Receptor desensitization is accomplished by accelerated endocytosis and degradation of ligand-receptor complexes. An in vitro reconstituted system indicates that Cbl adaptor proteins directly control downregulation of the receptor for the epidermal growth factor (EGFR) by recruiting ubiquitin-activating and -conjugating enzymes. We infer a sequential process initiated by autophosphorylation of EGFR at a previously identified lysosome-targeting motif that subsequently recruits Cbl. This is followed by tyrosine phosphorylation of c-Cbl at a site flanking its RING finger, which enables receptor ubiquitination and degradation. Whereas all three members of the Cbl family can enhance ubiquitination, two oncogenic Cbl variants, whose RING fingers are defective and phosphorylation sites are missing, are unable to desensitize EGFR. Our study identifies Cbl proteins as components of the ubiquitin ligation machinery and implies that they similarly suppress many other signaling pathways.

The ErbB family includes two receptors, ErbB‐1 and ErbB‐3, that respectively bind to epidermal growth factor and Neu differentiation factor, and an orphan receptor, ErbB‐2. Unlike ErbB‐1 and ErbB‐2, the intrinsic tyrosine kinase of ErbB‐3 is catalytically impaired. By using interleukin‐3‐dependent cells that ectopically express the three ErbB proteins or their combinations, we found that ErbB‐3 is devoid of any biological activity but both ErbB‐1 and ErbB‐2 can reconstitute its extremely potent mitogenic activity. Transactivation of ErbB‐3 correlates with heterodimer formation and is reflected in receptor phosphorylation and the transregulation of ligand affinity. Inter‐receptor interactions enable graded proliferative and survival signals: heterodimers are more potent than homodimers, and ErbB‐3‐containing complexes, especially the ErbB‐2/ErbB‐3 heterodimer, are more active than ErbB‐1 complexes. Nevertheless, ErbB‐1 signaling displays dominance over ErbB‐3 when the two receptors are coexpressed. Although all receptor combinations activate the mitogen‐activated protein kinases ERK and c‐Jun kinase, they differ in their rate of endocytosis and in coupling to intervening signaling proteins. It is conceivable that combinatorial receptor interactions diversify signal transduction and confer double regulation, in cis and in trans, of the superior mitogenic activity of the kinase‐defective ErbB‐3.

Ligand-induced activation of receptor tyrosine kinases (RTK) results in the initiation of diverse cellular pathways, including proliferation, differentiation and cell migration. The ErbB family of RTKs represents a model for signal diversification through the formation of homo-and heterodimeric receptor complexes. Each dimeric receptor complex will initiate a distinct signaling pathway by recruiting a different set of Src homology 2-(SH2-) containing effector proteins. Further complexity is added due to the existence of an oncogenic receptor that enhances and stabilizes dimerization but has no ligand (ErbB-2), and a receptor that can recruit novel SH-2-containing proteins, but is itself devoid of kinase activity (ErbB-3). The resulting signaling network has important implications for embryonic development and malignant transformation.

, is a seco-steroid hormone that binds with high affinity to a nuclear receptor, the vitamin D 3 receptor (VDR). This receptor selectively associates with recognition sequences in the promoter region of target genes, thereby regulating the transcription of those genes. The principal functions of 1,25(OH) 2 D 3 are the stimulation of intestinal calcium and phosphorus absorption, mediation of bone remodeling, and conservation of minerals in the kidney (for reviews, see references 46 and 63). In addition to its action in these tissues, however, 1,25(OH) 2 D 3 has been found in skin, testes, breast, muscle, pancreas, endocrine glands, thymus, and bone marrow, suggesting additional regulatory functions for the hormone. Notably, 1,25(OH) 2 D 3 appears to play an important role in modulating the growth of cells of the immune system: the hormone can induce the differentiation of myeloid leukemia cells along a monocyte/ macrophage lineage (1, 4, 6, 49) and can inhibit T-lymphocyte proliferation and activation both in vivo and in vitro (8,10,37,43,44). Interleukin-2 (IL-2), gamma interferon, and granulocytemacrophage colony-stimulating factor (GM-CSF) mRNA levels all decrease after T cells are exposed to 1,25(OH) 2 D 3 , but the molecular mechanisms mediating these changes have not been fully described (9,58,(66)(67)(68)(69)79).T-cell activation is a key step in the initiation of an immunological response. Upon receipt of the appropriate stimulus, a complex signaling cascade is initiated, resulting in cell proliferation and secretion of cytokines that enhance the immune response. One of the first genes to be expressed postactivation is the IL-2 gene. The lymphokine IL-2 exerts its influence by interacting with the IL-2 receptor on the surface of activated T cells, and this interaction is required for progression through the cell cycle (transition from G 1 to S phase). The kinetics of IL-2 induction are quite rapid, with transcripts detectable within 30 to 45 min after activation. A complex enhancer, which includes 275 bp just downstream from the transcription start site (Ϫ52 to Ϫ326), has been exhaustively studied (16,24,75). Binding sites for several ubiquitous and T-cell-specific transcription factors were defined in this region, and the proteins that bind to these sites, including Oct-1, AP1, NF-B, and NFATp/c, have been identified (65).Several agents, including the drugs cyclosporin A and FK506 (18, 51), as well as glucocorticoids (25,80,81) and retinoids (13, 19) appear to act as immunosuppressors by targeting IL-2 expression. 1,25(OH) 2 D 3 inhibits the entry of activated T cells into S phase (68); similar blocks at this point in the cell cycle have been demonstrated for other inhibitors of IL-2 synthesis, such as the synthetic glucocorticoid dexamethasone (7), further implicating IL-2 as a target for vitamin D 3 's immunosuppressive effect.These observations demonstrate an important role for 1,25(OH) 2 D 3 in the immunomodulation of T lymphocytes but not the actual mechanism by which this regulation is carried out....

Ligand-induced activation of surface receptors, including the epidermal growth factor receptor (EGFR), is followed by a desensitization process involving endocytosis and receptor degradation. c-Cbl, a tyrosine phosphorylation substrate shared by several signaling pathways, accelerates desensitization by recruiting EGFR and increasing receptor polyubiquitination. Here we demonstrate that the RING type zinc finger of c-Cbl is essential for ubiquitination and subsequent desensitization of EGFR. Mutagenesis of a single cysteine residue impaired the ability of c-Cbl to enhance both downregulation and ubiquitination of EGFR in living cells, although the mutant retained binding to the activated receptor. Consequently, the mutant form of c-Cbl acquired a dominant inhibitory function and lost the ability to inhibit signaling downstream to EGFR. In vitro reconstitution of EGFR ubiquitination implies that the RING finger plays an essential direct role in ubiquitin ligation. Our results attribute to the RING finger of cCbl a causative role in endocytic sorting of EGFR and desensitization of signal transduction.We have reported recently that c-Cbl can accelerate the rate of EGFR 1 degradation by increasing conjugation of polyubiquitin to an endocytosed EGFR (1). Consistent with sorting to degradation, genetic studies in Caenorhabditis elegans attributed a negative regulatory role to Sli-1, the c-Cbl ortholog of worms (2). This function is reminiscent of the action of c-Cbl downstream to the PGDFR (3), and it depends on a catalytically intact kinase domain of EGFR. The present study addressed the structural motif of c-Cbl involved in ubiquitination of EGFR. Although the 120-kDa c-Cbl protein possesses no identifiable catalytic activity, its NH 2 -terminal half carries a novel phosphotyrosine-binding domain (PYB) (4), that presumably mediates binding to the activated receptor (5). The carboxyl terminus of c-Cbl comprises several proline-rich tracts that allow constitutive binding of Src homology 3 (SH3)-containing proteins, such as Grb2, Nck, and the Cbl-associated protein, CAP (reviewed in Ref. 6). In addition, the COOH terminus is involved in inducible interactions with SH2-containing proteins, such as the regulatory subunit of the phosphoinositide 3-kinase, and the guanine nucleotide exchange protein Vav. An evolutionarily conserved RING-type zinc finger domain separates the adaptor domains of c-Cbl. RING fingers (RFs) are zinc-binding domains that differ from other zinc finger motifs in terms of structure and the zinc ligation scheme (reviewed in Ref. 7). Noteworthy is the fact that the two other family members of c-Cbl carry an intact RF (8, 9), but oncogenic Cbl variants are defective in the RF (10, 11). Our present study concentrated on the role the RF motif plays in Cbl-mediated degradation of EGFR. EXPERIMENTAL PROCEDURESMaterials, Buffers, and Antibodies-EGF was purchased from Sigma, and radioactive materials were from Amersham Pharmacia Biotech (Buckinghamshire, United Kingdom). Rabbit anti-c-Cbl (C-15) antibodies, ...

The tumor suppressor gene 101 (tsg101) regulates vesicular trafficking processes in yeast and mammals. We report a novel protein, Tal (Tsg101-associated ligase), whose RING finger is necessary for multiple monoubiquitylation of Tsg101. Bivalent binding of Tsg101 to a tandem tetrapeptide motif (PTAP) and to a central region of Tal is essential for Tal-mediated ubiquitylation of Tsg101. By studying endocytosis of the epidermal growth factor receptor and egress of the human immunodeficiency virus, we conclude that Tal regulates a Tsg101-associated complex responsible for the sorting of cargo into cytoplasm-containing vesicles that bud at the multivesicular body and at the plasma membrane.[Keywords: Endocytosis; growth factor; HIV; ubiquitin; signal transduction] Supplemental material is available at http://www.genesdev.org.

Transregulation of the epidermal growth factor receptor (EGFR) by protein kinase C (PKC) serves as a model for heterologous desensitization of receptor tyrosine kinases, but the underlying mechanism remained unknown. By using c-Cbl-induced ubiquitination of EGFR as a marker for transfer from early to late endosomes, we provide evidence that PKC can inhibit this process. In parallel, receptor down-regulation and degradation are significantly reduced. The inhibitory effects of PKC are mediated by a single threonine residue (threonine 654) of EGFR, which serves as a major PKC phosphorylation site. Biochemical and morphological analyses indicate that threonine-phosphorylated EGFR molecules undergo normal internalization, but instead of sorting to lysosomal degradation, they recycle back to the cell surface. In conclusion, by sorting EGFR to the recycling endosome, heterologous desensitization restrains ligand-induced down-regulation of EGFR.

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