Abstract
Cell growth and proliferation are two diverse processes yet always linked. Akt1, a serine/threonine kinase, is a multi-functional protein implicated in regulation of cell growth, survival and proliferation. Though it has a role in G1/S progression, the manner by which Akt1 controls cell cycle and blends cell growth with proliferation is not well explored. In this study, we characterize the Akt1 interactome as the cell cycle progresses from G0 to G1/S and G2 phase. For this, Akt1-overexpressing HEK293 cells were subjected to AP-MS. To distinguish between individual cell cycle stages, cells were cultured in the light, medium and heavy labelled SILAC media. We obtained 213 interacting partners of Akt1 from these studies. GO classification revealed that a significant number of proteins fall into functional classes related to cell growth or cell cycle processes. Of these, 32 proteins showed varying association with Akt1 in different cell cycle stages. Further analyses uncovered a subset of proteins showing counteracting effects so as to tune stage-specific progression through the cycle. Thus, our study provides some novel perspectives on Akt1-mediated regulation of the cell cycle and offers the framework for a detailed resolution of the downstream cellular mechanisms that are mediated by this kinase.
Highlights
The mammalian cell cycle consists of an ordered series of events and is a highly coordinated and regulated process[1]
To resolve between the individual cell cycle stages, we used the technique of selective isotope labelling of amino acids in cell culture (SILAC)
The serine/threonine kinase Akt/PKB is a central signalling node in all eukaryotic cells and represents the most important kinase at the core of human physiology and disease[4]. It functions downstream of growth factors, oncogenes and cell stress and is best known for promoting cell survival and growth[6,7]
Summary
The mammalian cell cycle consists of an ordered series of events and is a highly coordinated and regulated process[1]. To resolve between the individual cell cycle stages, we used the technique of selective isotope labelling of amino acids in cell culture (SILAC) These studies identified 213 proteins to interact either directly or indirectly with Akt[1]. Subsequent experiments revealed that at least a significant proportion of the proteins that associated with Akt[1] in a dynamic manner were involved in influencing progression of cells through the cycle. These proteins appeared to exert counteracting effects so as to tune stage-specific progression and, thereby, the overall population doubling time (PDT) of the cells. In addition to shedding some novel perspectives on Akt1-mediated regulation of the cell cycle, our studies provide the framework for a detailed resolution of the downstream cellular mechanisms that are mediated by this kinase
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