Mapping growth-factor-modulated Akt signaling dynamics

Abstract

Growth factors alter cellular behavior through shared signaling cascades, raising the question of how specificity is achieved. Here, we have determined how growth factor actions are encoded into Akt signaling dynamics by real-time tracking of a fluorescent sensor. In individual cells, Akt activity was encoded in an analog pattern, with similar latencies (∼2 min) and half-maximal peak response times (range of 5-8 min). Yet, different growth factors promoted dosedependent and heterogeneous changes in signaling dynamics. Insulin treatment caused sustained Akt activity, whereas EGF or PDGF-AA promoted transient signaling; PDGF-BB produced sustained responses at higher concentrations, but short-term effects at low doses, actions that were independent of the PDGF-α receptor. Transient responses to EGF were caused by negative feedback at the receptor level, as a second treatment yielded minimal responses, whereas parallel exposure to IGF-I caused full Akt activation. Small-molecule inhibitors reduced PDGF-BB signaling to transient responses, but only decreased the magnitude of IGF-I actions. Our observations reveal distinctions among growth factors that use shared components, and allow us to capture the consequences of receptor-specific regulatory mechanisms on Akt signaling.

Dorsal ruffles enhance activation of Akt by growth factors

“…The timing of CDR formation corresponds to the transient activation of Akt by EGF and PDGF and may reflect a transient amplification of PI3K within CDRs. Sustained activation of Akt observed in response to PDGF (Gross and Rotwein, 2016) could reflect an additional cytoskeleton-independent mechanism of PIP 3 generation. The timing of Akt phosphorylation responses to EGF (early and transient) and the absence of significant staining of pAkt on macropinosomes suggests that CDRs are the principal location of Akt phosphorylation, rather than fully formed macropinosomes.…”

“…The kinetics of Akt phosphorylation differ in response to different ligands and to different concentrations of ligands (Gross and Rotwein, 2016). The timing of CDR formation corresponds to the transient activation of Akt by EGF and PDGF and may reflect a transient amplification of PI3K within CDRs.…”

“…Different patterns of signaling to Akt have been described for cellular responses to PDGF and EGF. Flow cytometric analysis of signaling to Akt in response to PDGF and EGF revealed differences in the kinetics and duration of cellular responses (Gross and Rotwein, 2016), which indicates different and heterogeneous cellular responses to the two growth factors. This suggests that morphological comparisons of signaling by PDGF and EGF might reveal underlying mechanistic differences.…”

Newt cells secrete extracellular vesicles with therapeutic bioactivity in mammalian cardiomyocytes

“…Insulin-mediated AKT activation occurs rapidly via cell surface receptor signalling [64] and served as a positive control for our Western blots on NRVMs. Unlike insulin, which activated AKT phosphorylation persistently, A1-CM transiently increased AKT phosphorylation at 1 h. Such transient responses are not atypical for the AKT signalling pathway [65,66]; here, the observed time course may reflect kinetics of EV uptake and/or dissemination of signalling cargo materials within NRVMs, but we have not investigated these possibilities. In addition to AKT activation, we also observed increased transcript levels of AKT itself as well as other pathway-associated genes.…”

Cracking the context-specific PI3K signaling code

“…Different PH domains may bind either one or both lipid species, leading to translocation of the fluorescent reporter from the cytosol to the plasma membrane [for a comprehensive review on these sensors, see (64)]. (D) Fluorescent FOXO-based nucleocytoplasmic translocation reporters are commonly used in dynamic single-cell studies of PI3K signaling (62,(155)(156)(157). However, FOXO proteins are only responsible for a subset of PI3K-dependent phenotypes (158).…”