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Pelle Modulates dFoxO-Mediated Cell Death in Drosophila.

Wu C, Chen Y, Wang F, Chen C, Zhang S, Li C, Li W, Wu S, Xue L - PLoS Genet. (2015)

Bottom Line: Interleukin-1 receptor-associated kinases (IRAKs) are crucial mediators of the IL-1R/TLR signaling pathways that regulate the immune and inflammation response in mammals.Finally, Pll physically interacts with dFoxO and phosphorylates dFoxO directly.This study not only identifies a previously unknown physiological function of pll in cell death, but also shed light on the mechanism of IRAKs in cell survival/death during tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Interventional Radiology, Shanghai 10th People's Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Science and Technology, Tongji University, Shanghai, China.

ABSTRACT
Interleukin-1 receptor-associated kinases (IRAKs) are crucial mediators of the IL-1R/TLR signaling pathways that regulate the immune and inflammation response in mammals. Recent studies also suggest a critical role of IRAKs in tumor development, though the underlying mechanism remains elusive. Pelle is the sole Drosophila IRAK homolog implicated in the conserved Toll pathway that regulates Dorsal/Ventral patterning, innate immune response, muscle development and axon guidance. Here we report a novel function of pll in modulating apoptotic cell death, which is independent of the Toll pathway. We found that loss of pll results in reduced size in wing tissue, which is caused by a reduction in cell number but not cell size. Depletion of pll up-regulates the transcription of pro-apoptotic genes, and triggers caspase activation and cell death. The transcription factor dFoxO is required for loss-of-pll induced cell death. Furthermore, loss of pll activates dFoxO, promotes its translocation from cytoplasm to nucleus, and up-regulates the transcription of its target gene Thor/4E-BP. Finally, Pll physically interacts with dFoxO and phosphorylates dFoxO directly. This study not only identifies a previously unknown physiological function of pll in cell death, but also shed light on the mechanism of IRAKs in cell survival/death during tumorigenesis.

No MeSH data available.


Related in: MedlinePlus

Pll binds to and phosphorylates dFoxO.(A) Expression of Pll induced mobility shift of dFoxO, which was abolished by CIP treatment. Cell lysis was prepared from S2R+ cells expressing Flag-dFoxO alone or with HA-Pelle, treated with or without calf intestine phosphatase (CIP), followed by western blotting. (B) Kinase assay shows Pll phosphorylates the N- and C- terminal parts of dFoxO. (C) Co-immunoprecipitation experiment shows dFoxO interacts with Pelle in S2R+ cells. (D) GST pull-down assay shows direct binding between bacterially expressed His–dFoxO and GST–Pll in vitro.
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pgen.1005589.g010: Pll binds to and phosphorylates dFoxO.(A) Expression of Pll induced mobility shift of dFoxO, which was abolished by CIP treatment. Cell lysis was prepared from S2R+ cells expressing Flag-dFoxO alone or with HA-Pelle, treated with or without calf intestine phosphatase (CIP), followed by western blotting. (B) Kinase assay shows Pll phosphorylates the N- and C- terminal parts of dFoxO. (C) Co-immunoprecipitation experiment shows dFoxO interacts with Pelle in S2R+ cells. (D) GST pull-down assay shows direct binding between bacterially expressed His–dFoxO and GST–Pll in vitro.

Mentions: To investigate the underlying mechanism by which Pll regulates dFoxO activity, we expressed HA-tagged Pll (HA-Pll) and Flag-tagged dFoxO (Flag-dFoxO) in Drosophila S2R+ cells, and examined the phosphorylation of dFoxO through Calf Intestine Phosphatase (CIP) assay. We found that co-expression of Pll resulted in a mobility shift of dFoxO, which was abolished upon CIP treatment (Fig 10A), suggesting that dFoxO could be phosphorylated by Pll. Next, we performed the in vitro kinase assay to confirm the phosphorylation of dFoxO by Pll. Since Pll could be auto-phosphorylated (Fig 10B), to better distinguish the phosphorylated dFoxO from auto-phosphorylated Pll, we divided the full-length dFoxO (1-622aa) into two segments: the N-terminal (NT, 1-304aa) and the C-terminal (CT, 305-622aa). Indeed, bacterially purified GST-Pll was able to phosphorylate dFoxO in vitro, heavily on the NT and lightly on the CT (Fig 10B), suggesting Pll might phosphorylate dFoxO at multiple sites, most of which are located in the N-terminal half of dFoxO. Furthermore, Co-IP assay demonstrated a physical interaction between HA-Pll and Flag-dFoxO in S2R+ cells (Fig 10C). Finally, we performed the GST-pulldown assay and confirmed a direct binding between Pll and dFoxO (Fig 10D). Together, these data suggest that Pll could phosphorylate dFoxO through direct interaction, thus negatively regulates dFoxO activity by preventing its nuclear translocation.


Pelle Modulates dFoxO-Mediated Cell Death in Drosophila.

Wu C, Chen Y, Wang F, Chen C, Zhang S, Li C, Li W, Wu S, Xue L - PLoS Genet. (2015)

Pll binds to and phosphorylates dFoxO.(A) Expression of Pll induced mobility shift of dFoxO, which was abolished by CIP treatment. Cell lysis was prepared from S2R+ cells expressing Flag-dFoxO alone or with HA-Pelle, treated with or without calf intestine phosphatase (CIP), followed by western blotting. (B) Kinase assay shows Pll phosphorylates the N- and C- terminal parts of dFoxO. (C) Co-immunoprecipitation experiment shows dFoxO interacts with Pelle in S2R+ cells. (D) GST pull-down assay shows direct binding between bacterially expressed His–dFoxO and GST–Pll in vitro.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4608839&req=5

pgen.1005589.g010: Pll binds to and phosphorylates dFoxO.(A) Expression of Pll induced mobility shift of dFoxO, which was abolished by CIP treatment. Cell lysis was prepared from S2R+ cells expressing Flag-dFoxO alone or with HA-Pelle, treated with or without calf intestine phosphatase (CIP), followed by western blotting. (B) Kinase assay shows Pll phosphorylates the N- and C- terminal parts of dFoxO. (C) Co-immunoprecipitation experiment shows dFoxO interacts with Pelle in S2R+ cells. (D) GST pull-down assay shows direct binding between bacterially expressed His–dFoxO and GST–Pll in vitro.
Mentions: To investigate the underlying mechanism by which Pll regulates dFoxO activity, we expressed HA-tagged Pll (HA-Pll) and Flag-tagged dFoxO (Flag-dFoxO) in Drosophila S2R+ cells, and examined the phosphorylation of dFoxO through Calf Intestine Phosphatase (CIP) assay. We found that co-expression of Pll resulted in a mobility shift of dFoxO, which was abolished upon CIP treatment (Fig 10A), suggesting that dFoxO could be phosphorylated by Pll. Next, we performed the in vitro kinase assay to confirm the phosphorylation of dFoxO by Pll. Since Pll could be auto-phosphorylated (Fig 10B), to better distinguish the phosphorylated dFoxO from auto-phosphorylated Pll, we divided the full-length dFoxO (1-622aa) into two segments: the N-terminal (NT, 1-304aa) and the C-terminal (CT, 305-622aa). Indeed, bacterially purified GST-Pll was able to phosphorylate dFoxO in vitro, heavily on the NT and lightly on the CT (Fig 10B), suggesting Pll might phosphorylate dFoxO at multiple sites, most of which are located in the N-terminal half of dFoxO. Furthermore, Co-IP assay demonstrated a physical interaction between HA-Pll and Flag-dFoxO in S2R+ cells (Fig 10C). Finally, we performed the GST-pulldown assay and confirmed a direct binding between Pll and dFoxO (Fig 10D). Together, these data suggest that Pll could phosphorylate dFoxO through direct interaction, thus negatively regulates dFoxO activity by preventing its nuclear translocation.

Bottom Line: Interleukin-1 receptor-associated kinases (IRAKs) are crucial mediators of the IL-1R/TLR signaling pathways that regulate the immune and inflammation response in mammals.Finally, Pll physically interacts with dFoxO and phosphorylates dFoxO directly.This study not only identifies a previously unknown physiological function of pll in cell death, but also shed light on the mechanism of IRAKs in cell survival/death during tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Interventional Radiology, Shanghai 10th People's Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Science and Technology, Tongji University, Shanghai, China.

ABSTRACT
Interleukin-1 receptor-associated kinases (IRAKs) are crucial mediators of the IL-1R/TLR signaling pathways that regulate the immune and inflammation response in mammals. Recent studies also suggest a critical role of IRAKs in tumor development, though the underlying mechanism remains elusive. Pelle is the sole Drosophila IRAK homolog implicated in the conserved Toll pathway that regulates Dorsal/Ventral patterning, innate immune response, muscle development and axon guidance. Here we report a novel function of pll in modulating apoptotic cell death, which is independent of the Toll pathway. We found that loss of pll results in reduced size in wing tissue, which is caused by a reduction in cell number but not cell size. Depletion of pll up-regulates the transcription of pro-apoptotic genes, and triggers caspase activation and cell death. The transcription factor dFoxO is required for loss-of-pll induced cell death. Furthermore, loss of pll activates dFoxO, promotes its translocation from cytoplasm to nucleus, and up-regulates the transcription of its target gene Thor/4E-BP. Finally, Pll physically interacts with dFoxO and phosphorylates dFoxO directly. This study not only identifies a previously unknown physiological function of pll in cell death, but also shed light on the mechanism of IRAKs in cell survival/death during tumorigenesis.

No MeSH data available.


Related in: MedlinePlus