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p53- and ERK7-dependent ribosome surveillance response regulates Drosophila insulin-like peptide secretion.

Hasygar K, Hietakangas V - PLoS Genet. (2014)

Bottom Line: A downstream effector of this growth inhibitory response is an atypical MAP kinase ERK7 (ERK8/MAPK15), which is upregulated in the IPCs following impaired ribosome biogenesis as well as starvation.Moreover, we provide evidence that p53 and ERK7 contribute to the inhibition of dILP secretion upon starvation.Thus, we conclude that a cell autonomous ribosome surveillance response, which leads to upregulation of ERK7, inhibits dILP secretion to impede tissue growth under limiting dietary conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences & Institute of Biotechnology, University of Helsinki, Helsinki, Finland.

ABSTRACT
Insulin-like signalling is a conserved mechanism that coordinates animal growth and metabolism with nutrient status. In Drosophila, insulin-producing median neurosecretory cells (IPCs) regulate larval growth by secreting insulin-like peptides (dILPs) in a diet-dependent manner. Previous studies have shown that nutrition affects dILP secretion through humoral signals derived from the fat body. Here we uncover a novel mechanism that operates cell autonomously in the IPCs to regulate dILP secretion. We observed that impairment of ribosome biogenesis specifically in the IPCs strongly inhibits dILP secretion, which consequently leads to reduced body size and a delay in larval development. This response is dependent on p53, a known surveillance factor for ribosome biogenesis. A downstream effector of this growth inhibitory response is an atypical MAP kinase ERK7 (ERK8/MAPK15), which is upregulated in the IPCs following impaired ribosome biogenesis as well as starvation. We show that ERK7 is sufficient and essential to inhibit dILP secretion upon impaired ribosome biogenesis, and it acts epistatically to p53. Moreover, we provide evidence that p53 and ERK7 contribute to the inhibition of dILP secretion upon starvation. Thus, we conclude that a cell autonomous ribosome surveillance response, which leads to upregulation of ERK7, inhibits dILP secretion to impede tissue growth under limiting dietary conditions.

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erk7 mRNA levels are elevated upon impaired ribosome biogenesis or starvation.(A) ERK7 mRNA levels in the IPCs were detected by in situ hybridization upon IPC-specific knockdown of Rio2 (dILP2-Gal4) or starvation. (B, C) Quantitative RT-PCR analysis of relative erk7 mRNA expression upon ubiquitous knockdown (Tub-G80TS; Tub-Gal4) of Rio1 and Rio2 (B), or dMyc (C) in whole larvae. (D) Relative erk7 mRNA levels upon feeding and starvation in whole w1118 larvae. Error bars represent standard deviation (N = 3, ≥5 larvae/group). actin was used as an internal reference. *p<0.05, **p<0.01 (Student's t-test).
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pgen-1004764-g004: erk7 mRNA levels are elevated upon impaired ribosome biogenesis or starvation.(A) ERK7 mRNA levels in the IPCs were detected by in situ hybridization upon IPC-specific knockdown of Rio2 (dILP2-Gal4) or starvation. (B, C) Quantitative RT-PCR analysis of relative erk7 mRNA expression upon ubiquitous knockdown (Tub-G80TS; Tub-Gal4) of Rio1 and Rio2 (B), or dMyc (C) in whole larvae. (D) Relative erk7 mRNA levels upon feeding and starvation in whole w1118 larvae. Error bars represent standard deviation (N = 3, ≥5 larvae/group). actin was used as an internal reference. *p<0.05, **p<0.01 (Student's t-test).

Mentions: Identification of p53 as an essential inhibitor of dILP2 secretion implied that ribosome biogenesis regulates dILP secretion through an active surveillance mechanism, rather than through an indirect mechanism following reduced translation. To directly couple ribosome surveillance to secretion we turned our attention to known regulators of the secretory pathway. It was recently shown that an atypical MAP kinase called ERK7 (also known as ERK8 or MAPK15) inhibits secretion upon serum and amino acid starvation in Drosophila S2 cells [38]. ERK7 was shown to phosphorylate Sec16 and thus prevent its recruitment to ER exit sites, consequently preventing the export of the secretory cargo. Therefore, we wanted to explore, whether ERK7 is regulated in response to inhibited ribosome biogenesis. In order to do so, we used in situ hybridization of larval brain. erk7 expression was undetectable in fed control larvae, whereas overexpression of transgenic ERK7 by dILP2-Gal4 led to a strong IPC-specific signal (Figure S7). As a negative control we used an erk7 sense probe for the ERK7 overexpressing samples (Figure S7). After confirming the specificity of the in situ hybridization, we analysed erk7 expression upon disturbed ribosome biogenesis. Intriguingly, we observed that erk7 mRNA levels were clearly elevated in the IPCs following Rio2 depletion using dILP2-Gal4 (Figure 4A), implying that ERK7 is upregulated upon ribosome surveillance in the IPCs. As ribosome biogenesis is inhibited upon starvation [35], we hypothesized that starvation might also cause elevated expression of erk7, which indeed was the case (Figure 4A). Notably, in the context of brain tissue, the starvation-induced upregulation appeared specific to the IPCs, since erk7 mRNA remained undetectable in other brain areas. To confirm the regulation of erk7 mRNA by an independent method, we used quantitative RT-PCR on mRNA samples isolated from whole larvae. Disturbance of ribosome biogenesis by ubiquitous depletion of Rio1, Rio2 and Myc led to elevated erk7 expression (Figure 4B, C). Similarly, starvation caused significant upregulation of erk7 mRNA levels in the whole larval samples (Figure 4D). In sum, our data shows that erk7 gene expression is elevated upon impaired ribosome biogenesis and starvation, suggesting a possible role for ERK7 in the regulation of dILP secretion during these conditions.


p53- and ERK7-dependent ribosome surveillance response regulates Drosophila insulin-like peptide secretion.

Hasygar K, Hietakangas V - PLoS Genet. (2014)

erk7 mRNA levels are elevated upon impaired ribosome biogenesis or starvation.(A) ERK7 mRNA levels in the IPCs were detected by in situ hybridization upon IPC-specific knockdown of Rio2 (dILP2-Gal4) or starvation. (B, C) Quantitative RT-PCR analysis of relative erk7 mRNA expression upon ubiquitous knockdown (Tub-G80TS; Tub-Gal4) of Rio1 and Rio2 (B), or dMyc (C) in whole larvae. (D) Relative erk7 mRNA levels upon feeding and starvation in whole w1118 larvae. Error bars represent standard deviation (N = 3, ≥5 larvae/group). actin was used as an internal reference. *p<0.05, **p<0.01 (Student's t-test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230838&req=5

pgen-1004764-g004: erk7 mRNA levels are elevated upon impaired ribosome biogenesis or starvation.(A) ERK7 mRNA levels in the IPCs were detected by in situ hybridization upon IPC-specific knockdown of Rio2 (dILP2-Gal4) or starvation. (B, C) Quantitative RT-PCR analysis of relative erk7 mRNA expression upon ubiquitous knockdown (Tub-G80TS; Tub-Gal4) of Rio1 and Rio2 (B), or dMyc (C) in whole larvae. (D) Relative erk7 mRNA levels upon feeding and starvation in whole w1118 larvae. Error bars represent standard deviation (N = 3, ≥5 larvae/group). actin was used as an internal reference. *p<0.05, **p<0.01 (Student's t-test).
Mentions: Identification of p53 as an essential inhibitor of dILP2 secretion implied that ribosome biogenesis regulates dILP secretion through an active surveillance mechanism, rather than through an indirect mechanism following reduced translation. To directly couple ribosome surveillance to secretion we turned our attention to known regulators of the secretory pathway. It was recently shown that an atypical MAP kinase called ERK7 (also known as ERK8 or MAPK15) inhibits secretion upon serum and amino acid starvation in Drosophila S2 cells [38]. ERK7 was shown to phosphorylate Sec16 and thus prevent its recruitment to ER exit sites, consequently preventing the export of the secretory cargo. Therefore, we wanted to explore, whether ERK7 is regulated in response to inhibited ribosome biogenesis. In order to do so, we used in situ hybridization of larval brain. erk7 expression was undetectable in fed control larvae, whereas overexpression of transgenic ERK7 by dILP2-Gal4 led to a strong IPC-specific signal (Figure S7). As a negative control we used an erk7 sense probe for the ERK7 overexpressing samples (Figure S7). After confirming the specificity of the in situ hybridization, we analysed erk7 expression upon disturbed ribosome biogenesis. Intriguingly, we observed that erk7 mRNA levels were clearly elevated in the IPCs following Rio2 depletion using dILP2-Gal4 (Figure 4A), implying that ERK7 is upregulated upon ribosome surveillance in the IPCs. As ribosome biogenesis is inhibited upon starvation [35], we hypothesized that starvation might also cause elevated expression of erk7, which indeed was the case (Figure 4A). Notably, in the context of brain tissue, the starvation-induced upregulation appeared specific to the IPCs, since erk7 mRNA remained undetectable in other brain areas. To confirm the regulation of erk7 mRNA by an independent method, we used quantitative RT-PCR on mRNA samples isolated from whole larvae. Disturbance of ribosome biogenesis by ubiquitous depletion of Rio1, Rio2 and Myc led to elevated erk7 expression (Figure 4B, C). Similarly, starvation caused significant upregulation of erk7 mRNA levels in the whole larval samples (Figure 4D). In sum, our data shows that erk7 gene expression is elevated upon impaired ribosome biogenesis and starvation, suggesting a possible role for ERK7 in the regulation of dILP secretion during these conditions.

Bottom Line: A downstream effector of this growth inhibitory response is an atypical MAP kinase ERK7 (ERK8/MAPK15), which is upregulated in the IPCs following impaired ribosome biogenesis as well as starvation.Moreover, we provide evidence that p53 and ERK7 contribute to the inhibition of dILP secretion upon starvation.Thus, we conclude that a cell autonomous ribosome surveillance response, which leads to upregulation of ERK7, inhibits dILP secretion to impede tissue growth under limiting dietary conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences & Institute of Biotechnology, University of Helsinki, Helsinki, Finland.

ABSTRACT
Insulin-like signalling is a conserved mechanism that coordinates animal growth and metabolism with nutrient status. In Drosophila, insulin-producing median neurosecretory cells (IPCs) regulate larval growth by secreting insulin-like peptides (dILPs) in a diet-dependent manner. Previous studies have shown that nutrition affects dILP secretion through humoral signals derived from the fat body. Here we uncover a novel mechanism that operates cell autonomously in the IPCs to regulate dILP secretion. We observed that impairment of ribosome biogenesis specifically in the IPCs strongly inhibits dILP secretion, which consequently leads to reduced body size and a delay in larval development. This response is dependent on p53, a known surveillance factor for ribosome biogenesis. A downstream effector of this growth inhibitory response is an atypical MAP kinase ERK7 (ERK8/MAPK15), which is upregulated in the IPCs following impaired ribosome biogenesis as well as starvation. We show that ERK7 is sufficient and essential to inhibit dILP secretion upon impaired ribosome biogenesis, and it acts epistatically to p53. Moreover, we provide evidence that p53 and ERK7 contribute to the inhibition of dILP secretion upon starvation. Thus, we conclude that a cell autonomous ribosome surveillance response, which leads to upregulation of ERK7, inhibits dILP secretion to impede tissue growth under limiting dietary conditions.

Show MeSH
Related in: MedlinePlus