Akt phosphorylates both Tsc1 and Tsc2 in Drosophila, but neither phosphorylation is required for normal animal growth - PubMed (original) (raw)

Akt phosphorylates both Tsc1 and Tsc2 in Drosophila, but neither phosphorylation is required for normal animal growth

Sibylle Schleich et al. PLoS One. 2009.

Abstract

Akt, an essential component of the insulin pathway, is a potent inducer of tissue growth. One of Akt's phosphorylation targets is Tsc2, an inhibitor of the anabolic kinase TOR. This could account for part of Akt's growth promoting activity. Although phosphorylation of Tsc2 by Akt does occur in vivo, and under certain circumstances can lead to reduced Tsc2 activity, the functional significance of this event is unclear since flies lacking Akt phosphorylation sites on Tsc2 are viable and normal in size and growth rate. Since Drosophila Tsc1, the obligate partner of Tsc2, has an Akt phosphorylation motif that is not conserved in mammals, we investigate here whether Akt redundantly phosphorylates the Tsc complex on Tsc1 and Tsc2. We provide evidence that Akt phosphorylates Tsc1 at Ser533. We show that flies lacking Akt phosphorylation sites on Tsc1 alone, or on both Tsc1 and Tsc2 concurrently, are viable and normal in size. This shows that phosphorylation of the Tsc1/2 complex by Akt is not required for Akt to activate TORC1 and to promote tissue growth in Drosophila.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Drosophila Tsc1 is phosphorylated by Akt on Ser533.

(A) Phosphorylation of Tsc1 increases with insulin treatment. S2 cells transfected with constructs to express myc-Tsc1 and His-Tsc2 were treated without insulin (0 min) or with insulin (10 µg/mL) for indicated times (20, 40 or 60 min). Cells were then lysed and myc-Tsc1 immunoprecipitated using anti-myc antibody. Immunoprecipitates were probed with anti-myc antibody as a loading control, and anti-Phospho-(Ser/Thr) Akt Substrate antibody to detect phosphorylation of Tsc1. (Ser533 is part of an Akt phosphorylation consensus motif). (B) Tsc1 is phosphorylated on Ser533 in response to insulin treatment. Untransfected S2 cells (-) or S2 cells transfected with constructs to express either myc-Tsc1WT (“WT”) or myc-Tsc1S533A (“S533A”) together with His-Tsc2 were treated with or without insulin (10 µg/mL) for 1 hour prior to lysis and immunoprecipitation with anti-myc antibody. Immunoprecipitates were probed with anti-myc antibody as a loading control, and anti-Phospho-(Ser/Thr) Akt Substrate antibody to detect phosphorylation of Tsc1. (C) Knockdown of Akt abrogates the increase in phosphorylation of Tsc1 on Ser533 induced by insulin treatment. S2 cells transfected with expression constructs for myc-Tsc1WT and His-Tsc2 were treated with control dsRNA or Akt dsRNA for 4 days prior to insulin treatment (10 µg/mL for 1 hour), lysis and anti-myc immunoprecipitation. Immunoprecipitates were probed with anti-myc as a control and anti Phospho-(Ser/Thr) Akt Substrate antibody to detect phosphorylation of Tsc1 Ser533. Despite efficient knockdown of Akt (seen by lack of Akt protein and S6K phosphorylation in lanes 3 and 4), anti Phospho-(Ser/Thr) Akt Substrate antibody displays background binding in total cell lysates, as previously reported also by others.

Figure 2

Figure 2. Flies lacking Akt phosphorylation sites on Tsc1 and Tsc2 are viable and normal in size.

(A) Expression levels of myc-Tsc1 in fly lines homozygous for the Tsc129 mutation, rescued by ubiquitous expression of Tsc1WT, Tsc1S533A or Tsc1S533D, or flies homozygous for both the Tsc129 and Tsc2192 mutations rescued to viability by ubiquitous expression of both Tsc1S533A and Tsc2T437A/S924A/T1054A/T1518A (“Tsc1S533A,Tsc24A”). (B,C,D) Survival rates (B), pupation curves (C) and relative adult wing sizes (D) of animals seeded as L1 larvae under controlled growth conditions for genotypes w1118 (“w1118”), Tsc129 homozygotes rescued by ubiquitous expression of Tsc1WT (“WT”), Tsc1S533A (“S533A”) or Tsc1S533D(“S533D”), or flies homozygous for both the Tsc129 and Tsc2192 mutations rescued to viability by ubiquitous expression of both Tsc1S533A and Tsc2T437A/S924A/T1054A/T1518A (“double”).

Figure 3

Figure 3. Flies lacking Akt phosphorylation sites on both Tsc1 and Tsc2 are slightly lean.

Triglyceride levels normalized to total body protein for Tsc129 homozygotes rescued by ubiquitous expression of Tsc1WT (“WT”), Tsc1S533A (“S533A”) or Tsc1S533D(“S533D”), or flies homozygous for both the Tsc129 and Tsc2192 mutations rescued to viability by expression of both Tsc1S533A and Tsc2T437A/S924A/T1054A/T1518A (“double”). * indicates statistical significance (ttest = 0.01).

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