A Mouse with a Loss-of-function Mutation in the c-Cbl TKB Domain Shows Perturbed Thymocyte Signaling without Enhancing the Activity of the ZAP-70 Tyrosine Kinase

The unique tyrosine kinase binding (TKB) domain of Cbl targets phosphorylated tyrosines on activated protein tyrosine kinases (PTKs); this targeting is considered essential for Cbl proteins to negatively regulate PTKs. Here, a loss-of-function mutation (G304E) in the c-Cbl TKB domain, first identified in Caenorhabditis elegans, was introduced into a mouse and its effects in thymocytes and T cells were studied. In marked contrast to the c-Cbl knockout mouse, we found no evidence of enhanced activity of the ZAP-70 PTK in thymocytes from the TKB domain mutant mouse. This finding contradicts the accepted mechanism of c-Cbl–mediated negative regulation, which requires TKB domain targeting of phosphotyrosine 292 in ZAP-70. However, the TKB domain mutant mouse does show aspects of enhanced signaling that parallel those of the c-Cbl knockout mouse, but these involve the constitutive activation of Rac and not enhanced PTK activity. Furthermore, the enhanced signaling in CD4+CD8+ double positive thymocytes appears to be compensated by the selective down-regulation of CD3 on mature thymocytes and peripheral T cells from both strains of mutant c-Cbl mice

The E3 ubiquitin ligase c-Cbl restricts development and functions of hematopoietic stem cells

Hematopoietic stem cells (HSCs) are multipotent progenitors that give rise to all types of blood cells. In the present study, we document that HSC development and functions are negatively regulated by the E3 ubiquitin ligase c-Cbl (casitas B-cell lymphoma). HSCs of c-Cbl−/− mice exhibit augmented pool size, hyperproliferation, greater competence, and enhanced long-term repopulating capacity. Our mechanistic studies identified that c-Cbl−/− HSCs are hyperresponsive to thrombopoietin (TPO) and display elevated levels of STAT5 phosphorylation, thus leading to increased c-Myc expression. In essence, our data unequivocally identify c-Cbl as a novel negative regulator of developmental and functional properties of HSCs

Visualizing the role of Cbl-b in control of islet-reactive CD4 T cells and susceptibility to type 1 diabetes

The E3 ubiquitin ligase Cbl-b regulates T cell activation thresholds and has been associated with protecting against type 1 diabetes, but its invivo role in the process of self-tolerance has not been examined at the level of potentially autoaggressive CD4+ T cells. In this study, we visualize the consequences of Cbl-b deficiency on self-tolerance to lysozyme Ag expressed in transgenic mice under control of the insulin promoter (insHEL). By tracing the fate of pancreatic islet-reactive CD4+ T cells in prediabetic 3A9-TCR x insHEL double-transgenic mice, we find that Cbl-b deficiency contrasts with AIRE or IL-2 deficiency, because it does not affect thymic negative selection of islet-reactive CD4+ cells or the numbers of islet-specific CD4+ or CD4+ Foxp3+ T cells in the periphery, although it decreased differentiation of inducible regulatory T cells from TGF-β-treated 3A9-TCR cells in vitro. When removed from regulatory T cells and placed in culture, Cblb-deficient islet-reactive CD4+ cells reveal a capacity to proliferate to HEL Ag that is repressed in wild-type cells. This latent failure of T cell anergy is, nevertheless, controlled in vivo in prediabetic mice so that islet-reactive CD4+ cells in the spleen and the pancreatic lymph node of Cblb-deficient mice show no evidence of increased activation or proliferation in situ. Cblb deficiency subsequently precipitated diabetes in most TCR:insHEL animals by 15 wk of age. These results reveal a role for peripheral T cell anergy in organ-specific self-tolerance and illuminate the interplay between Cblb-dependent anergy and other mechanisms for preventing organ-specific autoimmunity

c-Cbl–deficient mice have reduced adiposity, higher energy expenditure, and improved peripheral insulin action

Casitas b-lineage lymphoma (c-Cbl) is an E3 ubiquitin ligase that has an important role in regulating the degradation of cell surface receptors. In the present study we have examined the role of c-Cbl in whole-body energy homeostasis. c-Cbl(–/–) mice exhibited a profound increase in whole-body energy expenditure as determined by increased core temperature and whole-body oxygen consumption. As a consequence, these mice displayed a decrease in adiposity, primarily due to a reduction in cell size despite an increase in food intake. These changes were accompanied by a significant increase in activity (2- to 3-fold). In addition, c-Cbl(–/–) mice displayed a marked improvement in whole-body insulin action, primarily due to changes in muscle metabolism. We observed increased protein levels of the insulin receptor (4-fold) and uncoupling protein-3 (2-fold) in skeletal muscle and a significant increase in the phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase. These findings suggest that c-Cbl plays an integral role in whole-body fuel homeostasis by regulating whole-body energy expenditure and insulin action