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Published on: May 23, 2025
Quantitative analysis of Akt phosphorylation and activity in response to EGF and insulin treatment
Neil Kumar1, Raffi Afeyan, Sarah Sheppard
1Department of Chemical Engineering, MIT, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. nkumar@mit.edu
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 across diverse treatment conditions. We performed quantitative Western blots and kinase activity assays on lysates generated during a 2h time course from two cell lines treated with either EGF or insulin. From the resulting approximately 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 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 S473 as proxies for kinase activity and suggests the coregulation of Akt phosphorylation and dephosphorylation.
Insights
Measuring Akt kinase activity using phosphorylation at threonine 308 (T308) and serine 473 (S473) is common. However, this study reveals these sites accurately reflect EGF-stimulated activity but not insulin-stimulated activity.
Area of Science:
- Cellular signaling and molecular biology
- Biochemistry and enzymology
- Cancer research and molecular pathology
Background:
- The protein kinase Akt is a key regulator of cell functions, with its dysregulation linked to pathologies like cancer.
- Akt activation is traditionally assessed via phosphorylation at threonine 308 (T308) and serine 473 (S473), particularly in response to insulin.
- Emerging evidence suggests other phosphorylation sites, including tyrosine phosphorylation, contribute to Akt activation.
Purpose of the Study:
- To quantitatively assess the reliability of T308 and S473 phosphorylation as surrogates for Akt kinase activity under various stimulation conditions.
- To investigate potential ligand-dependent differences in Akt activation mechanisms.
- To explore the correlation between phosphorylation and dephosphorylation dynamics at T308 and S473.
Main Methods:
- Quantitative Western blotting to measure phosphorylation levels at T308 and S473.
- Akt kinase activity assays.
- Time-course analysis of two cell lines treated with epidermal growth factor (EGF) or insulin.
Main Results:
- T308 and S473 phosphorylation accurately correlated with quantitative changes in Akt activity in EGF-stimulated cells.
- This correlation was not observed in insulin-stimulated cells.
- A strong correlation between the initiation of phosphorylation and dephosphorylation was found for both sites across most conditions, despite differing regulatory pathways.
Conclusions:
- The use of T308 and S473 phosphorylation as proxies for Akt kinase activity is ligand-dependent, with limitations in insulin-stimulated conditions.
- Akt phosphorylation and dephosphorylation dynamics appear to be coregulated, even when mediated by distinct kinases and phosphatases.
- Quantitative analysis provides critical insights into the nuanced regulation of Akt activation.
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