Vav Rho Family GTPases and MAP Kinases

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Vav proteins are major players in numerous downstream NK cell activation events and have been the subject of intense research in recent years. The Vav proteins are GEFs that activate certain Rho family GTPases by promoting the exchange of enzyme-bound GDP for GTP [45]. Rho family GTPases serve as molecular switches that are "on" when bound with GTP, but "off" when the nucleotide is hydrolyzed to GDP. The best-characterized subgroups of the Rho family are Rho (three members), Rac (four members), and Cdc42 (five members) [148]. By stimulating Rho family GTPases, Vav activation impacts upon a wide range of NK cell processes, including adhesion, cytoskeletal polarization toward the target cell, cytolytic granule release, transcriptional regulation, and cytokine production [45,60,164].

Vav-mediated activation of Rho family GTPases ultimately leads to the activation of members of the MAPK family, namely ERK, p38, and c-Jun N-terminal kinase (JNK). Evidence indicates that the p38 and ERK subgroups are important for IFNy production and cytotoxicity, whereas JNK is dispensable for the cytolytic response [38,108,115,162,174,175]. Importantly, the effector phase of the NK cell cytotoxicity response has been shown to require ERK activation that is mediated through Rac1 instead of its typical upstream effector, Ras [14,79,175]. This Rac1^Pak^MEK^ERK pathway was shown to be critical for polarization of cytolytic granules toward target cells [14,60, 79, 175]. In line with these studies, activation of Rac GTPases in NK cells with a pharmacological agent was recently shown to dramatically reorganize actin dynamics, increase target cell adhesion, and enhance cytotoxicity [109]. In addition to their roles in the cytolytic response, activated MAPKs can be transported to the nucleus to regulate transcription [36,177]. A growing body of evidence demonstrates selective transcriptional regulation events resulting from the activation of specific MAPKs [138,177].

The Vav family GEFs are believed to be recruited to the plasma membrane by the binding of their PH domain to PIP3, where they become activated [68], but evidence also exists for PI3K-independent Vav activation [16, 51, 73]. In addition to stimulation through ITAM and YINM signaling,

Vav proteins can also be activated by adhesion-related integrins [137]. In a quiescent cell, Vav exists in an inactive state where it is folded back upon itself in a hairpinlike conformation by an interaction between regulatory ty-rosine residues and other amino acids within the Dbl homology domain. When the regulatory tyrosines become phosphorylated, these interactions are broken and Vav unfolds into its active conformation [5, 15]. (For an excellent review of the structure and function of Vav domains, see [164].) Three members of the Vav family have been identified and to a certain extent characterized. Vav-1 is restricted to hematopoietic cells, but Vav-2 and Vav-3 are ubiquitously expressed [26]. Vav-1 is sometimes referred to simply as Vav, but here we will specify Vav-1 in reference to the first member discovered and use Vav when referring to all three family members collectively.

Recent experiments with mice deficient in one or more Vav family members have demonstrated specific functional linkage to distinct TM accessory proteins. These experiments showed Vav-1 to be essential for NKG2D/DAP10 (YINM)-mediated signaling, whereas DAP12 (ITAM)-mediated signals are transduced by Vav-2 and Vav-3 [31]. The fact that loss of both Vav-2 and Vav-3 was required to abolish signals through DAP12 suggests that these two GEFs function in a redundant fashion in ITAM-dependent NK cell signaling pathways. This correlates well with the observations that DAP12- but not DAP10-derived signals induce IFNy secretion [196] and that IFNy secretion by NK cells is independent of VAV-1 [42]. Further evidence for alternative methods of Vav activation was provided by Riteau et al., who showed that Vav-2 can become activated in NK cells by a signal originating from the p2 integrin LFA-1 [137]. Although clear distinctions that account for functional differences between Vav family members are not yet well established, many of the downstream impacts mediated through Rho family GTPases are outlined in the following discussion and schematized in Figs. 2 and 3.

Vav-1 has been shown to function as a GEF for Rac members [Rac-1 [46], Rac-2 [145], RhoG [145]], Cdc42 [191], and possibly RhoA [129]. Vav-2 and Vav-3 have been shown to activate Rac members (Rac-1, RhoG) and RhoA [121,122]. There are numerous conflicting reports as to whether Vav-2 is also a GEF for Cdc42 [2, 99, 122, 145, 176]. The Rac, Rho, and Cdc42 subgroups of GTPases impart distinct effects on the actin cytoskeleton as described below. Rac-1, Cdc42, and RhoA can also stimulate transcription through NF-kB [132]. Furthermore, Vav-1 itself has been shown to migrate directly to the nucleus to associate with and facilitate transcription by nuclear factor for activation of T cells (NF-AT) and NF-kB family members [72].

As previously mentioned, Rac-1 initiates a signaling cascade that is important during NK cell cytolytic responses by directly stimulating p21-activated

Itam Rac Pak Mek Erk

Fig. 3a, b Selective impacts of receptors coupled to YINM- or ITAM-containing receptor complexes on subtypes of Vav and downstream impacts of the major subgroups of Rho family GTPases that are stimulated by Vav. a Receptors coupled to DAP10 (containing a YINM motif) and DAP12 (containing an ITAM) activate distinct subsets of Vav subtypes as designated by the arrows. b Three major Rho family subgroups (Rac, Cdc42, and Rho) can be activated by Vav subtypes to stimulate distinct downstream cascades that lead to radically different impacts on the actin cytoskeleton. In addition, all three Rho subgroups can impact upon NF-kB to regulate transcription. Major effectors in downstream signaling cascades are designated with arrows

Fig. 3a, b Selective impacts of receptors coupled to YINM- or ITAM-containing receptor complexes on subtypes of Vav and downstream impacts of the major subgroups of Rho family GTPases that are stimulated by Vav. a Receptors coupled to DAP10 (containing a YINM motif) and DAP12 (containing an ITAM) activate distinct subsets of Vav subtypes as designated by the arrows. b Three major Rho family subgroups (Rac, Cdc42, and Rho) can be activated by Vav subtypes to stimulate distinct downstream cascades that lead to radically different impacts on the actin cytoskeleton. In addition, all three Rho subgroups can impact upon NF-kB to regulate transcription. Major effectors in downstream signaling cascades are designated with arrows kinase (Pak), which activates MEK1 to stimulate ERK [79,175]. Activation of Rac-1 brings about morphological changes in the actin cytoskeleton that produce lamellipodia and ruffles [136]. Lamellipodia are the actin-rich leading edges of motile cells, and ruffles are dorsal circular structures. Rac-2 is also known to act through ERK and p38 and to promote actin polymerization in T cells [186] and granule exocytosis in neutrophils [1]. Rho-G also generates lamellipodia and ruffles, but in a more site-specific manner than Rac-1 [176].

RhoA has been shown in NK cells to activate Rho-associated kinase (ROCK), which subsequently phosphorylates and activates LIM-kinase (LIMK) [103]. Active LIMK inhibits the actin-depolymerizing protein cofilin. Disruption of cofilin function leads to the formation of focal adhesions and stress fibers that are involved in cell adhesion and motility [103]. Integrity of the RhoA^ROCK^LIMK pathway has been shown to be essential for recruitment of lipid rafts and actin polymerization at the cNKIS and for target cytotoxicity [103].

Members of the Cdc42 subgroup of the Rho family recruit WASP. WASP facilitates Arp2/3-mediated actin nucleation, which promotes growth of elongated cytoskeletal protrusions called filopodia [128]. Although a clear role for Cdc42 in NK cell function has not been defined, WASP is important in cytotoxicity responses [66, 127], as mentioned above. Like Rac, Cdc42 can also activate Pak and its downstream signaling events [148].

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