Quantitative analysis of Akt phosphorylation and activity in response to EGF and insulin treatment

Abstract

The protein kinase Akt is a critical regulator of cell function and its overexpression and activation have been functionally linked to numerous pathologies such as cancer. Previous reports regarding the mechanism regulating Akt's activation have revealed two phosphorylation events, at threonine 308 (T308) and serine 473 (S473), as necessary for the full activation of the kinase in response to insulin. For this reason and because of the availability of phospho-specific antibodies to both T308 and S473, many studies that focus on Akt's role in governing cell function rely on the measurement of these two sites to understand changes in kinase activity. Recent evidence, however, suggests the involvement of other phosphorylation sites; for example, in Src-transformed and epidermal growth factor (EGF)-treated cells, tyrosine phosphorylation has been found important for full kinase activation. In this study, we probed the quantitative reliability of using S473 and/or T308 phosphorylation as surrogates for Akt kinase activity acress diverse treatment conditions. We performed quantitative western blots and kinase activity assays on lysates generated during a two hour time course from two cell lines treated with either EGF or insulin. From the resulting ∼250 quantitative measurements of phosphorylation and activity, we found that both T308 and S473 phosphorylation accurately captured quantitative changes in EGF-stimulated cells, but not in insulinstimulated cells. Morever, in all but one condition studied, we found a tight correlation between the onset of phosphorylation and dephosphorylation for both sites, despite the fact that they do not share common kinase-or phosphatase-mediated regulation. In sum, using a quantitative approach to study Akt activation identified ligand-dependent limits for the use of T308 or S473 as proxies for kinase activity and suggests the coregulation of Akt phosphorylation and dephosphorylation.

Aging‐induced Akt activation involves in aging‐related pathologies and Aβ‐induced toxicity

“…Furthermore, increased EGFR, but not InR, mediates these effects in the neurons, at least in affecting animal longevity. In fact, other works have also reported that downstream signaling that EGFR activates Akt is not exactly the same as InR actives Akt (Borisov et al., ; Kumar, Afeyan, Sheppard, Harms & Lauffenburger, ; Roudabush, Pierce, Maudsley, Khan & Luttrell, ). Although the molecular reason behind the differential functions of Akt in different cell types is unclear, the cell type‐specific molecular property of Akt may provide a means of differentially and precisely regulating the functions of various areas of the brain under different physiological conditions.…”

Proinsulin protects against age-related cognitive loss through anti-inflammatory convergent pathways

“…2A). Our long-term, sustained, in vivo treatment displayed a well-established sign of activation of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway (Kumar et al, 2007). Two-way ANOVA demonstrated an effect of the factor treatment for Akt Ser473 levels [F(1,22) = 12.2, P = 0.002].…”

Low‐power laser irradiation promotes cell proliferation by activating PI3K/Akt pathway

“…Its activation can be initiated by various extracellular signals that turn on PI3K (Engelman et al, 2006). After stimulus affecting on its respective receptor (Kumar et al, 2007), PI3K is activated and then phosphorylates inositol lipids (Bevan, 2001). This results in the production of PI3,4,5P3 and PI3,4P2 at the plasma membrane (Cantrell, 2001).…”