Ras-related C3 Botulinum Toxin Substrate (Rac) and Src Family Kinases (SFK) Are Proximal and Essential for Phosphatidylinositol 3-Kinase (PI3K) Activation in Natural Killer (NK) Cell-mediated Direct Cytotoxicity against Cryptococcus neoformans

Abstract

The activity of Rac in leukocytes is essential for immunity. However, its role in NK cell-mediated anti-microbial signaling remains unclear. In this study, we investigated the role of Rac in NK cell mediated anti-cryptococcal killing. We found that Cryptococcus neoformans independently activates both Rac and SFK pathways in NK cells, and unlike in tumor killing, Cryptococcus initiated a novel Rac 3 PI3K 3 Erk cytotoxicity cascade. Remarkably, Rac was not required for conjugate formation, despite its essential role in NK cytotoxicity against C. neoformans. Taken together, our data show that, unlike observations with tumor cells, NK cells use a novel Rac cytotoxicity pathway in conjunction with SFK, to kill C. neoformans. NK3 cells are a subset of lymphocytes that have well characterized anti-tumor and anti-viral activity. However, it is now apparent that NK cells also have the capacity to kill microbial targets, which contribute to host defense, and differ in important ways from tumor killing (1). Receptor-mediated NK antitumor signaling depends on sequential activation of PI3K 3 Rac 3 Erk (2); however, because the receptors required for cryptococcal killing differ from those required for tumor killing (3), we predicted that anti-microbial signaling would also differ. Our previous studies have shown that a PI3K 3 Erk signaling pathway was essential for cryptococcal killing (4). Because other laboratories showed that PI3K can activate Erk via either Rac or phospholipase C␥ (PLC␥), we sought to examine the pathway used in NK cell-mediated cryptococcal killing by investigating both PI3K 3 Rac 3 Erk and PI3K 3 PLC␥ 3 Erk pathways (5-9).Rac is a subfamily of Ras homology (Rho) GTPases consisting of Rac1, Rac2, Rac3, and RhoG. They are activated by guanine nucleotide exchange factors by exchanging GDP for GTP (reviewed in Ref. 10). Activation of Rac in NK cells is crucial for conjugate formation between NK and tumor targets (11,12). During tumor killing, Rac-GTP regulates actin remodeling by activating downstream effectors such as WAVE (WASP family verprolin-homologous protein), SRA1 (specifically RAC1-associated protein 1), and p21-activated kinases (PAKs) (13-16). In addition to its important role in cytoskeletal modulation, Rac plays a role in the activation of Erk and NK cell-mediated tumor cytotoxicity (9). Against Raji tumor targets, the human NK cell line, NK92 and primary NK cells required Rac to initiate a PAK1 3 Mek1/2 3 Erk1/2 signaling cascade that allows for cytotoxic granule mobilization and release (2). Rac activation was found to be dependent on PI3K, because NK92 cells transfected with a constitutively active Rac were able to continue killing tumor targets in the presence of PI3K inhibitors, whereas control cells were not (2).In addition to Rac, PLC␥ can also become activated by PI3K and lead to Erk activation. PLC␥ is best known for the production of diacyl glycerol and inositol 3-phosphate, which activates downstream kinases and regulates intracellular calcium (reviewed in Ref. 17). In certain cells,...

Granule-Dependent Natural Killer Cell Cytotoxicity to Fungal Pathogens

“…Unlike in tumor killing where Rac is activated by SFK and is downstream of PI3K, both Rac and SFK were found not to activate each other, but both were essential for PI3K activation in NK cell-mediated killing of Cryptococcus (15). In fact, Rac was found to be upstream of PI3K and was required for the activation of the PI3K → Erk signaling pathway during NK cell antifungal activity (15), thereby suggesting two separate pathways for PI3K activation (Figure 2). This interesting finding that Rac is upstream of PI3K needs to be confirmed by others.…”

“…Similar to tumor killing, fungal killing, demonstrated using Cryptococcus , depended on PI3K → Erk signaling (35). However, unlike tumor killing, PLCγ was not required for cryptococcal killing (15). Further, in the context of tumor killing, and depending on the activating receptor involved, Src family kinases (SFKs) either directly activated PI3K, leading to Rac and Erk activation, or recruited Vav1 to activate Rac (Figure 1).…”

“…Similar to tumor killing, two SFKs, Fyn and Lyn, redundantly mediated NK cell anticryptococcal activity by activating PI3K and Erk1/2 (41). Also, SFK was required to form NK cell-cryptococcal conjugates (15), and NKp30 was required for NK cell–fungal conjugate formation and PI3K–Erk signaling (7), making it likely that NKp30 activated SFK. Unlike in tumor killing where Rac is activated by SFK and is downstream of PI3K, both Rac and SFK were found not to activate each other, but both were essential for PI3K activation in NK cell-mediated killing of Cryptococcus (15).…”

“…Also, SFK was required to form NK cell-cryptococcal conjugates (15), and NKp30 was required for NK cell–fungal conjugate formation and PI3K–Erk signaling (7), making it likely that NKp30 activated SFK. Unlike in tumor killing where Rac is activated by SFK and is downstream of PI3K, both Rac and SFK were found not to activate each other, but both were essential for PI3K activation in NK cell-mediated killing of Cryptococcus (15). In fact, Rac was found to be upstream of PI3K and was required for the activation of the PI3K → Erk signaling pathway during NK cell antifungal activity (15), thereby suggesting two separate pathways for PI3K activation (Figure 2).…”

“…NK cells also indirectly mediated fungal elimination by secreting IFN-gamma to either mediate fungicidal activity of murine peritoneal exudate cells against C. neoformans (11) or mediate phagocytosis of C. albicans by splenic macrophages (12) or secrete GM-CSF to promote neutrophil phagocytosis of C. albicans (13). It has also been shown that the receptor and signaling pathway used for cryptococcal killing differs from that used for tumor killing (14, 15). This review will focus on the recent insights into activating receptor-mediated signaling and granule trafficking during NK cell killing of fungi compared to that of tumor cells.…”