Abstract
The small GTPase Rac participates in various cellular events such as cytoskeletal reorganization. It has remained, however, largely unknown about intracellular signaling pathways for Rac activation because of the lack of a simple and reliable assay to estimate the activation. Here we describe a novel method to detect the GTP-bound, active Rac in cells by pulling it down with the Rac-binding domain of the protein kinase PAK. Experiments using this method reveal that stimulation of human neutrophils with the Gi-coupled receptor agonists N-formyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4 (LTB4) leads to a rapid and transient increase in the GTP-bound state of Rac2, whereas phorbol myristate acetate (PMA) causes a slow but more sustained activation of Rac2. Pretreatment of cells with pertussis toxin results in defective activation of Rac2 in response to fMLP and LTB4, indicating that coupling of the receptors to Gi plays a crucial role in the activation. Furthermore, the phosphoinositide 3-kinase (PI3K) inhibitors wortmannin and LY294002 block Rac2 activation elicited by the receptor agonists, but not that by PMA. Thus the Gi-coupled receptors likely mediate Rac2 activation via PI3K, whereas PMA activates Rac2 in a PI3K-independent manner.
Highlights
The small GTPases Rac1 and Rac2 are members of the Rho subfamily of the Ras-related GTP-binding proteins and serve as a molecular switch cycling between an active GTP-bound and an inactive GDP-bound state [1,2,3]
We have developed here a novel assay to estimate activation of endogenous Rac in cells, by pulling down the GTP-bound, active Rac with the Rac-binding domain (RBD) of the protein kinase PAK2 expressed as a GST fusion
Rac2 Is Activated in Human Neutrophils Stimulated with fMLP, leukotriene B4 (LTB4), and phorbol myristate acetate (PMA)—After human neutrophils were stimulated with the Gi-coupled receptor agonist fMLP and lysed, Rac was precipitated with GST-PAK2-RBD bound to glutathione-Sepharose beads and identified by Western blot with a polyclonal antibody against Rac2
Summary
The small GTPases Rac1 and Rac2 are members of the Rho subfamily of the Ras-related GTP-binding proteins and serve as a molecular switch cycling between an active GTP-bound and an inactive GDP-bound state [1,2,3]. We show here that Rac2, the predominant isoform in human neutrophils [29], is rapidly and transiently converted to the GTP-bound, active state, in response to the Gi-coupled receptor agonists fMLP and LTB4.
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