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

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 5473, 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 5473 and/or T308 phosphorylation as surrogates for Akt kinase activity across diverse treatment conditions. We performed quantitative Western blots and kinase activity assays on lysates generated during a 2 h time course from two cell lines treated with either EGF or insulin. From the resulting similar to 250 quantitative measurements of phosphorylation and activity, we found that both T308 and 5473 phosphorylation accurately captured quantitative changes in EGF-stimulated cells, but not in insulin-stimulated cells. Moreover, 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 5473 as proxies for kinase activity and suggests the coregulation of Akt phosphorylation and dephosphorylation. (c) 2006 Elsevier Inc. All rights reserved.