Elsevier

Cellular Signalling

Volume 22, Issue 3, March 2010, Pages 377-385
Cellular Signalling

c-Cbl acts as a mediator of Src-induced activation of the PI3K-Akt signal transduction pathway during TRAIL treatment

https://doi.org/10.1016/j.cellsig.2009.10.007Get rights and content

Abstract

We have previously observed that TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces acquired TRAIL resistance by increasing Akt phosphorylation and Bcl-xL expression. In this study, we report that Src, c-Cbl, and PI3K are involved in the phosphorylation of Akt during TRAIL treatment. Data from immunoprecipitation and immunoblotting assay reveal that Src interacts with c-Cbl and PI3K. Data from immune complex kinase assay demonstrate that Src can directly phosphorylate c-Cbl and PI3K p85 subunit protein. Data from gene knockdown experiments with an RNA interference (RNAi) technique show that c-Cbl is involved in the interaction between Src and PI3K p85 during TRAIL treatment, playing an important role in TRAIL-induced Akt phosphorylation. Taken together, c-Cbl may act as a mediator to regulate the Src-PI3K-Akt signal transduction pathway during TRAIL treatment.

Introduction

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a type II integral membrane protein belonging to the TNF family. Like Fas ligand (FasL) and TNF-Ī±, the c-terminal extracellular region of TRAIL (amino acids 114ā€“281) exhibits a homotrimeric subunit structureĀ [1]. However, unlike FasL and TNF-Ī±, TRAIL induces apoptosis in a variety of tumor cell lines more efficiently than in normal cells [2]. The apoptotic signal of TRAIL is transduced by binding to the death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5), which are members of the TNF receptor superfamily. Ligation of TRAIL to its receptors results in trimerization of the receptor and clustering of the receptor's intracellular death domain (DD), leading to the formation of the death-inducing signaling complex (DISC). Trimerization of the receptors leads to the recruitment of an adaptor molecule, Fas-associated death domain (FADD), and subsequent binding and activation of caspase-8 and -10. Activated caspase-8 and -10 then cleave caspase-3, which in turn leads to cleavage of the death substrate. Despite TRAIL's potential as an anticancer agent both in vitro and in vivo, some cancer cells that were originally sensitive to TRAIL-induced apoptosis can become resistant after repeated exposure (acquired resistance) [3], [4], suggesting that the physiological role of TRAIL is more complex than merely activating caspase-dependent apoptosis of cancer cells [5]. For example, it was reported that TRAIL stimulated the anti-apoptotic PI3K/Akt pathway in endothelial cells [5], [6] and fibroblast cells [7] as well as that TRAIL-induced PI3K/Akt and NF-ĪŗB activation in Jurkat T leukemia cells [8]. These results imply that, depending on circumstances, TRAIL can function as a cytokine of either cell death or cell survival, similar to NF-ĪŗB [9]. In the pathway of cell survival, Akt has been known to be activated by phosphorylation at threonine 308 and serine 473 in response to various growth factors through a pathway that requires PI3K-dependent generation of PI(3,4,5)P3 [10]. PIP3 facilitates the recruitment of Akt to the plasma membrane through binding with the pleckstrin homology (PH) domain of Akt. At the plasma membrane, Akt is activated by phosphoinositide-dependent kinase-1 (PDK-1) at threonine 308 and becomes fully activated after phosphorylation within the carboxy-terminus at serine 473 [11], [12].

Previously, we reported that DU-145 prostate cancer cells develop acquired TRAIL resistance after TRAIL treatment, and that phosphorylated Akt (pAkt) and its downstream member Bcl-xL are involved in the process of acquired resistance [4]. However, how Akt phosphorylation is increased during development of acquisition of TRAIL resistance has not yet been clearly understood. The main point is that, as stated by Trauzold et al. [13], TRAIL and TRAIL death receptors do not only stimulate apoptosis but also engage non-apoptotic signaling pathways leading to activation of survival-related signals. One of the well known nondeath signaling pathways induced by TRAIL is through TNF receptor-associated protein with death domain (TRADD), receptor-interacting protein (RIP) and TNF receptor-associated factor 2 (TRAF2) [14], which are nondeath signaling modulatory adaptors that interact with the ligand's homotrimerized receptors, and lead to the activation of kinase cascades resulting in activation of NF-ĪŗB and the mitogen-activated protein kinases [15]. However, activation of Akt, upstream of NF-ĪŗB [16], [17], has not been well defined.

Recently, we observed that acquired TRAIL resistance is developed through degradation of TRAIL receptors as well as increased Bcl-xL expression, demonstrating that degradation of TRAIL receptors is mediated by c-Cbl (Casitas B-lineage lymphoma) during TRAIL treatment [4], [18]. However, the mechanism of increased Akt phosphorylation during TRAIL treatment which is another cause of acquired resistance has not yet been clearly explained. Here, we demonstrate that c-Cbl is also responsible for TRAIL-induced Akt phosphorylation through Src-PI3K activation. Src is activated during TRAIL treatment, followed by phosphorylation of PI3K and c-Cbl. c-Cbl may act as a scaffolding molecule for phosphorylation of PI3K by Src.

We recently observed that c-Cbl functions in the degradation of TRAIL receptors as an E3 ligase [18]. In addition to this E3 ligase activity, many cellular events mediated or regulated by c-Cbl protein are dependent on its adaptor functions, suggesting c-Cbl's diverse and sometimes opposing roles in the regulation of signal transduction in response to different stimulation [19]. For example, association of the distal proline-rich sequences of c-Cbl and the SH3 domain of the p85 subunit of PI3 kinase is responsible for the activation of PI3-kinase by EGF stimulation [20], [21], [22].

In this paper we demonstrate that c-Cbl plays an important role in the TRAIL-induced activation of the Src-PI3K-Akt signal transduction pathway.

Section snippets

Cell culture and survival assay

Human prostate adenocarcinoma DU-145 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA) and 26Ā mM sodium bicarbonate for monolayer cell culture. The cells were maintained in a humidified atmosphere containing 5% CO2 and air at 37Ā Ā°C.

Reagents and antibodies

Anti-caspase 8, anti-phosphoS473-Akt, anti-Akt, anti-phosphoTyr416-Src, anti-Src, anti-phosphoTyr731-c-Cbl, and anti-c-Cbl were purchased from Cell Signaling (Beverly, MA, USA).

Akt and Src phosphorylation during TRAIL treatment

We previously reported that acquired TRAIL resistance is developed by multimode such as increasing Akt phosphorylation and Bcl-xL expressionĀ [4]. It is well known that Bcl-xL expression is upregulated by activated (phosphorylated) Akt [23]. However, the remaining question is how TRAIL treatment increases phosphorylation of Akt. In this study, we hypothesized that Src, one of the non-receptor tyrosine kinase, is responsible for Akt phosphorylation during TRAIL treatment. To examine this

Discussion

In this study we observed that Src is responsible for TRAIL-induced activation of the PI3K-Akt signal transduction pathway. Interestingly, c-Cbl acts as a mediator in the Src-PI3K-Akt signal transduction pathway. Previous studies have shown that c-Cbl has multiple functions in the cells [28]. One of functions is to be involved in the degradation of TRAIL receptors [18]; c-Cbl acts as an E3 ligase and plays an important role in the development of acquired TRAIL resistance through the degradation

Acknowledgements

This work was supported by the following grants: NCI grant funds (CA121395 and CA140554: J. J. S., Y.J. L.) and (CA113263: M.A.A., D.L.B., Y.J. L.), and this research was also supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0071809; J.J. Song) and a faculty research grant of Yonsei University College of Medicine for 2009-0113; J.J. Song) and a grant from the Ministry of

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