Abnormalities induced by the mutant gene, lpr. Patterns of disease and expression of murine leukemia viruses in SJL/J mice homozygous and heterozygous for lpr.

SJL/J mice heterozygous or homozygous for the lpr mutation were compared with SJL/J-+/+ mice for longevity, histopathology, antigenic characteristics of lymphocytes and expression of murine leukemia viruses (MuLV). In comparison to +/+ mice, lpr homozygotes had a markedly shortened life span, died with infiltrative pulmonary disease, but little or no renal disease, and expressed high levels of infectious ecotropic MuLV in lymphoid tissues. SJL-lpr/+ mice had a life span intermediate between SJL-+/+ and -lpr/lpr mice, died with lymphomas that histologically resembled the neoplasms of +/+ mice, and sometimes expressed high levels of ecotropic MuLV. The lymphomas of lpr/+ could be transplanted to +/+ recipients in 78% of cases, and continuous in vitro lines were established from some of them. Similar effects on virus expression or lymphoma development were not observed in other strains homozygous or heterozygous for the lpr mutation. These results indicate that the diseases expressed by mice homozygous for the lpr mutation are highly strain-dependent, and that this gene can have an effect in the heterozygous state in SJL mice.

The E mu-myc transgenic mouse. A model for high-incidence spontaneous lymphoma and leukemia of early B cells.

Mice transgenic for a c-myc gene driven by the IgH enhancer (E mu-myc) were shown to almost invariably develop lymphomas, 90% succumbing in the first 5 mo of life. The tumors typically presented as rapidly progressive lymphadenopathy with thymic involvement and were highly malignant by transplantation assay. Morphologically, they were lymphoblastic lymphomas, usually accompanied by lymphoid leukemia and granulocytosis, and were distinct from the tumors that arose much later in 37% of nontransgenic mice of the same (C57BL/6 x SJL)F2 genetic background. Cell-surface markers on 31 E mu-myc tumors identified 52% as pre-B lymphomas, 29% as mixed pre-B and B lymphomas, and 19% as B lymphomas. The tumors appeared to arise at random from a population of pre-B cells expanded by constitutive expression of the myc transgene. A majority of the animals initiated malignancy at the rate of 17% per week. The rate at which the cycling, benign pre-B cells spontaneously convert to malignancy was estimated to about 10(-10) per cell per generation. A transient leukocytosis identified in young E mu-myc mice was developed into a rapid assay for inheritance of the transgene.

Expression of the 6C3 antigen on murine hematopoietic neoplasms. Association with expression of abl, ras, fes, src, erbB, and Cas NS-1 oncogenes but not with myc.

The monoclonal antibody 6C3 was used to test a wide variety of murine hematopoietic neoplasms for cell surface expression of a 160 kD glycoprotein (gp160(6C3)) previously shown to be expressed by neoplastic pre-B and some B lymphocytes transformed by Abelson murine leukemia virus (A-MuLV). This antigen was expressed on many pre-B and B cell lymphomas, but not on A-MuLV-transformed fibroblasts, T cell lymphomas, or myelomonocytic leukemias, gp160(6C3) was expressed by most early B-lineage spontaneous tumors, and early B tumors induced by replication-defective MuLV-containing oncogenes the products of which are associated with the cytoplasmic aspect of the plasma membrane, i.e., fes, abl, H-ras, bas, src, erbB, and Cas NS-1. By comparison, none of the early B lineage lymphomas induced by the "nuclear" oncogene avian v-myc MuLV, or arising in mice transgenic for a murine c-myc gene, or later B cell lineage stages bearing translocations of the c-myc locus expressed this antigen.

Phosphotyrosine binding domain-dependent upregulation of the platelet-derived growth factor receptor alpha signaling cascade by transforming mutants of Cbl: implications for Cbl's function and oncogenicity.

Recent studies have demonstrated that Cbl, the 120-kDa protein product of the c-cbl proto-oncogene, serves as a substrate of a number of receptor-coupled tyrosine kinases and forms complexes with SH3 and SH2 domain-containing proteins, pointing to its role in signal transduction. Based on genetic evidence that the Caenorhabditis elegans Cbl homolog, SLI-1, functions as a negative regulator of the LET-23 receptor tyrosine kinase and our demonstration that Cbl's evolutionarily conserved N-terminal transforming region (Cbl-N; residues 1 to 357) harbors a phosphotyrosine binding (PTB) domain that binds to activated ZAP-70 tyrosine kinase, we examined the possibility that oncogenic Cbl mutants may activate mitogenic signaling by deregulating cellular tyrosine kinase machinery. Here, we show that expression of Cbl-N and two other transforming Cbl mutants (CblY368 delta and Cbl366-382 delta or Cb170Z), but not wild-type Cbl, in NIH 3T3 fibroblasts leads to enhancement of endogenous tyrosine kinase signaling. We identified platelet-derived growth factor receptor alpha (PDGFR alpha) as one target of mutant Cbl-induced deregulation. In mutant Cbl transfectants, PDGFR alpha was hyperphosphorylated and constitutively complexed with a number of SH2 domain-containing proteins. PDGFR alpha hyperphosphorylation and enhanced proliferation of mutant Cbl-transfected NIH 3T3 cells were drastically reduced upon serum starvation, and PDGF-AA substituted for the maintenance of these traits. PDGF-AA stimulation of serum-starved Cbl transfectants induced the in vivo association of transfected Cbl proteins with PDGFR alpha. In vitro, Cbl-N directly bound to PDGFR alpha derived from PDGF-AA-stimulated cells but not to that from unstimulated cells, and this binding was abrogated by a point mutation (G306E) corresponding to a loss-of-function mutation in SLI-1. The Cbl-N/G306E mutant protein, which failed to induce enhanced growth and transformation of NIH 3T3 cells, also failed to induce hyperphosphorylation of PDGFR alpha. Altogether, these findings identify a novel mechanism of Cbl's physiological function and oncogenesis, involving its PTB domain-dependent direct interaction with cellular tyrosine kinases.

Tissue hyperplasia and enhanced T-cell signalling via ZAP-70 in c-Cbl-deficient mice.

The c-Cbl protein is tyrosine phosphorylated and forms complexes with a wide range of signalling partners in response to various growth factors. How c-Cbl interacts with proteins, such as Grb2, phosphatidylinositol 3-kinase, and phosphorylated receptors, is well understood, but its role in these complexes is unclear. Recently, the Caenorhabditis elegans Cbl homolog, Sli-1, was shown to act as a negative regulator of epidermal growth factor receptor signalling. This finding forced a reassessment of the role of Cbl proteins and highlighted the desirability of testing genetically whether c-Cbl acts as a negative regulator of mammalian signalling. Here we investigate the role of c-Cbl in development and homeostasis in mice by targeted disruption of the c-Cbl locus. c-Cbl-deficient mice were viable, fertile, and outwardly normal in appearance. Bone development and remodelling also appeared normal in c-Cbl mutants, despite a previously reported requirement for c-Cbl in osteoclast function. However, consistent with a high level of expression of c-Cbl in the hemopoietic compartment, c-Cbl-deficient mice displayed marked changes in their hemopoietic profiles, including altered T-cell receptor expression, lymphoid hyperplasia, and primary splenic extramedullary hemopoiesis. The mammary fat pads of mutant female mice also showed increased ductal density and branching compared to those of their wild-type littermates, indicating an unanticipated role for c-Cbl in regulating mammary growth. Collectively, the hyperplastic histological changes seen in c-Cbl mutant mice are indicative of a normal role for c-Cbl in negatively regulating signalling events that control cell growth. Consistent with this view, we observed greatly increased intracellular protein tyrosine phosphorylation in thymocytes following CD3epsilon cross-linking. In particular, phosphorylation of ZAP-70 kinase in thymocytes was uncoupled from a requirement for CD4-mediated Lck activation. This study provides the first biochemical characterization of any organism that is deficient in a member of this unique protein family. Our findings demonstrate critical roles for c-Cbl in hemopoiesis and in controlling cellular proliferation and signalling by the Syk/ZAP-70 family of protein kinases.

The Cbl proto-oncogene product negatively regulates the Src-family tyrosine kinase Fyn by enhancing its degradation.

Fyn is a prototype Src-family tyrosine kinase that plays specific roles in neural development, keratinocyte differentiation, and lymphocyte activation, as well as roles redundant with other Src-family kinases. Similar to other Src-family kinases, efficient regulation of Fyn is achieved through intramolecular binding of its SH3 and SH2 domains to conserved regulatory regions. We have investigated the possibility that the tyrosine kinase regulatory protein Cbl provides a complementary mechanism of Fyn regulation. We show that Cbl overexpression in 293T embryonic kidney and Jurkat T-lymphocyte cells led to a dramatic reduction in the active pool of Fyn; this was seen as a reduction in Fyn autophosphorylation, reduced phosphorylation of in vivo substrates, and inhibition of transcription from a Src-family kinase response element linked to a luciferase reporter. Importantly, a Fyn mutant (FynY528F) relieved of intramolecular repression was still negatively regulated by Cbl. The Cbl-dependent negative regulation of Fyn did not appear to be mediated by inhibition of Fyn kinase activity but was correlated with enhanced protein turnover. Consistent with such a mechanism, elevated levels of Fyn protein were observed in cell lines derived from Cbl(-/-) mice compared to those in wild-type controls. The effects of Cbl on Fyn were not observed when the 70ZCbl mutant protein was analyzed. Taken together, these observations implicate Cbl as a component in the negative regulation of Fyn and potentially other Src-family kinases, especially following kinase activation. These results also suggest that protein degradation may be a general mechanism for Cbl-mediated negative regulation of activated tyrosine kinases.

Histologic and cell surface antigen studies of hematopoietic tumors induced by Cas-Br-M murine leukemia virus.

Cas-Br-M, a cloned ecotropic murine leukemia virus (MuLV) of wild mouse origin that induces both neurogenic hindlimb paralysis and lymphomas, was injected into NFS/N inbred mice neonatally. Then the mice were observed for the development of neurologic disease and tumors. All mice manifested neurologic abnormalities by 6 months of age, and 58% of the animals died with hematopoietic neoplasms. The tumors included T- and B-cell lymphomas, lymphoblastic lymphoma, erythroleukemias, myelogenous leukemias, and a megakaryocytic leukemia. Cas-Br-M thus appeared to be unique among ecotropic MuLV in inducing a wide spectrum of hematopoietic tumors.

The protein product of the c-cbl oncogene rapidly complexes with the EGF receptor and is tyrosine phosphorylated following EGF stimulation.

The cbl oncogene was first identified as part of a transforming retrovirus which arose in a mouse pre-B cell lymphoma. Its protein product, p120cbl, is cytoplasmic and has several distinctive domains including a highly basic region, a RING finger motif and a large proline-rich domain. A mutation to cbl in the 70Z/3 pre-B cell lymphoma produces an oncogenic protein which exhibits a marked enhancement of tyrosine phosphorylation. Parallel studies have demonstrated that p120cbl is a substrate of protein tyrosine kinases activated by engagement of the T cell antigen receptor and that cbl is phosphorylated by oncogenic forms of the Abl tyrosine kinase. A genetic analysis of the Caenorhabditis elegans cbl homologue, sli-1, demonstrates that sli-1 negatively regulates the LET-23 tyrosine kinase receptor. Here we show that p120cbl is rapidly phosphorylated on tyrosine residues following EGF stimulation and that it forms an inducible complex with the receptor. Our results also show that the oncogenic 70Z/3 form of cbl has enhanced binding to the EGF receptor and that peptides spanning the proline-rich region bind a range SH3 domains. These findings are consistent with a conserved role for cbl/sli-1 proteins in mammals and nematodes.

EGF receptor binding and transformation by v-cbl is ablated by the introduction of a loss-of-function mutation from the Caenorhabditis elegans sli-1 gene.

The 120 kD product of the c-cbl oncogene is rapidly tyrosine phosphorylated and recruited to the EGF receptor following ligand binding. Cbl's oncogenic potential is activated by a large carboxy-terminal truncation that generated v-cbl and removes the Ring finger and proline-rich SH3-binding domains. Here we show that this truncation reveals a novel and highly conserved domain that can interact directly with the EGF receptor in a phosphorylation dependent manner. Furthermore we demonstrate that the v-cbl domain is not utilized by c-cbl for recruitment to the receptor since this binding property is not evident in c-cbl constructs with proline domain deletions, and it is only revealed following deletion of the Ring finger. We also analyse a loss-of-function mutation from the C. elegans homologue, sli-1, and show that the corresponding mutation in v-cbl ablates transformation and EGF receptor association. Thus our findings provide further evidence that v-cbl possesses a novel and evolutionarily conserved phosphotyrosine binding domain and that the dual capability of EGF receptor binding by cbl involves two distinct mechanisms. In addition these findings raise the possibility that v-cbl may transform by competing with c-cbl for phosphorylated binding sites on activated receptor complexes.

Tyrosine kinase activity of the EGF receptor is enhanced by the expression of oncogenic 70Z-Cbl.

The 120 kD product of the c-Cbl oncogene is a prominent substrate of protein tyrosine kinases that lacks a known catalytic activity but possesses an array of binding sites for cytoplasmic signalling proteins. An oncogenic form of Cbl was recently identified in the 70Z/3 pre-B cell lymphoma which has a small deletion at the N-terminus of the Ring finger domain. This form of Cbl, termed 70Z-Cbl, exhibits an enhanced level of tyrosine phosphorylation compared with c-Cbl. Here we demonstrate that the expression of 70Z-Cbl induces a tenfold enhancement in the kinase activity of the EGF receptor in serum-starved and EGF-stimulated cells. In serum-starved cells this results in EGF receptor autophosphorylation and the recruitment of Grb2, Shc and Sos1 but does not induce a corresponding increase in MAP kinase activity. Furthermore the expression of 70Z-Cbl greatly enhances EGF-induced tyrosine phosphorylation of the protein tyrosine phosphatase SHP-2. We also show that the Cbl/EGF receptor complex is predominantly associated with CrkII and is distinct to the Grb2/Shc/Sos1 complex that associates with the EGF receptor. These findings therefore demonstrate a biochemical effect of an oncogenic Cbl protein and support predictions from C. elegans that Cbl functions as regulator of receptor tyrosine kinases.

A rearrangement of the c-cbl proto-oncogene in HUT78 T-lymphoma cells results in a truncated protein.

The murine Cas NS-1 retrovirus carries the v-cbl oncogene and induces pro-B, pre-B and myeloid tumors in mice inoculated at birth. The human homolog of v-cbl is located on chromosome 11q23.3, which is in the region of translocation breakpoints in a range of acute leukemias. The sequencing of the human and murine c-cbl proto-oncogenes has revealed v-cbl as a markedly truncated form of c-cbl that encompasses 40% of the amino terminus. This truncation deletes a proline- and serine/threonine-rich domain and a putative leucine zipper. In this study we describe the nature of a truncated form of c-cbl present in the cutaneous T-cell lymphoma line HUT78. In these cells a genetic rearrangement involving unknown sequences and Alu repeat sequences results in the insertion of a premature stop codon that removes 259 amino acids from the carboxy terminus. This results in the translation of a 72 kDa protein, compared with the 120 kDa protein encoded by full-length c-cbl, which lacks a portion of the proline-rich domain and the entire leucine zipper.

Tyrosine phosphorylation of p120cbl in BCR/abl transformed hematopoietic cells mediates enhanced association with phosphatidylinositol 3-kinase.

Increased tyrosine kinase activity of abl oncogene in Philadelphia chromosome positive-leukemic cells leads to activation of p21ras and phosphatidylinositol 3'-kinase (PI 3-Kinase). The mechanism of activation of these signaling pathways is not understood, but numerous studies have focused on the identification and characterization of downstream substrates of BCR/abl tyrosine kinase as potential mediators of oncogenic signaling. It was recently found that the 120 kDa protein product of the c-cbl proto-oncogene is highly tyrosine phosphorylated and associates with BCR/abl in transformed hematopoietic cells. We have characterized further cbl's involvement in BCR/abl mediated tumorigenesis using growth factor independent BCR/abl transformed BaF3 cells. Our experiments show that, in contrast to other cell types, the in vivo interaction of cbl with GRB2 and p85 is significantly enhanced in BCR/abl transformed BaF3 cells and that tyrosine phosphorylation of cbl leads to a direct interaction with GRB2, p85 and abl SH2 domains. A 14-fold increase in cbl associated PI 3-kinase activity in BCR/abl transformed cells suggests that the binding of p85 SH2 domains to tyrosine phosphorylated cbl may contribute to PI 3-kinase activation. Domain analysis studies indicate that both SH3 domains of GRB2 bind to the proline rich region of cbl in quiescent BaF3 cells, whereas GRB2 SH2 domain interacts with a non-contiguous sequence of cbl in transformed cells. Although the interaction of cbl with GRB2 in transformed cells was facilitated by binding of GRB2 to BCR/abl, phosphorylation of cbl and its interaction with p190 BCR/abl remained unaltered in BaF3 cells transformed by p190Y177F BCR/abl mutant which is unable to bind GRB2. The current information and the data presented here suggest that, although cbl lacks src homology domains, it represents a novel intermediate protein which, by interaction with key SH-containing adaptor proteins, may participate in regulation of the Ras and PI 3-kinase pathways in BCR/abl transformed hematopoietic cells.

Oncogenic forms of Cbl abrogate the anchorage requirement but not the growth factor requirement for proliferation.

Recent studies have demonstrated that Cbl, the 120 kDa protein product of the c-cbl proto-oncogene, becomes tyrosine phosphorylated in response to stimulation of growth factor receptors and upon integrin-mediated cell adhesion. As a result, Cbl forms complexes with SH2 and SH3 domain-containing proteins, pointing to its role in signal transduction. The cellular form of Cbl can be rendered into transforming by naturally occurring or engineered mutations to its amino acid sequence. To gain insight into the mechanisms how oncogenic forms of Cbl render cells tumorigenic and what the function of the cellular Cbl might be, we have undertaken an analysis of NIH3T3 cells transfected with wild-type and oncogenic forms of Cbl. We demonstrate that unlike cellular Cbl, the mutant forms of Cbl are tyrosine phosphorylated in an adhesion-independent manner and interact with and activate SH2-containing signaling molecules in both suspended and adherent cells. Our data further show that oncogenic forms of Cbl induce anchorage-independent but serum-dependent growth. These results support the view that transformation by oncogenic forms of Cbl results from constitutive activation of integrin-dependent, rather than growth factor-dependent signaling events and, as a corollary, suggest that cellular Cbl might be a functionally important mediator of integrin signaling.

The sequences of the human and mouse c-cbl proto-oncogenes show v-cbl was generated by a large truncation encompassing a proline-rich domain and a leucine zipper-like motif.

The murine Cas NS-1 retrovirus carries the v-cbl oncogene and induces pre-B cell lymphomas and myeloid leukemias. The cellular homolog of v-cbl has been identified in mouse and human DNA, and was recently mapped to mouse chromosome 9 and human chromosome 11q23. To determine the coding sequences of the human and mouse c-cbl proto-oncogenes cDNA clones were isolated from libraries prepared from the human T cell leukemia lines CCRF-CEM and HUT 78, and the mouse pre-B cell line 70Z/3. DNA sequencing revealed an open reading frame encoding 906 amino acids in the human cDNAs and 896 amino acids in 70Z/3. The sequence showed that v-cbl is a markedly truncated form of murine c-cbl containing 355 N-terminal amino acids. The nucleotide sequence of v-cbl is identical to murine c-cbl in this region, and the human sequence has only five amino acid changes in the v-cbl portion. The most notable features of the sequence which was lost in the generation of v-cbl is a C-terminal leucine zipper and a stretch of 208 amino acids containing 23% proline and 19% serine/threonine residues. This proline-rich sequence has similarities to the transcriptional activation domains of some transcription factors, and v-cbl's transforming potential may be due to the loss of this region and the leucine zipper.

The two major sites of cbl tyrosine phosphorylation in abl-transformed cells select the crkL SH2 domain.

We recently found that the 120-kD protein product of the c-cbl oncogene is tyrosine phosphorylated in tumor cells generated by bcr-abl or v-abl and that p120cbl will associate with these proteins in vivo. We also found an oncogenic form of cbl protein in the 70Z/3 pre-B cell lymphoma which exhibits deregulated tyrosine phosphorylation. These findings have led us to broaden our study of cbl's involvement in abl-mediated tumorigenesis. Here we show by immunodepletion that cbl is the major 120-kD tyrosine phosphorylated protein in cells which express activated forms of the abl oncogene. We also demonstrate that tyrosine phosphorylation of pl20cbl in bcr-abl transformed cells does not alter its subcellular localization. In addition we show that the oncogenic 7OZ/3 form of cbl exhibits enhanced tyrosine phosphorylation in v-abl infected cells and that cbl is heavily tyrosine phosphorylated in hemopoietic cells transformed by v-src. Finally this study identifies two sites that are essential for the tyrosine phosphorylation of cbl in abl-transformed cells. These sites conform to the preferred abl kinase substrate sequence of YXXP and we show that following phosphorylation they mediate an association with the crkL SH2 domain.

Expression of c-cbl proto-oncogene is modulated during differentiation but not during induction of proliferation.

The proto-oncogene c-cbl is expressed as two mRNAs, ca. 10.5 and 3.1 kb, both of which appear to be functional inasmuch as both can be found on polyribosomes in tissues that express both mRNAs. The function of the 120 kDa c-cbl protein is not known, but its primary structure resembles that of a DNA-binding transcription factor with a basic region, a nuclear localization sequence, a zinc finger-like motif and a leucine zipper. To test whether expression of this protein resembles that of regulatory proteins, we studied expression of c-cbl mRNA and protein in differentiating cells and in proliferating cells, conditions in which expression of regulatory proteins commonly is modulated. Differentiation of both erythroleukemia cells and teratocarcinoma cells showed a decrease in c-cbl expression, with kinetics similar to those of transcription factors that are immediate early response genes. Unlike early response genes, however, c-cbl mRNA showed a very long half life in B lymphocytes. Further, in fibroblasts and spleen cells that were induced to proliferate, c-cbl mRNA expression did not change, and expression of c-cbl protein did not change during any stage of the cell cycle. These characteristics indicate that c-cbl does not belong to the immediate early response type of transcription factor. Yet when c-cbl is truncated, as in v-cbl, the protein does enter the nucleus and bind DNA, and it contributes to neoplastic transformation of B lymphocytes and fibroblasts. These findings indicate that the regulation of the c-cbl proto-oncogene is different from that of the proto-oncogenes identified to date and suggest that c-cbl belongs to a new class of proto-oncogenes.