Dissociation between the translocation and the activation of Akt in fMLP-stimulated human neutrophils—effect of prostaglandin E2

Abstract

PGE(2) and other cAMP-elevating agents are known to down-regulate most functions stimulated by fMLP in human polymorphonuclear neutrophils. We reported previously that the inhibitory potential of PGE(2) resides in its capacity to suppress fMLP-stimulated PI-3Kgamma activation via the PGE(2) receptor EP(2) and hence, to decrease phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P(3)] formation. Akt activity is stimulated by fMLP through phosphorylation on threonine 308 (Thr308) and serine 473 (Ser473) by 3-phosphoinositide-dependent kinase 1 (PDK1) and MAPK-AP kinase (APK)-APK-2 (MAPKAPK-2), respectively, in a PI-3K-dependent manner. Despite the suppression of fMLP-induced PI-3Kgamma activation observed in the presence of PGE(2), we show that Akt is fully phosphorylated on Thr308 and Ser473. However, fMLP-induced Akt translocation is decreased markedly in this context. PGE(2) does not affect the phosphorylation of MAPKAPK-2 but decreases the translocation of PDK1 induced by fMLP. Other cAMP-elevating agents such as adenosine (Ado) similarly block the fMLP-induced PI-3Kgamma activation process but do not inhibit Akt phosphorylation. However, Akt activity stimulated by fMLP is down-regulated slightly by agonists that elevate cAMP levels. Whereas protein kinase A is not involved in the maintenance of Akt phosphorylation, it is required for the inhibition of Akt translocation by PGE(2). Moreover, inhibition of fMLP-stimulated PI-3Kdelta activity by the selective inhibitor IC87114 only partially affects the late phase of Akt phosphorylation in the presence of PGE(2). Taken together, these results suggest that cAMP-elevating agents, such as PGE(2) or Ado, are able to induce an alternative mechanism of Akt activation by fMLP in which the translocation of Akt to PI(3,4,5)P(3)-enriched membranes is not required prior to its phosphorylation.

Vesicular trafficking through cortical actin during exocytosis is regulated by the Rab27a effector JFC1/Slp1 and the RhoA-GTPase–activating protein Gem-interacting protein

“…Alternatively, JFC1 conformational changes by phosphorylation may not affect binding to GMIP but may instead facilitate RhoA-GAP activation and a concomitant decrease of active RhoA at the granule-surrounding areas. A possible role for JFC1 regulation by phosphorylation during exocytosis in granulocytes is supported by previous studies showing that fMLF-mediated neutrophil stimulation induces Akt activation (Burelout et al , 2007), and that JFC1 is an Akt substrate in vivo (Johnson et al , 2005b). Also, JFC1-dependent activation of the RhoA-GAP of GMIP may induce not only inactivation but also redistribution of RhoA toward cellular compartments other than secretory organelles in which RhoA may again become active.…”

VIP differentially activates β2 integrins, CR1, and matrix metalloproteinase-9 in human monocytes through cAMP/PKA, EPAC, and PI-3K signaling pathways via VIP receptor type 1 and FPRL1

“…Monocyte exposure to these different inhibitors did not alter their viability, as measured by the propidium iodide exclusion test. Dose and time exposure was similar to the ones reported in the literature for LY294002 [46], U73122 [47], PTX [48], PD98059 and SB203580 [49], WRW4 [50], and H89 [51]. Activated monocytes used as positive controls consist in monocytes exposed to fMLP (0.1 M, 2 min) or LPS (1 g/ml, 10 min).…”

AKT-mediated regulation of polarization in differentiated human neutrophil-like HL-60 cells

“…2a), we next asked whether AKT activity was required for cell polarization. AKT activation stimulated by various agonists is PI3K-dependent, and its phosphorylation on Ser473 and Thr308 residues is usually considered an index of PI3K activation [8]; therefore, we investigated the changes in dHL-60 cell polarization and chemotaxis after dHL-60 cells were treated with the two PI3K and two AKT inhibitors. The AKT inhibitors, AKT inhibitor and Deguelin which show strong inhibition of AKT activity, but less inhibition of PI3K activity, were chosen to perform the following study.…”

“…In the PI3K-dependent signaling pathway, the PI3K-catalyzed formation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) results in recruitment of AKT to the cell membrane during neutrophil polarization [8]. AKT (also known as protein kinase B, PKB) is a serine/threonine protein kinase, and phosphorylation of AKT at both T308 and S473 is required for full kinase activity [9][10][11].…”