Signals Downstream of ITAMs

The ITAM is characterized by an amino acid sequence of Yxx(L/I/V)x(6-8) Yxx(L/I/V) [71]. When a receptor associated with an ITAM containing accessory protein binds to its ligand, the tyrosine residues in the ITAM become phosphorylated by a Src family kinase (e.g., Lyn, Lck, or Fyn). It is believed that this important phosphorylation event is primarily initiated by ligand interaction-mediated recruitment of these receptor-associated domains into lipid rafts at the c-SMAC, where Src family PTKs are resident. The bispho-sphorylated ITAM establishes a highly specific binding site for the tandem SH2 domains of the Syk family PTKs: spleen tyrosine kinase (Syk) and Ç-associated protein of 70 kDa (ZAP-70). Membrane recruitment of these PTKs and their subsequent activation by Src family kinases represent significant signal-initiating events that are analogous to those of numerous antigen and Fc receptors on lymphocytes [159]. Although either Syk or ZAP-70 is capable of binding to all three ITAM-containing TM accessory proteins, several reports have described preferential association of Syk with DAP12 and y, whereas ZAP-70 reportedly binds most tightly to Ç, as the name would imply [88,114,156].

Syk and ZAP-70 are critical PTKs for the function of ITAM-containing receptor complexes, and Syk has been shown to be required for human NK cell cytotoxicity [22, 80]. It was surprising, however, that NK cells from mice deficient in both of these PTKs still exhibited cytotoxicity toward a number of target cells [43]. At least part of the residual activation signaling in Syk-/ZAP-70-deficient NK cells is likely mediated through integrins, a receptor associated with DAP10, and the 2B4 receptor, all of which function independently of ITAMs, as will be discussed in subsequent sections [6, 8, 16, 146].

Phosphorylation of Syk or ZAP-70 initiates a cascade of downstream events that are fundamental to our understanding of general lymphocyte activation and important for initiating cytotoxicity and cytokine release by NK cells. Some of the major activating signal pathways emanating from ITAMs are outlined in Fig. 2. Initial substrates of Syk and ZAP-70 include phosphatidylinosi-tol 3-kinase (PI3K) and PLCy [79,80,160]. PLCy activation also requires phosphorylation by a Tec family PTK, such as Itk, which is activated by Syk family PTKs in T cells [105]. PLCy cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane to release inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 migrates to the endoplasmic reticulum to release stored calcium, and DAG activates PKC at the plasma membrane [82, 97]. Alternately, PI3K phosphorylates the 3'-inositol hydroxyl group of PIP2 to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3). PIP3 production creates a specific plasma membrane binding site for recruitment of certain signaling proteins containing pleckstrin homology (PH) domains, which include PLCy, Vav, Itk, and 3BP2. 3BP2 is an adaptor protein that brings PLCy and Vav together at the assembling signalosome [78, 107]. Syk-phosphorylated Vav activates GTPases of the Rhofamily [60], which will be covered in Sect. 6 below Activation of PLCy, PKC, Vav, and PI3K as well as elevation of cytosolic calcium concentrations are important events for initiation of the cytolytic hit by NK cells [19, 34,42,60, 79, 82,158-160,181].

Another substrate of Syk and ZAP-70 kinases is growth factor receptor-bound protein 2 (Grb2), which is an adaptor that recruits son of sevenless (SOS). SOS is a guanine nucleotide exchange factor (GEF) that activates the Ras family GTPases, which trigger the Ras^RAF^MEK^ERK cascade [59]. Extracellular signal-regulated kinase (ERK) is a mitogen-activated protein ki-nase (MAPK) involved in regulating growth, proliferation, and cytotoxicity. ERK is activated by ITAM-coupled receptors and integrins in NK cells and important for granule-mediated cytotoxicity and IFNy production [27, 29, 108, 115, 163, 174]. One report, however, showed that ERK does not contribute to conjugation with target cells or reorganization of actin or tubulin cytoskeleton in NK cells [163]. It was recently reported that abnormal NK

Activating Receptor

DAP 12 TCRÇ FcRy

DAP 12 TCRÇ FcRy

Cell Cytokine Production

Cytokine Production

Fig. 2 Signaling cascades downstream from ITAM-coupled receptors. This model diagrams the major downstream intracellular events emanating from an engaged NK cell-activating receptor that is noncovalently associated with a disulfide-linked (SS) dimer of ITAM-containing TM accessory proteins. Single arrows label activation impacts or motility of molecules, double-headed arrow designates physical interaction, dash-lined arrows specify links to effector functions in another portion of the cell surface plasma membrane (gray bar) for clarity, p signifies a phosphorylation event, and gray boxes highlight ultimate biological responses. Rounded notches in individual effector modules represent SH2 or PH domains that interact with tyrosine phospho-rylated proteins or PIP3, respectively. Abbreviations correspond with the text

Cytokine Production

Fig. 2 Signaling cascades downstream from ITAM-coupled receptors. This model diagrams the major downstream intracellular events emanating from an engaged NK cell-activating receptor that is noncovalently associated with a disulfide-linked (SS) dimer of ITAM-containing TM accessory proteins. Single arrows label activation impacts or motility of molecules, double-headed arrow designates physical interaction, dash-lined arrows specify links to effector functions in another portion of the cell surface plasma membrane (gray bar) for clarity, p signifies a phosphorylation event, and gray boxes highlight ultimate biological responses. Rounded notches in individual effector modules represent SH2 or PH domains that interact with tyrosine phospho-rylated proteins or PIP3, respectively. Abbreviations correspond with the text cells from patients with lymphoproliferative disease of granular lymphocytes (NK-LDGL) exhibit constitutive Ras and ERK activation that contributes to their accumulation [52].

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