Signaling requirements for translocation of P-Rex1, a key Rac2 exchange factor involved in chemoattractant-stimulated human neutrophil function

Abstract

PI 3,4,5-trisphosphate [PI(3,4,5)P3; PIP3]-dependent Rac exchanger 1 (P-Rex1) is a Rac-specific guanine nucleotide exchange factor abundant in neutrophils and myeloid cells. As a selective catalyst for Rac2 activation, P-Rex1 serves as an important regulator of human neutrophil NADPH oxidase activity and chemotaxis in response to a variety of extracellular stimuli. The exchange activity of P-Rex1 is synergistically activated by the binding of PIP3 and betagamma subunits of heterotrimeric G proteins in vitro, suggesting that the association of P-Rex1 with membranes is a prerequisite for cellular activation. However, the spatial regulation of endogenous P-Rex1 has not been well defined, particularly in human neutrophils activated through G protein-coupled receptors. Upon stimulation of neutrophil chemoattractant receptors, we observed that P-Rex1 translocated from cytoplasm to the leading edge of polarized cells in a G protein betagamma subunit- and PIP3-dependent manner, where it colocalized with F-actin and its substrate, Rac2. Redistribution of P-Rex1 to the leading edge was also dependent on tyrosine kinase activity and was modulated by cell adhesion. Furthermore, we observed that activation of cAMP-dependent protein kinase A (PKA), which phosphorylates and inactivates P-Rex1, inhibited its translocation. Our data indicate that endogenous P-Rex1 translocates to areas of Rac2 and cytoskeletal activation at the leading edge in response to chemoattractant stimuli in human neutrophils and that this translocation can be negatively modulated by activation of PKA and by cell adhesion.

PREX1 Protein Function Is Negatively Regulated Downstream of Receptor Tyrosine Kinase Activation by p21-activated Kinases (PAKs)

“…A recent study showed that Rac binds to a PKA regulatory subunit and that PKA phosphorylates and activates PAK, setting up a model where PKA binds Rac-GTP to help in the stimulation of PAK and its downstream signals (45). It is also important to note that PREX1 is both activated by isoproterenol in cells and negatively regulated by PKA in vitro (10,31,32,46). One possible model that incorporates all of these data is that, after GPCR stimulation, G␤␥ signals to PREX1 to activate Rac and G␣ signals to PKA to help in the activation of PAK by Rac-GTP, and then these two events lead to PAK-mediated negative feedback on PREX1 to turn off Rac (Fig.…”

Small Molecule Disruption of G Protein βγ Subunit Signaling Inhibits Neutrophil Chemotaxis and Inflammation

“…For example, M119 blocks membrane translocation of P-Rex, a PIP 3 -and G␤␥-regulated Rac2 exchange factor, in human neutrophils, which could be due in part to directly blocking P-Rex binding to G␤␥ in addition to blocking PIP 3 production by PI3-kinase (Zhao et al, 2007). It has recently been shown that Ras is required for full PI3-kinase ␥ activation in neutrophils (Suire et al, 2006).…”

“…Other previous work showed that M119 inhibited P-Rex1 activation by fMLP suggesting the compounds could inhibit Rac activation (Zhao et al, 2007). We determined whether G␤␥-binding compounds would inhibit receptor-dependent activation of Rac1.…”

The 3BP2 Adapter Protein Is Required for Chemoattractant-Mediated Neutrophil Activation

“…Moreover, P-Rex1 is required for neutrophil migration rate and superoxide production in response to fMLF (31,84). P-Rex1 polarizes to the leading edge of neutrophils during chemotaxis in a region where phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) accumulates following activation of class I PI3K (26,88,89). PRex1 activation requires the coincident input of PIP 3 phospholipids and Gbg dimers (83), suggesting that its localization in proximity to these two agonists at the leading edge may be a requirement for its full activation.…”