Adenylylated Proteins in Mammalian Cell Lysates

Detection of Adenylylated Proteins in Mammalian Cell Lysates 

The example of adenylylation of Rabi described here demonstrates the importance of a combined structural biological/biochemical/chemical approach to identifying and unraveling biological mechanisms, in this case a PTM. It also shows how this can lead to development of techniques for wider scale application to address the question of the prevalence of the particular modification. The basic principle here is that a thorough biochemical characterization has led to the ability to design and synthesize probe molecules and analytical techniques for this purpose. 

and Itzen, A. (2013) Modulation of small GTPases by Legionella. Curr. Top. Microbiol. Immunol., 376, 117-133. 

Aktories, K. (2011) Bacterial protein toxins that modify host regulatory GTPases. Nat. Rev. Microbiol., 9 (7), 487 -498. 

Muller, M.P., Peters, H., Bliimer, J., Blankenfeldt, W., Goody, R.S., and Itzen, A. (2010) The Legionella effector protein DrrA AMPylates the membrane traffic regulator Rablb. Science, 329 (5994), 946-949. 

---

Figure 9.5 Detection of adenylylated proteins in mammalian cell lysates. Preparative adenylylated Rabi and Cdc42 (0.1 jg each) have been exogenously added to buffer or 100 jg of Cos7 cell lysate. A biotinylated a-Tyr-Rabl-AMP antibody has been immobilized on magnetic streptavidin beads and used to pull down the Rabi-AMP and...

---