Dynamics in Signaling Events

Signaling processes are dynamic. This inherent property has not yet been fully taken into account when linear signal transduction pathways are established and interaction networks are being interpreted. Taking dynamic aspects of protein-protein interactions and modifications into account is a big challenge to the biologist, as experiments need to be planned carefully and good controls need to be considered.

The first proteomic approach to address the temporal aspects of protein phosphorylation cascades came from the field of EGF signaling. Using metabolically labeled cell cultures, phosphorylated proteins were immuno-precipitated and quantified by mass spectrometry at different times after EGF stimulation (Blagoev et al. 2004; Olsen et al. 2006). The results indicate a clear hierarchy of phosphorylation events, starting with autophosphorylation of the EGF receptor and progressing to the MAP kinase signaling pathways within 10 min. In addition, at least in mammalian cells, early phosphoryla-tion events are tyrosine phosphorylations, while later ones are in serine and threonine phosphorylation.

In similar experiments using liquid cultures of Arabidopsis seedlings, the phosphorylation status of membrane proteins was analyzed at different time points of sucrose resupply after a 2-day starvation period (Niittyla et al. 2007). The results reveal novel protein phosphorylation sites involved in sucrose response as well as changes in phosphorylation level of well-characterized proteins, such as the plasma membrane ATPase or phosphoenol-pyruvate carboxylase. These initial experiments will provide a broad basis for the identification of candidate proteins that may be involved in a particular signaling pathway.

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