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.

Antibodies that coengage activating FcγRs expressed by tumor-associated leukocytes facilitate the selective elimination of intratumoral T cells.

Decreased activity of osteoblasts (OBs) contributes to osteolytic lesions in multiple myeloma (MM). IntroductionA cardinal clinical feature of multiple myeloma (MM) is the presence of osteolytic bone lesions. Myeloma cells disrupt the delicate balance between bone formation and bone resorption. 1,2 Various clinical observations 3 and experimental studies 4,5 have linked the level of MM bone disease with disease burden. Increased osteoclastic activity and its molecular basis have long been considered a primary pathogenic event in MM bone disease. However, a molecular basis for the well recognized lack of osteoblast (OB) function, specifically DKK-1, in the MM bone disease has only recently been described. 6,7 Canonical Wnt pathway plays an important role in controlling proliferation, differentiation, and survival of OB. [8][9][10][11] Previous studies have reported high expression levels of the canonical Wnt inhibitor DKK1 and osteolytic bone lesions in various tumor types including breast, 12,13 neuroblastoma, 14 esophageal, and lung cancer, 15 and conversely enhanced OB activity and osteoblastic bone lesions associated with decreased DKK1 levels in prostate and colon cancers. [16][17][18] In MM, high serum DKK1 levels were correlated with focal bone lesions. 19 The DKK1 produced by MM cells can inhibit the differentiation of OB precursor cells 19 and bone formation in vitro 20 through a DKK1-mediated attenuation of Wnt3a-induced stabilization of ␤-catenin. 21 These findings confirm DKK1 as an important regulator of bone formation in the bone microenvironment. The importance of DKK1 secretion in diseases associated with bone destruction is reinforced by a recent study showing that DKK1 mediates the bone destructive effects of rheumatoid arthritis and that a neutralizing antibody to DKK1 could inhibit the bone destructive process in that disease. 22 There is also emerging evidence that the cellular bone compartment affects MM cell growth and progression. This is supported by the observation that osteoclasts can support long-term survival and proliferation of primary MM cells, 23,24 and OB may impede MM cell growth. 7,25 Thus, targeting these cellular elements may also favorably affect disease control. Therefore, we have evaluated DKK1 as a therapeutic target in MM in the context of the bone marrow (BM) microenvironment, analyzing the effect of a human DKK1 neutralizing antibody (BHQ880). We show that this clinically applicable antibody increases OB function and number and also has anti-MM effect when evaluated in the presence of the BM milieu. Methods ReagentsBHQ880 is a phage-derived DKK1 neutralizing human immunoglobulin G1 (IgG1) antibody (provided by Novartis, Cambridge, MA). BHQ880 has a high affinity for and can neutralize both human DKK1 and murine DKK1. IgG1 isotype antibody was used as control. CellsBone marrow mononuclear cells (BMMNCs) and primary MM cells were isolated using Ficoll-Hypaque density gradient sedimentation from BM Submitted November 25, 2008; accepted April 20, 2009. Prepublished online ...

The Cbl proteins are a family of proteins found in metazoans from nematodes to vertebrates. These proteins have several highly conserved domains including an N-terminal tyrosine kinase binding (TKB) 1 domain and a RING finger (1-9). The three mammalian Cbl proteins,2,[6][7][8], are tyrosine-phosphorylated upon activation of a wide variety of growth factor receptors, and they associate with many signaling proteins via SH2 and SH3 interactions (reviewed in Ref. 10 and 11). These diverse interactions modulate signaling through many pathways (10,11). Recent work has shown that c-Cbl-and Cblb-deficient mice have hyperplastic tissues, consistent with a negative regulatory role in cellular proliferation for Cbl proteins (12-15). Together, these data indicate that the Cbl proteins are important regulators of intracellular signaling and consequently of cell function and development.Cbl proteins are negative regulators of epidermal growth factor receptor (EGFR) signaling. This was first shown by genetic studies in Caenorhabditis elegans, which demonstrated that Sli-1 (the C. elegans Cbl homologue) is a negative regulator of the Let-23 receptor tyrosine kinase (the EGFR homologue) in vulva development (3, 16). The Drosophila Cbl protein (D-Cbl) has been shown to associate with the EGFR, and overexpression of D-Cbl in the eye of Drosophila embryos inhibits EGFR-dependent photoreceptor cell development (4, 5). Several studies have shown that mammalian Cbl proteins become phosphorylated and recruited to the EGFR upon stimulation (11, 17) and that they inhibit EGFR function (7, 18 -20).The mechanism underlying the negative regulation of activated tyrosine kinases by Cbl proteins has recently been described. Cbl proteins function as ubiquitin protein ligases, which mediate the ubiquitination of activated tyrosine kinases including the EGFR and target them for degradation (20 -31). Ubiquitination of proteins occurs via the sequential activation and conjugation of ubiquitin to target proteins by the ubiquitinactivating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin protein ligase (E3) (32). The E3 confers specificity to the ubiquitination process. An increasing number of RING finger proteins has been demonstrated to function as E3 proteins or as part of E3 complexes, and in each of them the RING finger is essential to this activity (33-43). The highly conserved TKB and RING finger domains of Cbl proteins are essential and sufficient for their E3 activity, and together these domains target the ubiquitination of activated tyrosine kinases such as the EGFR (20 -31).Here, we show that EGF activation induces a coordinated degradation of the EGFR, Cbl proteins, and other proteins of the EGFR signaling complex. These results suggest that Cbl proteins regulate degradation of multiple proteins in the active EGFR-signaling complex. EXPERIMENTAL PROCEDURESExpression Constructs-The expression plasmid for HA epitopetagged Cbl-b, c-Cbl, and the control vector (pCEFL) have been previously described (18). HA epitope-tagged C...