ZAP-70 by these two highly related proteins results in opposite effects on NF-AT activation. An explanation is not obvious. However, another study also noted opposite effects of c-Cbl and Cbl-b53. In these experiments, Cbl-b inhibited EGF-induced proliferation whereas the overexpression of c-Cbl had no effect, even though both seem equally effective in directing EGF receptor multi-ubiquitylation and downregulation18. These experiments are difficult to reconcile as both proteins would be expected to have functions conserved with SLI-1. Indeed, the fact that c-Cbl and Cbl-b deficient mice are viable and fertile but the loss of both is embryonic lethal suggests that they have important overlapping functions (H. Gu, M. Murphy and D.
Bowtell, personal communication). c-Cbl also might contribute to the generation of ANERGIC T cells in which Fyn is constitutively activated and mediates the tyrosine phosphorylation of c-Cbl. As Y700 and Y774 in c-Cbl are docking sites for CrkL, which forms a complex with the guanine nucleotide exchange factor C3G to catalyse the exchange of GTP for Rap1, it has been proposed that the predominance of Rap1–GTP over Ras–GTP in these cells is mediated by c-Cbl, and this results in the blockade of interleukin-2 transcription leading to anergy83. Cbl-deficient mice
Although c-Cbl- and Cbl-b-deficient mice are generally healthy and do not show developmental abnormalities, they do show phenotypic alterations, the most marked being associated with thymocyte and peripheral T-cell activation, respectively. Thymocytes in c-Cbl–/– mice show a marked activation of ZAP-70 in response to TCR stimulation, in contrast to wild-type thymocytes, which
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
VOLUME 2 | APRIL 2001 | 2 9 9
© 2001 Macmillan Magazines Ltd