Using flow cytometry, we investigated the effect of TLR agonists on human polymorphonuclear neutrophil (PMN) apoptosis in whole blood. LPS (TLR4), peptidoglycan (TLR2), R-848 (TLR7/8), and CpG-DNA (TLR9) were equally effective at delaying spontaneous apoptosis of PMN, while PamCSK4 (TLR1/2), macrophage-activating lipopeptide-2 (TLR2/6), flagellin (TLR5), and loxoribine (TLR7) were less effective or inactive. TLR agonists found to delay apoptosis also extended the functional life span of PMN. Analysis of signaling pathways revealed that the antiapoptotic effect of TLR agonists required NF-κB and PI3K activation. Furthermore, analysis of intact cells by flow cytometry showed that TLR agonists delaying PMN apoptosis increased phosphorylation of Akt, a major target of PI3K. This effect was associated with a PI3K-dependent increase in heat shock protein 27 phosphorylation, which has been reported to play a key role in PMN survival. Finally, the TLR-induced delay in PMN apoptosis was associated with increased levels of Mcl-1 and A1, which are antiapoptotic members of the Bcl-2 family. These effects were reversed by PI3K and NF-κB inhibitors, respectively. TLR activation also led to PI3K-dependent phosphorylation of the proapoptotic protein Bad. Taken together, our results strongly suggest a role of NF-κB and PI3K in TLR-induced PMN survival, leading to modulation of Bcl-2 family molecules.

G␣-interacting protein (GAIP) is a regulator of G protein signaling (RGS) that accelerates the rate of GTP, EC 50 ‫؍‬ 3.16 ؎ 0.12 M) or to GAIP phosphorylated by other Ser/Thr protein kinases (protein kinase C, casein kinase II). This stimulation and the phosphorylation of GAIP by Erk2 were abrogated when serine at position 151 in the RGS domain was substituted by an alanine residue using site-directed mutagenesis. Furthermore, the lysosomalautophagic pathway was not stimulated in S151A-GAIP mutant-expressing cells when compared with wild-type GAIP-expressing cells. These results demonstrate that the GTPase-activating protein activity of GAIP is stimulated by Erk2 phosphorylation. They also suggested that Erk1/2 and GAIP are engaged in the signaling control of a major catabolic pathway in intestinal derived cells. Regulators of G protein signaling proteins (RGS)1 are a family of proteins that control the activity of trimeric G proteins (1-4). More than 20 mammalian RGS proteins have been identified generally by reference to a conserved domain of about 115 amino acid residues known as the RGS box (5). RGS proteins are involved in modulating a variety of cell functions such as proliferation, differentiation, response to neurotransmitters, membrane trafficking, and embryonic development (4, 6). RGS act as negative regulators of several G proteins by accelerating the rate of GTP hydrolysis by the G␣ proteins, thereby promoting their association with the ␤␥ dimer (7-9). This GTPaseactivating protein (GAP) activity is engaged in the desensitization of signaling by the trimeric G proteins, but it can also speed up the transmission of signals in some cases (10, 11). Recently, the key role of RGS in the regulation of G proteincoupled receptor signaling has been demonstrated in vivo (12, 13). However, recent evidence supports the notion that RGS proteins may be engaged in functions distinct from the regulation of G protein-activity (14).GAIP (G␣-interacting protein) is an RGS protein, which is known to interact with G␣ i3 protein (15). GAIP has been located to the Golgi apparatus membrane and newly budding Golgi vesicles (16,17) and associated with clathrin-coated vesicles (18), suggesting its potential role in vesicular transport.Recently, it has been demonstrated that posttranslational modifications of RGS can modulate their properties. Palmitoylation of conserved cysteines in RGS boxes has been shown to modify the GAP activity of RGS4 and RGS10 (19). In addition, phosphorylation has been reported to influence the stability and the membrane association of the yeast RGS Sst2 and human GAIP, respectively (20,21). However, it is not known whether or not phosphorylation could modulate the GAP activity of RGS.In the present work, we show that the phosphorylation of GAIP is in part dependent upon the activation of the Erk1/2 MAP kinases in the human intestinal HT-29 cells, and both of these events were sensitive to PKC inhibitors and amino acids. Using a panel of Ser/Thr protein kinases in an in vitro assay, we demonstrate th...

Generation of superoxide anion by the multiprotein complex NADPH phagocyte oxidase is accompanied by extensive phosphorylation of its 47-kDa protein component, p47phox, a major cytosolic component of this oxidase. Protein kinase C ζ (PKC ζ), an atypical PKC isoform expressed abundantly in human polymorphonuclear leukocytes (PMN), translocates to the PMN plasma membrane upon stimulation by the chemoattractant fMLP. We investigated the role of PKC ζ in p47phox phosphorylation and in superoxide anion production by human PMN. In vitro incubation of recombinant p47phox with recombinant PKC ζ induced a time- and concentration-dependent phosphorylation of p47phox with an apparent Km value of 2 μM. Phosphopeptide mapping analysis of p47phox showed that PKC ζ phosphorylated fewer selective sites in comparison to “conventional” PKCs. Serine 303/304 and serine 315 were identified as targets of PKC ζ by site-directed mutagenesis. Stimulation of PMN by fMLP induced a rapid and sustained plasma membrane translocation of PKC ζ that correlated to that of p47phox. A cell-permeant-specific peptide antagonist of PKC ζ inhibited both fMLP-induced phosphorylation of p47phox and its membrane translocation. The antagonist also inhibited the fMLP-induced production of oxidant (IC50 of 10 μM), but not that induced by PMA. The inhibition of PKC ζ expression in HL-60 neutrophil-like cells using antisense oligonucleotides (5 and 10 μM) inhibited fMLP-promoted oxidant production (27 and 50%, respectively), but not that induced by PMA. In conclusion, p47phox is a substrate for PKC ζ and participates in the signaling cascade between fMLP receptors and NADPH oxidase activation.

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