Papers by Christine Thien
Biochemical and Biophysical Research Communications, Nov 7, 1997
The protein product of c-cbl proto-oncogene is known to interact with several proteins, including... more The protein product of c-cbl proto-oncogene is known to interact with several proteins, including Grb2, Crk and PI3 kinase, and is thought to regulate signalling by many cell surface receptors. The precise function of c-Cbl in these pathways is not clear, although a genetic analysis in Caenorhabditis elegans suggests that c-Cbl is a negative regulator of the epidermal growth factor receptor. Here we describe a yeast two hybrid screen performed with c-Cbl in an attempt to further elucidate its role in signal transduction. The screen identified interactions involving c-Cbl and two 14-3-3 isoforms, cytokeratin 18, human unconventional myosin IC, and a recently identified SH3 domain containing protein, SH3 P17. We have used the yeast two hybrid assay to localise regions of c-Cbl required for its interaction with each of the proteins. Interaction with 14-3-3 is demonstrated in mammalian cell extracts.
Diabetes, Dec 1, 2006
Casitas b-lineage lymphoma (c-Cbl) is a multiadaptor protein with E3-ubiquitin ligase activity re... more Casitas b-lineage lymphoma (c-Cbl) is a multiadaptor protein with E3-ubiquitin ligase activity residing within its RING finger domain. We have previously reported that c-Cbl-deficient mice exhibit elevated energy expenditure, reduced adiposity, and improved insulin action. In this study, we examined mice expressing c-Cbl protein with a loss-of-function mutation within the RING finger domain (c-Cbl A/Ϫ mice). Compared with control animals, c-Cbl A/Ϫ mice display a phenotype that includes reduced adiposity, despite greater food intake; reduced circulating insulin, leptin, and triglyceride levels; and improved glucose tolerance. c-Cbl A/Ϫ mice also display elevated oxygen consumption (13%) and are protected against high-fat diet-induced obesity and insulin resistance. Unlike c-Cbl A/Ϫ mice, mice expressing a mutant c-Cbl with the phosphatidylinositol (PI) 3-kinase binding domain ablated (c-Cbl F/F mice) exhibited an insulin sensitivity, body composition, and energy expenditure similar to that of wild-type animals. These results indicate that c-Cbl ubiquitin ligase activity, but not c-Cbl-dependent activation of PI 3-kinase, plays a key role in the regulation of whole-body energy metabolism.
The Journal of Immunology
Recent studies indicate that c-Cbl and its oncogenic variants can modulate the activity of protei... more Recent studies indicate that c-Cbl and its oncogenic variants can modulate the activity of protein tyrosine kinases. This finding is supported by studies showing that c-Cbl interacts directly with a negative regulatory tyrosine in ZAP-70, and that the levels of tyrosine-phosphorylated ZAP-70 and numerous other proteins are increased in TCR-stimulated thymocytes from c-Cbl-deficient mice. Here, we demonstrate that this enhanced phosphorylation of ZAP-70 and that of two substrates, LAT and SLP-76, is not due to altered protein levels but is the consequence of two separate events. First, we find increased expression of tyrosinephosphorylated TCR chain in c-Cbl-deficient thymocytes, which results in a higher level of -chain-associated ZAP-70 that is initially accessible for activation. Thus, more ZAP-70 is activated and more of its substrates (LAT and SLP-76) become tyrosinephosphorylated after TCR stimulation. However, an additional mechanism of ZAP-70 regulation is evident at a later time poststimulation. At this time, ZAP-70 from both normal and c-Cbl ؊/؊ thymocytes becomes hyperphosphorylated; however, only in normal thymocytes does this correlate with ZAP-70 down-regulation and a diminished ability to phosphorylate LAT and SLP-76. In contrast, c-Cbl-deficient thymocytes display altered phosphorylation kinetics, for which LAT phosphorylation is increased and SLP-76 phosphorylation is sustained. Thus, the ability to down-regulate the phosphorylation of two ZAP-70 substrates is impaired in c-Cbl ؊/؊ thymocytes. These findings provide evidence that c-Cbl is involved in the negative regulation of the phosphorylation of LAT and SLP-76 by ZAP-70.
Journal of orthopaedic research : official publication of the Orthopaedic Research Society, Jan 29, 2015
Physiotherapy is one of the effective treatments for tendinopathy, whereby symptoms are relieved ... more Physiotherapy is one of the effective treatments for tendinopathy, whereby symptoms are relieved by changing the biomechanical environment of the pathological tendon. However, the underlying mechanism remains unclear. In this study, we first established a model of progressive tendinopathy-like degeneration in the rabbit Achilles. Following ex vivo loading deprivation culture in a bioreactor system for 6 and 12 days, tendons exhibited progressive degenerative changes, abnormal collagen type III production, increased cell apoptosis and weakened mechanical properties. When intervention was applied at day 7 for another 6 days by using cyclic tensile mechanical stimulation (6% strain, 0.25Hz, 8h/day) in a bioreactor the pathological changes and mechanical properties were almost restored to levels seen in healthy tendon. Our results indicated that a proper biomechanical environment was able to rescue early-stage pathological changes by increased collagen type I production, decreased colla...
The Journal of experimental medicine, Jan 17, 2003
The unique tyrosine kinase binding (TKB) domain of Cbl targets phosphorylated tyrosines on activa... more 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 p...
Molecular and cellular biology, 2000
Fyn is a prototype Src-family tyrosine kinase that plays specific roles in neural development, ke... more 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-depen...
Molecular and cellular biology, 1998
The c-Cbl protein is tyrosine phosphorylated and forms complexes with a wide range of signalling ... more 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...
PLoS ONE, 2014
This study aimed to determine whether the multi-kinase inhibitor dasatinib would provide an effec... more This study aimed to determine whether the multi-kinase inhibitor dasatinib would provide an effective therapy for myeloproliferative diseases (MPDs) involving c-Cbl mutations. These mutations, which occur in the RING finger and linker domains, abolish the ability of c-Cbl to function as an E3 ubiquitin ligase and downregulate activated protein tyrosine kinases. Here we analyzed the effects of dasatinib in a c-Cbl RING finger mutant mouse that develops an MPD with a phenotype similar to the human MPDs. The mice are characterized by enhanced tyrosine kinase signaling resulting in an expansion of hematopoietic stem cells, multipotent progenitors and cells within the myeloid lineage. Since c-Cbl is a negative regulator of c-Kit and Src signaling we reasoned that dasatinib, which targets these kinases, would be an effective therapy. Furthermore, two recent studies showed dasatinib to be effective in inhibiting the in vitro growth of cells from leukemia patients with c-Cbl RING finger and linker domain mutations. Surprisingly we found that dasatinib did not provide an effective therapy for c-Cbl RING finger mutant mice since it did not suppress any of the hematopoietic lineages that promote MPD development. Thus we conclude that dasatinib may not be an appropriate therapy for leukemia patients with c-Cbl mutations. We did however find that dasatinib caused a marked reduction of pre-B cells and immature B cells which correlated with a loss of Src activity. This study is therefore the first to provide a detailed characterization of in vivo effects of dasatinib in a hematopoietic disorder that is driven by protein tyrosine kinases other than BCR-ABL.
Experimental hematology, Jan 19, 2014
Mutations in the Fms-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase (RTK) occur frequentl... more Mutations in the Fms-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase (RTK) occur frequently in acute myeloid leukemia (AML), with the most common involving internal tandem duplication (ITD) within the juxtamembrane domain. Fms-like tyrosine kinase 3-ITD mutations result in a mislocalized and constitutively activated receptor, which aberrantly phosphorylates signal transducer and activator of transcription 5 (STAT5) and upregulates the expression of its target genes. c-Cbl is an E3 ubiquitin ligase that negatively regulates RTKs, including FLT3, but whether it can downregulate mislocalized FLT3-ITD remains to be resolved. To help clarify this, we combined a FLT3-ITD mutation with a loss-of-function mutation in the RING finger domain of c-Cbl that abolishes its E3 ligase activity. Mice transplanted with hematopoietic stem cells expressing both mutations rapidly develop myeloid leukemia, indicating strong cooperation between the two. Although the c-Cbl mutation was shown to caus...
Radiation Research, 2004
The mechanism of UV-radiation-induced EGF receptor (EGFR) internalization remains to be establish... more The mechanism of UV-radiation-induced EGF receptor (EGFR) internalization remains to be established. In the present study, we found UV-radiation-mediated internalization of the EGFR to be dependent on the cytoplasmic carboxy-terminal region. UV radiation was unable to induce internalization of EGFR carboxy-terminal truncation mutants where all or four of the five major autophosphorylation sites were missing (963- and 1028-EGFR, respectively). Mutational removal of serine residues 1046, 1047, 1057 and 1142 within the carboxy-terminal receptor region was also sufficient to abolish UV-radiation-induced internalization of the EGFR. Furthermore, the UV-radiation-induced internalization was abrogated for an EGFR mutated in tyrosine 1045 (Y1045F), the major c-Cbl binding site. However, UV radiation did not induce phosphorylation at tyrosine 1045, in contrast to the prominent phosphorylation induced by EGF. Our results suggest a mechanism for UV-radiation-induced internalization of EGFR involving a conformational change that is dependent on structural elements formed by specific serine and tyrosine residues in the carboxy-terminal domain.
Nature Immunology, 2009
Apoptosis is central to the interaction between pathogenic mycobacteria and host macrophages. Cas... more Apoptosis is central to the interaction between pathogenic mycobacteria and host macrophages. Caspase-8-dependent apoptosis of infected macrophages, which requires activation of the mitogen-activated protein (MAP) kinase p38, lowers the spread of mycobacteria. Here we establish a link between the release of tumor necrosis factor (TNF) and mycobacteria-mediated macrophage apoptosis. TNF activated a pathway involving the kinases ASK1, p38 and c-Abl. This pathway led to phosphorylation of FLIP S , which facilitated its interaction with the E3 ubiquitin ligase c-Cbl. This interaction triggered proteasomal degradation of FLIP S , which promoted activation of caspase-8 and apoptosis. Our findings identify a previously unappreciated signaling pathway needed for Mycobacterium tuberculosis-triggered macrophage cell death.
Journal of Clinical Investigation, 2005
The authors regret the error. Corrigendum Plague: the mysterious past and terrifying future of th... more The authors regret the error. Corrigendum Plague: the mysterious past and terrifying future of the world's most dangerous disease Brendan Wren Original citation: J. Clin. Invest. 115:4 (2005).
Journal of Biological Chemistry, 2010
The ability of thymocytes to assess T cell receptor (TCR) signaling strength and to initiate the ... more The ability of thymocytes to assess T cell receptor (TCR) signaling strength and to initiate the appropriate downstream response is crucial for determining their fate. We have previously shown that a c-Cbl RING finger mutant knock-in mouse, in which the E3 ubiquitin ligase activity of c-Cbl is inactivated, is highly sensitive to TCR-induced death signals that cause thymic deletion. This high intensity signal involves the enhanced tyrosine phosphorylation of the mutant c-Cbl protein promoting a marked increase in the activation of Akt. Here we show that this high-intensity signal in c-Cbl RING finger mutant thymocytes also promotes the enhanced induction of two mediators of TCR-directed thymocyte apoptosis, Nur77 and the pro-apoptotic Bcl-2 family member, Bim. In contrast, a knock-in mouse harbouring a mutation at Y737, the site in c-Cbl that activates phosphatidylinositol 3kinase (PI3K), shows reduced TCRmediated responses including suppression of Akt activation, a reduced induction of Nur77 and Bim and greater resistance to thymocyte death. These findings identify tyrosine-phosphorylated c-Cbl as a critical sensor of TCR signal strength that regulates the engagement of death-promoting signals.
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 2011
The E3 ubiquitin ligase Cbl-b regulates T cell activation thresholds and has been associated with... more The E3 ubiquitin ligase Cbl-b regulates T cell activation thresholds and has been associated with protecting against type 1 diabetes, but its in vivo 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 × 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-r...
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 2011
E3 ubiquitin ligases have been placed among the essential molecules involved in the regulation of... more E3 ubiquitin ligases have been placed among the essential molecules involved in the regulation of T cell functions and T cell tolerance. However, it has never been experimentally proven in vivo whether these functions indeed depend on the catalytic E3 ligase activity. The Casitas B-cell lymphoma (Cbl) family protein Cbl-b was the first E3 ubiquitin ligase directly implicated in the activation and tolerance of the peripheral T cell. In this study, we report that selective genetic inactivation of Cbl-b E3 ligase activity phenocopies the T cell responses observed when total Cbl-b is ablated, resulting in T cell hyperactivation, spontaneous autoimmunity, and impaired induction of T cell anergy in vivo. Moreover, mice carrying a Cbl-b E3 ligase-defective mutation spontaneously reject tumor cells that express human papilloma virus Ags. These data demonstrate for the first time, to our knowledge, that the catalytic function of an E3 ligase, Cbl-b, is essential for negative regulation of T ...
Pathology, 2011
The aim of this study was to investigate the incidence and characteristics of c-Cbl mutations in ... more The aim of this study was to investigate the incidence and characteristics of c-Cbl mutations in acute myeloid leukaemias (AMLs) from an Australian patient cohort. Two initial studies examining c-Cbl mutations in AML, one from Germany and one from the US, found vastly different incidences of mutations (0.6% compared to 33%, respectively). Therefore, it was important to determine the incidence and characteristics of c-Cbl mutations in a cohort of Australian AML patients.
Diabetes, 2006
Casitas b-lineage lymphoma (c-Cbl) is a multiadaptor protein with E3-ubiquitin ligase activity re... more Casitas b-lineage lymphoma (c-Cbl) is a multiadaptor protein with E3-ubiquitin ligase activity residing within its RING finger domain. We have previously reported that c-Cbl-deficient mice exhibit elevated energy expenditure, reduced adiposity, and improved insulin action. In this study, we examined mice expressing c-Cbl protein with a loss-of-function mutation within the RING finger domain (c-Cbl A/Ϫ mice). Compared with control animals, c-Cbl A/Ϫ mice display a phenotype that includes reduced adiposity, despite greater food intake; reduced circulating insulin, leptin, and triglyceride levels; and improved glucose tolerance. c-Cbl A/Ϫ mice also display elevated oxygen consumption (13%) and are protected against high-fat diet-induced obesity and insulin resistance. Unlike c-Cbl A/Ϫ mice, mice expressing a mutant c-Cbl with the phosphatidylinositol (PI) 3-kinase binding domain ablated (c-Cbl F/F mice) exhibited an insulin sensitivity, body composition, and energy expenditure similar to that of wild-type animals. These results indicate that c-Cbl ubiquitin ligase activity, but not c-Cbl-dependent activation of PI 3-kinase, plays a key role in the regulation of whole-body energy metabolism.
Blood, 2012
High levels of expression of wild-type Flt3 characterize many hematopoietic proliferative disease... more High levels of expression of wild-type Flt3 characterize many hematopoietic proliferative diseases and neoplasms, providing a potential therapeutic target. Using the c-Cbl RING finger mutant mouse as a model of a myeloproliferative disease (MPD) driven by wild-type Flt3, in the present study, we show that treatment with the Flt3 kinase inhibitor AC220 blocks MPD development by targeting Flt3(+) multipotent progenitors (MPPs). We found that daily administration of AC220 caused a marked reduction in Flt3 expression, induction of quiescence, and a significant loss of MPPs within 4 days. Unexpectedly, a robust Flt3 ligand-associated proliferative recovery response soon followed, preventing further loss of MPPs. However, continued AC220 treatment limited MPP recovery and maintained reduced, steady-state levels of cycling MPPs that express low levels of Flt3. Therefore, a finely tuned balance between the opposing forces of AC220 and Flt3 ligand production was established; whereas the Flt3 ligand blunted the inhibitory effects of AC220, the disease was held in remission for as long as therapy was continued. The net effect is a potent therapy indicating that patients with c-Cbl mutations, or those with similarly enhanced Flt3 signaling, may respond well to AC220 even after the induction of high levels of Flt3 ligand.
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Papers by Christine Thien