Synergistic Signals for Natural Cytotoxicity Are Required to Overcome Inhibition by c-Cbl Ubiquitin Ligase

Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institute of Health, Rockville, MD 20852, USA.
Immunity (Impact Factor: 21.56). 02/2010; 32(2):175-86. DOI: 10.1016/j.immuni.2010.02.004
Source: PubMed

ABSTRACT Natural killer (NK) cell cytotoxicity toward target cells depends on synergistic coactivation by NK cell receptors such as NKG2D and 2B4. How synergy occurs is not known. Synergistic phosphorylation of phospholipase PLC-gamma2, Ca(2+) mobilization, and degranulation triggered by NKG2D and 2B4 coengagement were blocked by Vav1 siRNA knockdown, but enhanced by knockdown of c-Cbl. c-Cbl inhibited Vav1-dependent signals, given that c-Cbl knockdown did not rescue the Vav1 defect. Moreover, c-Cbl knockdown and Vav1 overexpression each circumvented the necessity for synergy because NKG2D or 2B4 alone became sufficient for activation. Thus, synergy requires not strict complementation but, rather, strong Vav1 signals to overcome inhibition by c-Cbl. Inhibition of NK cell cytotoxicity by CD94-NKG2A binding to HLA-E on target cells was dominant over synergistic activation, even after c-Cbl knockdown. Therefore, NK cell activation by synergizing receptors is regulated at the level of Vav1 by a hierarchy of inhibitory mechanisms.

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Available from: Hun Sik Kim, Sep 26, 2015
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    • "Activation through EAT-2 is dependent on tyrosine phosphorylation of the tail of EAT-2 [27], [30]. Recruitment of Fyn by EAT-2 [11], [21], [27] may contribute to early phosphorylation events, however, an interaction between PLCγ1 and the tyrosine phosphorylated tail of EAT-2 [11], [27] suggests a mechanism of recruitment of PLCγ through SLAM family receptors (Figure 1) [9], [11], [27], [31], [32]. Furthermore, mouse NK cells lacking PLCγ2 fail to mobilize calcium and have impaired cytotoxicity through all activating receptors examined including CD244 [33]. "
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    ABSTRACT: SLAM family receptors regulate activation and inhibition in immunity through recruitment of activating and inhibitory SH2 domain containing proteins to immunoreceptor tyrosine based switch motifs (ITSMs). Binding of the adaptors, SAP and EAT-2 to ITSMs in the cytoplasmic regions of SLAM family receptors is important for activation. We analysed the fine specificity of SLAM family receptor phosphorylated ITSMs and the conserved tyrosine motif in EAT-2 for SH2 domain containing signalling proteins. Consistent with the literature describing dependence of CRACC (SLAMF7) on EAT-2, CRACC bound EAT-2 (KD = 0.003 μM) with approximately 2 orders of magnitude greater affinity than SAP (KD = 0.44 μM). RNA interference in cytotoxicity assays in NK92 cells showed dependence of CRACC on SAP in addition to EAT-2, indicating selectivity of SAP and EAT-2 may depend on the relative concentrations of the two adaptors. The concentration of SAP was four fold higher than EAT-2 in NK92 cells. Compared with SAP, the significance of EAT-2 recruitment and its downstream effectors are not well characterised. We identified PLCγ1 and PLCγ2 as principal binding partners for the EAT-2 tail. Both PLCγ1 and PLCγ2 are functionally important for cytotoxicity in NK92 cells through CD244 (SLAMF4), NTB-A (SLAMF6) and CRACC. Comparison of the specificity of SH2 domains from activating and inhibitory signalling mediators revealed a hierarchy of affinities for CD244 (SLAMF4) ITSMs. While binding of phosphatase SH2 domains to individual ITSMs of CD244 was weak compared with SAP or EAT-2, binding of tandem SH2 domains of SHP-2 to longer peptides containing tandem phosphorylated ITSMs in human CD244 increased the affinity ten fold. The concentration of the tyrosine phosphatase, SHP-2 was in the order of a magnitude higher than the adaptors, SAP and EAT-2. These data demonstrate a mechanism for direct recruitment of phosphatases in inhibitory signalling by ITSMs, while explaining competitive dominance of SAP and EAT-2.
    PLoS ONE 03/2014; 9(3):e92184. DOI:10.1371/journal.pone.0092184 · 3.23 Impact Factor
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    • "c-Cbl is induced upon engagement of an activating receptor (e.g., NKG2D), to dampen NK cell cytotoxicity, which in turn, leads to Vav1’s ubiquitination and proteosomal degradation. The loss of Vav1 decreases the levels of phosphorylated PLCγ and prevents NK cell degranulation [55]. To overcome the effects of c-Cbl’s inhibition of Vav1, NK cells require concomitant engagement of activating and co-activating receptors [55]. "
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    ABSTRACT: Natural killer (NK) cells' major role in the control of viruses is to eliminate established infected cells. The capacity of NK cells to kill virus-infected cells is dependent on the interactions between ligands on the infected cell and receptors on the NK cell surface. Because of the importance of ligand-receptor interactions in modulating the NK cell cytotoxic response, HIV has developed strategies to regulate various NK cell ligands making the infected cell surprisingly refractory to NK cell lysis. This is perplexing because the HIV-1 accessory protein Vpr induces expression of ligands for the NK cell activating receptor, NKG2D. In addition, the accessory protein Nef removes the inhibitory ligands HLA-A and -B. The reason for the ineffective killing by NK cells despite the strong potential to eliminate infected cells is due to HIV-1 Vpu's ability to down modulate the co-activation ligand, NTB-A, from the cell surface. Down modulation of NTB-A prevents efficient NK cell degranulation. This review will focus on the mechanisms through which the HIV-1 accessory proteins modulate their respective ligands, and its implication for NK cell killing of HIV-infected cells.
    Viruses 07/2011; 3(7):1091-111. DOI:10.3390/v3071091 · 3.35 Impact Factor
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    • "Even in the absence of NK cell stimulation by target cells or by cell surface receptors, some of the LG are close to the PM, as visualized by total internal reflection fluorescence (TIRF) microscopy [11], [12]. LG that are close to the PM may represent a functional pool available for release of cytolytic effectors, as degranulation by NK cells has been observed in the absence of granule polarization [13], [14]. "
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    ABSTRACT: Protocols were developed to automate image analysis and to track the movement of thousands of vesicular compartments in live cells. Algorithms were used to discriminate among different types of movement (e.g. random, caged, and directed). We applied these tools to investigate the steady-state distribution and movement of lytic granules (LG) in live natural killer (NK) cells by high-speed 3-dimensional (3D) spinning disc confocal and 2-dimensional total internal reflection fluorescence microscopy. Both mouse NK cells and a human NK cell line deficient in the small GTPase Rab27a were examined. The unbiased analysis of large datasets led to the following observations and conclusions. The majority of LG in the cytosol and at the plasma membrane of unstimulated NK cells are mobile. The use of inhibitors indicated that movement in the cytosol required microtubules but not actin, whereas movement at the plasma membrane required both. Rab27a deficiency resulted in fewer LG, and in a reduced fraction of mobile LG, at the plasma membrane. In contrast, loss of Rab27a increased the fraction of mobile LG and the extent of their movement in the cytosol. Therefore, in addition to its documented role in LG delivery to the plasma membrane, Rab27a may restrict LG movement in the cytosol.
    PLoS ONE 09/2010; 5(9):e12870. DOI:10.1371/journal.pone.0012870 · 3.23 Impact Factor
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