C-cbl Proto-oncogene Product Research Articles

Phosphorylation of c-Cbl protooncogene product following ethanol administration in rat cerebellum: possible involvement of Fyn kinase

We have previously shown that ethanol administration results in tyrosine phosphorylation of the 130 kDa protein in rat brain, and identified the protein as Cas, the crk-associated src substrate. In the present study, we demonstrate that Cbl of a 120 kDa protein is also tyrosine-phosphorylated in the cerebellum in response to ethanol administration. We also investigated whether Fyn kinase was involved in ethanol-induced Cbl phosphorylation. Immunoprecipitation experiments showed that the amount of coimmunoprecipitated Fyn kinase with an anti-Cbl antibody increased in extracts from ethanol-administered rats compared to those from saline-administered rats. Exogenous Fyn kinase was shown to phosphorylate on tyrosine residue(s) of Cbl from the cerebellum in vitro. Furthermore, Fyn kinase and Cbl were demonstrated immunohistochemically to be coexpressed in white matter in the cerebellum. These findings indicate that Cbl is tyrosine-phosphorylated in rat cerebellum in response to ethanol administration, and also raise the possibility that Fyn kinase may be involved in the process.

Negative regulation of EphA2 receptor by Cbl

The c-Cbl proto-oncogene product Cbl has emerged as a negative regulator of receptor and non-receptor tyrosine kinases, a function dependent on its recently identified ubiquitin ligase activity. Here, we report that EphA2, a member of Eph receptor tyrosine kinases is negatively regulated by Cbl. The negative regulation of EphA2 mediated by Cbl is dependent on the activity of EphA2, as the kinase inactive mutant of EphA2 cannot be regulated by Cbl. Moreover, a point mutation (G306E-Cbl) in TKB region of Cbl that has been reported to abolish Cbl binding to RTKs and non-receptor tyrosine kinases impaired the binding to active EphA2. The dominant negative mutant 70Z-Cbl, which has a 17-amino acids deletion in the N-boundary of the RING finger domain, defuncted negative regulatory function of Cbl to EphA2. These results demonstrate that the TKB domain and RING finger domain of Cbl are essential for this negative regulation.

C-Cbl protein in human cancer tissues is frequently tyrosine phosphorylated in a tumor-specific manner.

To search for the signaling pathway relevant to the tumorigenesis in human, we examined the expression and tyrosine phosphorylation of c-cbl proto-oncogene product, c-Cbl, in various human cancer cell lines and surgical specimens. In cells derived from human cancer, we found constitutive tyrosine phosphorylation of c-Cbl protein, whereas its phosphorylation was undetectable in control ECV304 cells. Expression and tyrosine phosphorylation of c-Cbl was also examined in various surgical specimens. Thirty-six surgical specimens obtained from human tumor tissues were studied: 9 gastric carcinomas, 10 colon carcinomas, 6 renal carcinomas, 2 hepatomas, 2 brain tumors, 2 uterus tumors, 1 breast carcinoma, 1 thyroid tumor, 1 bladder tumor and 2 lung carcinomas. We found tyrosine phosphorylation of c-Cbl protein in 12 cases (33%) of these tumor tissues in a tumor-specific manner. These results suggest the importance of c-Cbl signaling in tumorigenesis in human.

IFN-gamma activates the C3G/Rap1 signaling pathway.

IFN-gamma transduces signals by activating the IFN-gamma receptor-associated Jak-1 and Jak-2 kinases and by inducing tyrosine phosphorylation and activation of the Stat-1 transcriptional activator. We report that IFN-gamma activates a distinct signaling cascade involving the c-cbl protooncogene product, CrkL adapter, and small G protein Rap1. During treatment of NB-4 human cells with IFN-gamma, c-cbl protooncogene product is rapidly phosphorylated on tyrosine and provides a docking site for the src homology 2 domain of CrkL, which also undergoes IFN-gamma-dependent tyrosine phosphorylation. CrkL then regulates activation of the guanine exchange factor C3G, with which it interacts constitutively via its N terminus src homology 3 domain. This results in the IFN-gamma-dependent activation of Rap1, a protein known to exhibit tumor suppressor activity and mediate growth inhibitory responses. In a similar manner, Rap1 is also activated in response to treatment of cells with type I IFNs (IFN-alpha, IFN-beta), which also engage CrkL in their signaling pathways. On the other hand, IFN-gamma does not induce formation of nuclear CrkL-Stat5 DNA-binding complexes, which are induced by IFN-alpha and IFN-beta, indicating that pathways downstream of CrkL are differentially regulated by different IFN subtypes. Taken altogether, our data demonstrate that, in addition to activating the Stat pathway, IFN-gamma activates a distinct signaling cascade that may play an important role in the generation of its growth inhibitory effects on target cells.

Cloning and Characterization of a Novel Adaptor Protein, CIN85, That Interacts with c-Cbl

The c-Cbl protooncogene product is a prominent substrate of protein tyrosine kinases and is rapidly tyrosine-phosphorylated upon stimulation of a wide variety of cell-surface receptors. We have identified a novel c-Cbl-interacting protein termed CIN85 with a molecular mass of 85 kDa which shows similarity to adaptor proteins, CMS and CD2AP. CIN85 mRNA is expressed ubiquitously in normal human tissues and cancer cell lines analyzed. CIN85 was basally associated with c-Cbl. For interaction of CIN85 with c-Cbl, the second SH3 domain of CIN85 was shown to serve as a central player. The CIN85–c-Cbl association was enhanced shortly after stimulation of 293 cells with epidermal growth factor (EGF) and gradually diminished to a basal level, which correlated with a tyrosine phosphorylation level of c-Cbl. Our results suggest that CIN85 may play a specific role in the EGF receptor-mediated signaling cascade via its interaction with c-Cbl.

C-Cbl Is Tyrosine-Phosphorylated by Interleukin-4 and Enhances Mitogenic and Survival Signals of Interleukin-4 Receptor by Linking With the Phosphatidylinositol 3′-Kinase Pathway

AbstractInterleukin-4 (IL-4) is a cytokine that induces both proliferation and differentiation and suppresses apoptosis of B cells. Although IL-4 has been shown to activate the phosphatidylinositol 3′ (PI3)-kinase pathway, the role of PI3 kinase in the IL-4 receptor (IL-4R) signaling remains unclear. In this study, we demonstrated that c-Cbl proto-oncogene product is inducibly phosphorylated on tyrosine residues and is associated with the p85 subunit of PI3-kinase by IL-4 stimulation. Overexpression of c-Cbl enhances the PI3-kinase activity and, at the same time, mitogenic activity and survival of cells in the presence of IL-4. However, these effects of c-Cbl were abolished by wortmannin, a specific inhibitor for the PI3 kinase pathway, or by a point mutation at tyrosine 731 of c-Cbl, which is a major binding site for p85. These results indicate that c-Cbl plays a role in linking IL-4R with the PI3 kinase pathway and thus enhancing the mitogenic and survival signals.

C-Cbl Is Tyrosine-Phosphorylated by Interleukin-4 and Enhances Mitogenic and Survival Signals of Interleukin-4 Receptor by Linking With the Phosphatidylinositol 3′-Kinase Pathway

Interleukin-4 (IL-4) is a cytokine that induces both proliferation and differentiation and suppresses apoptosis of B cells. Although IL-4 has been shown to activate the phosphatidylinositol 3′ (PI3)-kinase pathway, the role of PI3 kinase in the IL-4 receptor (IL-4R) signaling remains unclear. In this study, we demonstrated that c-Cbl proto-oncogene product is inducibly phosphorylated on tyrosine residues and is associated with the p85 subunit of PI3-kinase by IL-4 stimulation. Overexpression of c-Cbl enhances the PI3-kinase activity and, at the same time, mitogenic activity and survival of cells in the presence of IL-4. However, these effects of c-Cbl were abolished by wortmannin, a specific inhibitor for the PI3 kinase pathway, or by a point mutation at tyrosine 731 of c-Cbl, which is a major binding site for p85. These results indicate that c-Cbl plays a role in linking IL-4R with the PI3 kinase pathway and thus enhancing the mitogenic and survival signals.

Cbl-b, a member of the Sli-1/c-Cbl protein family, inhibits Vav-mediated c-Jun N-terminal kinase activation.

We have used the yeast two-hybrid system to identify proteins that interact with Vav, a GDP/GTP exchange factor for the Rac-1 GTPase that plays an important role in cell signaling and oncogenic transformation. This experimental approach resulted in the isolation of Cbl-b, a signal transduction molecule highly related to the mammalian c-cbl proto-oncogene product and to the C. elegans Sli-1 protein, a negative regulator of the EGF-receptor-like Let23 protein. The interaction between Vav and Cbl-b requires the entire SH3-SH2-SH3 carboxy-terminal domain of Vav and a long stretch of proline-rich sequences present in the central region of Cbl-b. Stimulation of quiescent rodent fibroblasts with either epidermal or platelet-derived growth factors induces an increased affinity of Vav for Cbl-b and results in the subsequent formation of a Vav-dependent trimeric complex with the ligand-stimulated tyrosine kinase receptors. During this process, Vav, but not Cbl-b, becomes highly phosphorylated on tyrosine residues. Overexpression of Cbl-b inhibits the signal transduction pathway of Vav that leads to the stimulation of c-Jun N-terminal kinase. By contrast, expression of truncated Cbl-b proteins and of missense mutants analogous to those found in inactive Sli-1 proteins have no detectable effect on Vav activity. These results indicate that Vav and Cbl-b act coordinately in the first steps of tyrosine protein kinase receptor-mediated signaling and suggest that members of the Sli-1/Cbl family are also negative regulators of signal transduction in mammalian cells.

Ligation of the T cell antigen receptor induces tyrosine phosphorylation of p105CasL, a member of the p130Cas-related docking protein family, and its subsequent binding to the Src homology 2 domain of c-Crk.

p105CasL (CasL) is a recently identified signaling molecule closely related to the p130Cas (Crk-associated substrate) docking protein. CasL has a single Src homology (SH) 3 domain in its N-terminal portion followed by multiple consensus motifs for binding to SH2 domains. Like original p130Cas, CasL undergoes tyrosine phosphorylation upon integrin-mediated cell adhesion. In the present report, we provide direct evidence that CasL is also involved in T cell antigen receptor (TcR)-mediated signal transduction. In binding studies in vitro using glutathione-S-transferase fusion proteins, we have identified 105- and 120-kDa phosphotyrosyl proteins (pp105 and pp120, respectively) tightly bound to the SH2 domain of the Crk adapter protein in the H9 human T cell line after stimulation through the CD3/TcR complex. pp120, but not pp105, also bound to the SH3 of another adapter protein, Ash/Grb2. Immunoblotting with specific antibodies revealed that pp120 and pp105 were identical to the c-cbl proto-oncogene product (p120cbl) and CasL, respectively. Association between Crk and tyrosine-phosphorylated CasL after TcR stimulation was also confirmed in vivo. CasL phosphorylation induced by TcR ligation reached maximal levels within 2 min and rapidly declined thereafter, whereas the integrin-dependent response occurred slowly and was more prolonged. Finally, we demonstrated that Crk/CasL association occurred in peripheral blood T lymphocytes in response to TcR engagement. Our findings suggest that CasL is involved in T cell activation signals and resides at a point where two distinct receptor-mediated signaling pathways converge. This provides one mechanism by which integrins may mediate T cell co-stimulation.

Deficient Tyrosine Phosphorylation of c-Cbl and Associated Proteins in Phorbol Ester-resistant EL4 Mouse Thymoma Cells

Two tyrosine phosphoproteins in phorbol ester-sensitive EL4 (S-EL4) mouse thymoma cells have been identified as the p120 c-Cbl protooncogene product and the p85 subunit of phosphatidylinositol 3-kinase. Tyrosine phosphorylation of p120 and p85 increased rapidly after phorbol ester stimulation. Phorbol ester-resistant EL4 (R-EL4) cells expressed comparable amounts of c-Cbl and phosphatidylinositol 3-kinase protein but greatly diminished tyrosine phosphorylation. Co-immunoprecipitation experiments revealed complexes of c-Cbl with p85, and of p85 with the tyrosine kinase Lck in phorbol ester-stimulated S-EL4 but not in unstimulated S-EL4 or in R-EL4 cells. In vitro binding of c-Cbl with Lck SH2 or SH3 domains was detected in both S-EL4 and R-EL4 cells, suggesting that c-Cbl, p85, and Lck may form a ternary complex. In vitro kinase assays revealed phosphorylation of p85 by Lck only in phorbol ester-stimulated S-EL4 cells. Collectively, these results suggest that Cbl-p85 and Lck-p85 complexes may form in unstimulated S-EL4 and R-EL4 cells but were not detected due to absence of tyrosine phosphorylation of p85. Greatly decreased tyrosine phosphorylation of c-Cbl and p85 in the complexes may contribute to the failure of R-EL4 cells to respond to phorbol ester.

Antisense Repression of Proto-oncogene c-Cbl Enhances Activation of the JAK-STAT Pathway but Not the Ras Pathway in Epidermal Growth Factor Receptor Signaling

Many growth factors including epidermal growth factor (EGF) induce tyrosine phosphorylation of the c-Cbl proto-oncogene product, whose function, however, remains unclear. Recently, Sli-1, a Caenorhabditis elegans homologue of c-Cbl, was found to be a negative regulator of let-23-mediated vulval induction pathway, suggesting that c-Cbl may negatively regulate EGF receptor (EGFR)-mediated signaling. In this study, by an antisense RNA approach, we examined the effects of expression level of c-Cbl on EGFR signaling and showed that overexpression of c-Cbl reduces and antisense repression of c-Cbl enhances autophosphorylation of EGF receptors and activation of the JAK-STAT pathway. However, in contrast to the Sli-1 protein, the expressed amount of c-Cbl does not affect activation of the Ras pathway, suggesting that the EGFR-mediated signaling pathways are differently regulated by c-Cbl among nematodes and mammals.

The Proto-oncogene Product p120cbl Links c-Src and Phosphatidylinositol 3′-Kinase to the Integrin Signaling Pathway

Integrin-mediated cell adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation of intracellular proteins. We show in this report that p120(cbl) (Cbl), the 120-kDa c-cbl proto-oncogene product, becomes tyrosine-phosphorylated during integrin-mediated macrophage cell adhesion to extracellular matrix substrata and anti-integrin antibodies. This tyrosine phosphorylation does not occur when cells attach to polylysine, to which cells adhere in a nonspecific fashion. It also does not take place when adhesion-induced reorganization of the cytoskeleton is inhibited with cytochalasin D. In contrast to the rapid and transient tyrosine phosphorylation of Cbl by CSF-1 stimulation, tyrosine phosphorylation of Cbl by cell attachment was gradual and persistent. Tyrosine-phosphorylated Cbl was found to form complexes with the SH2 domain-containing signaling proteins Src and phosphatidylinositol 3-kinase; in vitro kinase assays demonstrated that these kinases were active in the Cbl complexes following integrin ligand binding. Furthermore, Cbl was found to translocate to the plasma membrane in response to cell adhesion to fibronectin. These observations suggest that Cbl serves as a docking protein and may transduce signals to downstream signaling pathways following integrin-mediated cell adhesion in macrophages.

Signal transduction via the CD38/NAD+ glycohydrolase.

The human cell surface CD38 molecule is a 46-kDa type-II transmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long Cys-rich C-terminal extracellular one. We previously demonstrated that an ecto-form NAD+ glycohydrolase (NADase) activity induced by all-trans retinoic acid in HL-60 cells is due to the extracellular domain of CD38. In the present study, we investigated a possible signal transduction mediated through CD38 in the retinoic acid-differentiated HL-60 cells with anti-CD38 monoclonal antibodies (mAbs). The addition of selected anti-CD38 mAbs to the cells induced rapid tyrosine phosphorylation of the cellular proteins with the molecular weights of 120,000, 87,000 and 77,000; the phosphorylated 120-kDa protein was identified as the c-cbl proto-oncogene product, p120c-cbl. Furthermore, the phosphorylated p 120c-cbl associated with the 85-kDa subunit of phosphatidylinositol 3-kinase. To determine the relationship between the amino acid sequence responsible for the NADase activity and epitopes recognized by the stimulatory mAbs, we produced its carboxy-terminal deletion mutants in COS-7 cells. The mutants with less than 15 amino acids deleted from the carboxyl terminus of the 300-amino acid wild-type molecule still maintained NADase activity, but those with more than 27 amino acids deleted did not. Introduction of site-directed mutation of a cysteine residue (Cys275), located in the 273-285 sequence, completely abolished the NADase activity. These CD38 mutants were also used for an epitope mapping of anti-CD38 mAbs. All the epitopes recognized by the mAbs inducing the tyrosine phosphorylation were mapped on the same Cys275-containing sequence of 273-285. Thus, the discrete carboxy-terminal sequence not only plays a key role in its ecto-NADase activity, but also contains the epitopes of the agonistic anti-CD38 mAbs for the transmembrane signaling. We also found that the agonistic mAbs markedly potentiate superoxide generation induced by the stimulation of G protein-coupled chemotactic receptors. Our results suggested that the stimulation of CD38 might generate an accessory signal(s) to enhance the G protein-mediated signaling, probably though the protein-tyrosine phosphorylation.

Biology and action of colony--stimulating factor-1.

Colony-stimulating factor-1 (CSF-1), also known as macrophage colony-stimulating factor, controls the survival, proliferation, and differentiation of mononuclear phagocytes and regulates cells of the females reproductive tract. It appears to play an autocrine and/or paracrine role in cancers of the ovary, endometrium, breast, and myeloid and lymphoid tissues. Through alternative mRNA splicing and differential post-translational proteolytic processing, CSF-1 can either be secreted into the circulation as a glycoprotein or chondroitin sulfate-containing proteoglycan or be expressed as a membrane-spanning glycoprotein on the surface of CSF-1-producing cells. Studies with the op/op mouse, which possesses an inactivating mutation in the CSF-1 gene, have established the central role of CSF-1 in directly regulating osteoclastogenesis and macrophage production. CSF-1 appears to preferentially regulate the development of macrophages found in tissues undergoing active morphogenesis and/or tissue remodeling. These CSF-1 dependent macrophages may, via putative trophic and/or scavenger functions, regulate characteristics such as dermal thickness, male fertility, and neural processing. Apart from its expression on mononuclear phagocytes and their precursors, CSF-1 receptor (CSF-1R) expression on certain nonmononuclear phagocytic cells in the female reproductive tract and studies in the op/op mouse indicate that CSF-1 plays important roles in female reproduction. Restoration of circulating CSF-1 to op/op mice has preliminarily defined target cell populations that are regulated either humorally or locally by the synthesis of cell-surface CSF-1 or by sequestration of the CSF-1 proteoglycan. The CSF-1R is a tyrosine kinase encoded by the c-fms proto-oncogene product. Studies by several groups have used cells expressing either the murine or human CSF-1R in fibroblasts to pinpoint the requirement of kinase activity and the importance of various receptor tyrosine phosphorylation sites for signaling pathways stimulated by CSF-1. To investigate post-CSF-1R signaling in the macrophage, proteins that are rapidly phosphorylated on tyrosine in response to CSF-1 have been identified, together with proteins associated with them. Studies on several of these proteins, including protein tyrosine phosphates 1C, the c-cbl proto-oncogene product, and protein tyrosine phosphatase-phi are discussed.

Regulation of the Association of p120cbl with Grb2 in Jurkat T Cells

The c-cbl protooncogene product (p120(cbl)) is a known substrate of multiple tyrosine kinases. It is found in complexes with critical signal transduction molecules, including the linker protein Grb2. Here, we demonstrate using an immobilized Grb2-binding peptide that the Grb2-p120(cbl) complex dissociates in vivo following engagement of the T-cell antigen receptor in Jurkat T-cells. The early kinetics of this dissociation correlate with the known time course of tyrosine phosphorylation of p120(cbl) and other substrates. This dissociation persists in vivo even when p120(cbl) becomes dephosphorylated to basal levels. However, this decreased association is not observed in protein overlay assays on nitrocellulose membranes in which a Grb2 fusion protein is used to detect p120(cbl) from stimulated or unstimulated cells. These data suggest that the tyrosine phosphorylation of p120(cbl) does not completely account for the regulation of its association with Grb2. Additionally, we used truncation mutations of p120(cbl) to map the p120(cbl)-Grb2 interaction to amino acids 481-528 of p120(cbl); this interaction is stronger in longer constructs that include additional proline-rich motifs. The in vivo regulation of the Grb2-p120(cbl) complex further supports the idea of a significant role for p120(cbl) in receptor-mediated signaling pathways.

Activation of Src-family tyrosine kinases during Fas-induced apoptosis.

Stimulation of several human and murine hematopoietically derived cell lines with anti-Fas antibodies induced increased tyrosine phosphorylation of a panel of proteins observed in whole-cell lysates. In the human T cell line Jurkat, the activity of a 56-kDa tyrosine kinase was likewise activated by anti-Fas antibodies. Immunoprecipitation studies of anti-Fas-stimulated human Jurkat and murine 2B4.11 T cells revealed activation of the Src-family tyrosine kinases Lck and Fyn. Fas receptor-induced tyrosine phosphorylation of p120 c-cbl proto-oncogene product was observed in Jurkat T cells. Pharmacological experiments demonstrated that pretreatment of Jurkat cells with tyrphostins inhibited Fas-induced apoptosis; likewise, Lck activity was inhibited by tyrphostins in a dose-dependent fashion. Finally, Lck derived from unstimulated Jurkat T cells formed stable complexes with the intracellular domain of the Fas receptor. These data are consistent with the notion that expression and activation of members of the Src-family kinases is required for Fas-induced cell death in T lymphocytes and consistent with recent findings demonstrating decreased Fas-mediated thymocytic death in Fyn-knockout mice.

Interactions of Cbl with Two Adaptor Proteins, Grb2 and Crk, upon T Cell Activation

Several recent studies have demonstrated that Grb2, composed entirely of SH2 and SH3 domains, serves as an adaptor protein in tyrosine kinase signaling pathways. Cb1, the protein product of c-cbl proto-oncogene, has been reported to be phosphorylated on tyrosine residues upon T cell receptor (TCR) engagement. Here we show that in unstimulated Jurkat cells Cbl is co-immunoprecipitated with monoclonal antibody against Grb2. However, in lymphocytes activated through the TCR, Cbl loses its ability to bind to Grb2 precipitated either with anti-Grb2 antibody or with an immobilized tyrosine phosphopeptide, Y1068-P, derived from the epidermal growth factor receptor. In vitro studies confirm that the ability of Cb1 to bind to both SH3 domains of Grb2 is strongly reduced in activated T lymphocytes. Investigation of the time course of Cbl dissociation from Grb2 reveals that it is transient and correlates with the kinetics of tyrosine phosphorylation of Cbl. Moreover, Cb1 is co-immunoprecipitated with Crk, another SH2/SH3 domain-containing protein, upon TCR stimulation. Tyrosine-phosphorylated Cbl binds exclusively to the SH2 domain of Crk. These results suggest that different adaptor proteins may have different roles in the regulation of c-cbl proto-oncogene product.

P120cbl Is a Major Substrate of Tyrosine Phosphorylation upon B Cell Antigen Receptor Stimulation and Interacts in Vivo with Fyn and Syk Tyrosine Kinases, Grb2 and Shc Adaptors, and the p85 Subunit of Phosphatidylinositol 3-Kinase

We and others have demonstrated that the c-cbl proto-oncogene product is one of the earliest targets of tyrosine phosphorylation upon T cell receptor stimulation. Given the similarities in the B and T lymphocyte antigen receptors, and the induction of pre-B leukemias in mice by the v-cbl oncogene, we examined the potential involvement of Cbl in B cell receptor signaling. We demonstrate prominent and early tyrosine phosphorylation of Cbl upon stimulation of human B cell lines through surface IgM. Cbl was associated in vivo with Fyn and, to a lesser extent, other Src family kinases. B cell activation also induced a prominent association of Cbl with Syk tyrosine kinase. A substantial fraction of Cbl was constitutively associated with Grb2 and this interaction was mediated by Grb2 SH3 domains. Tyrosine-phosphorylated Shc, which prominently associated with Grb2, was detected in association with Cbl in activated B cells. Thus, Grb2 and Shc adaptors, which associate with immunoreceptor tyrosine based activation motifs, may link Cbl to the B cell receptor. B cell activation also induced a prominent association between Cbl and the p85 subunit of phosphatidylinositol (PI) 3-kinase resulting in the association of a substantial fraction of PI 3-kinase activity with Cbl. Thus, Cbl is likely to play an important role to couple the B cell receptor to the PI 3-kinase pathway. Our results strongly suggest a role for p120cbl in signaling downstream of the B cell receptor and support the idea that Cbl participates in a general signal transduction function downstream of the immune cell surface receptors.

Tyrosine Phosphorylation of the c-cbl Proto-oncogene Product Mediated by Cell Surface Antigen CD38 in HL-60 Cells

The human cell surface antigen CD38 is a 46-kDa type II transmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long Cys-rich C-terminal extracellular one. We demonstrated previously that the extracellular domain of CD38 has NAD+ glycohydrolase (NADase) activity and that the ecto-form NADase activity induced in HL-60 cells during cell differentiation by retinoic acid is due to CD38. In the present study, we investigated the intracellular signaling mediated by CD38 in retinoic acid-differentiated HL-60 cells with an anti-CD38 monoclonal antibody. The addition of anti-CD38 monoclonal antibody to the cells induced rapid tyrosine phosphorylation of the cellular proteins with molecular weights of 120,000, 87,000, and 77,000. An increase in tyrosine kinase activity in the anti-phosphotyrosine immunoprecipitates of the cells was also observed after the addition of anti-CD38 monoclonal antibody. Moreover, one of the prominent tyrosine-phosphorylated proteins stimulated by the anti-CD38 monoclonal antibody was identified as the c-cbl proto-oncogene product, p120c-cbl. These results indicated that tyrosine phosphorylation of cellular proteins, including p120c-cbl, is possibly involved in transmembrane signaling mediated by CD38.

TPO/c-mpl Ligand Induces Tyrosine Phosphorylation of Multiple Cellular Proteins Including Proto-oncogene Products, Vav and c-Cbl, and Ras Signaling Molecules

Thrombopoietin (TPO)/c-mpl ligand is a hematopoietic stimulates proliferation and maturation of megakaryocytes. To analyze the downstream of the c-mpl product, we used a human megakaryoblastic cell line, Mo7e, that has been proved to be responsive to TPO in terms of DNA synthesis. In this study, we found that TPO treatment resulted in tyrosine phosphorylation of Jak-2 kinase. Moreover, it was revealed that several functional molecules involved in the Ras signaling pathway, Shc and Sos, were phosphorylated by treatment with TPO. Finally, tyrosine phosphorylation of the proto-oncogene products, Vav and c-Cbl, has been proved to be induced by TPO. These results suggest that TPO could activate several signaling pathways including the Jak/Stat pathway, the Ras pathway and possibly another pathway involving the c-Cbl proto-oncogene product.