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Cbl-c suppresses v-Src-induced transformation through ubiquitin-dependent protein degradation
Minsoo Kim1, Tohru Tezuka, Keiji Tanaka
1Division of Oncology, Department of Cancer Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Abstract:
The Cbl family proteins Cbl, Cbl-b, and Cbl-c/Cbl-3 are thought to regulate signaling through protein-tyrosine kinases, positively as scaffold proteins and negatively as ubiquitin ligases. However, the precise signaling pathways and target proteins for each Cbl family member are not well understood. Here we show that Src is a preferential target of Cbl-c for degradation. Although exogenous expression of all Cbl family proteins suppressed the anchorage-independent growth of v-Src-transformed NIH3T3 cells, only Cbl-c caused reversion of the refractile morphology. The level of v-Src protein was reduced by Cbl-c, possibly through a lysosome-dependent pathway. The TKB domain and RING finger of Cbl-c were important for its antioncogenic activity. Wild-type Cbl-c promoted ubiquitination of Src in 293T cells, whereas a RING finger mutant did not. Cbl-c bound specifically to Src phosphorylated at Tyr419. Furthermore, Cbl-c together with UbcH5 induced ubiquitination of Src in vitro. Importantly, the Tyr419 nonphosphorylated form of Src was not ubiquitinated by Cbl-c. Therefore, activated Src may be a direct target of Cbl-c in vivo. Our results suggest that Cbl and Cbl-b suppress v-Src-induced transformation through mechanisms distinct from that of Cbl-c.
Insights
Cbl-c targets activated Src for degradation, reverting v-Src-induced cell transformation. This Cbl family member ubiquitinates Src, unlike Cbl and Cbl-b, suggesting distinct anti-cancer mechanisms.
Area of Science:
- Molecular Biology
- Cell Signaling
- Oncogenesis
Background:
- Cbl family proteins (Cbl, Cbl-b, Cbl-c) regulate protein-tyrosine kinase signaling.
- Their precise roles and targets in signaling pathways remain unclear.
Purpose of the Study:
- To elucidate the specific signaling pathways and target proteins regulated by Cbl family members.
- To investigate the role of Cbl-c in regulating Src signaling and v-Src-induced cell transformation.
Main Methods:
- Investigated Cbl family protein function in v-Src-transformed NIH3T3 cells.
- Analyzed v-Src protein levels and morphology changes.
- Assessed protein ubiquitination and binding interactions using 293T cells and in vitro assays.
Main Results:
- Cbl-c preferentially targets Src for degradation, reducing v-Src protein levels.
- Cbl-c, but not Cbl or Cbl-b, reverted v-Src-induced cell morphology.
- Cbl-c's TKB domain and RING finger are crucial for its anti-oncogenic activity.
- Cbl-c directly ubiquitinates activated Src (phosphorylated at Tyr419) in vitro and in vivo.
Conclusions:
- Activated Src is a direct target of Cbl-c, mediated by ubiquitination.
- Cbl-c suppresses v-Src-induced transformation through a distinct mechanism involving Src degradation.
- Cbl and Cbl-b likely employ different pathways to inhibit v-Src-induced transformation.