Drosophila Tribbles Antagonizes Insulin Signaling-Mediated Growth and Metabolism via Interactions with Akt Kinase

Abstract

Drosophila Tribbles (Trbl) is the founding member of the Trib family of kinase-like docking proteins that modulate cell signaling during proliferation, migration and growth. In a wing misexpression screen for Trbl interacting proteins, we identified the Ser/Thr protein kinase Akt1. Given the central role of Akt1 in insulin signaling, we tested the function of Trbl in larval fat body, a tissue where rapid increases in size are exquisitely sensitive to insulin/insulin-like growth factor levels. Consistent with a role in antagonizing insulin-mediated growth, trbl RNAi knockdown in the fat body increased cell size, advanced the timing of pupation and increased levels of circulating triglyceride. Complementarily, overexpression of Trbl reduced fat body cell size, decreased overall larval size, delayed maturation and lowered levels of triglycerides, while circulating glucose levels increased. The conserved Trbl kinase domain is required for function in vivo and for interaction with Akt in a yeast two-hybrid assay. Consistent with direct regulation of Akt, overexpression of Trbl in the fat body decreased levels of activated Akt (pSer505-Akt) while misexpression of trbl RNAi increased phospho-Akt levels, and neither treatment affected total Akt levels. Trbl misexpression effectively suppressed Akt-mediated wing and muscle cell size increases and reduced phosphorylation of the Akt target FoxO (pSer256-FoxO). Taken together, these data show that Drosophila Trbl has a conserved role to bind Akt and block Akt-mediated insulin signaling, and implicate Trib proteins as novel sites of signaling pathway integration that link nutrient availability with cell growth and proliferation.

Skeletal Muscle Tissue Trib3 Links Obesity with Insulin Resistance by Autophagic Degradation of AKT2

“…Mechanistically, consistent with previous findings in hepatocytes [11,12,31], insulinresistant phenotypes observed in Trib3TG mice appear to be caused by an inhibition of AKT signaling by Trib3. Similar to that in hepatic Trib3 overexpression, we also observed reduced AKT phosphorylation in skeletal muscle tissues of Trib3TG mice; however, this primarily contributed from the decreased total AKT protein level, particularly AKT2, rather than blocking the phosphorylation of AKT.…”

Tribbles in the 21st Century: The Evolving Roles of Tribbles Pseudokinases in Biology and Disease

“…Of major interest, Trbl mutant cells exhibit premature mitosis, leading to defective gastrulation based on an inability to degrade target proteins in a timely fashion. Trbl fly models continue to be instrumental in revealing requirements for the pseudokinase domain in biological signaling events that have been conserved in vertebrate eukaryotes 23, 24. For example, String is the Drosophila ortholog of the CDC25 dual-specificity phosphatases, which initiate eukaryotic S-phase and mitosis and are themselves regulated at various cell cycle checkpoints.…”

The Tribbles 2 (TRB2) pseudokinase binds to ATP and autophosphorylates in a metal-independent manner

“…Logical ‘back’ mutation of conserved non-canonical CASK amino acids leads to reinstatement of efficient metal-activated kinase activity, suggesting that CASK has evolved an atypical pseudokinase domain in order to regulate a specific function in the cell [24]. The four human Tribbles (TRB)-related pseudokinases [25] termed in the present study, TRB1, TRB2, TRB3 and SgK495 (sugen kinase 495), also lie in the CAMK sub-family and are most closely related to the Drosophila TRB pseudokinase, which controls targeted ubiquitination and degradation of specific transcription factors that co-ordinate cell cycle and morphogenesis in a pseudokinase domain-dependent manner [26–30]. …”