DAP12-mediated Signal Transduction in Natural Killer Cells

Abstract

The murine Ly49 family contains nine genes in two subgroups: the inhibitory receptors (Ly49A, B, C, E, F, G2, and I) and the noninhibitory receptors (Ly49D and H). Unlike their inhibitory counterparts, Ly49D and H do not contain immunoreceptor tyrosine-based inhibitory motifs but associate with a recently described coreceptor, DAP12, to transmit positive signals to natural killer (NK) cells. DAP12 is also expressed in myeloid cells, but the receptors coupled to it there are unknown. Here we document the signaling pathways of the Ly49D/ DAP12 complex in NK cells. We show that ligation of Ly49D results in 1) tyrosine phosphorylation of several substrates, including phospholipase C␥1, Cbl, and p44/ p42 mitogen-activated protein kinase, and 2) calcium mobilization. Moreover, we demonstrate that although human DAP12 reportedly binds the SH2 domains of both Syk and Zap-70, ligation of Ly49D leads to activation of Syk but not Zap-70. Consistent with this observation, Ly49D/DAP12-mediated calcium mobilization is blocked by dominant negative Syk but not by catalytically inactive Zap-70. These data demonstrate the dependence of DAP12-coupled receptors on Syk and suggest that the outcome of Ly49D/DAP12 engagement will be regulated by Cbl and culminate in the activation of transcription factors.

The immunomodulatory adapter proteins DAP12 and Fc receptor γ-chain (FcRγ) regulate development of functional osteoclasts through the Syk tyrosine kinase

“…In other cells, phosphorylation of ITAM tyrosines recruits and activates Syk through binding to its SH2 domains (2,3,11). We found that Syk is expressed in wild-type OCLs generated by 70 ng͞ml RANKL and 10 ng͞ml M-CSF in vitro, and it colocalizes with actin at the cell periphery ( Fig.…”

“…This observation suggests that other ITAM signaling adapter proteins, such as FcR␥, may also be involved in osteoclastogenesis. DAP12 and FcR␥ are both transmembrane adapter proteins with ITAM domains that couple to downstream pathways through the Syk tyrosine kinase (4,11). Thus, we studied mice doubly deficient in DAP12 and FcR␥ and examined the functional role of Syk in osteoclasts.…”

The Linker for Activation of B Cells (LAB)/Non-T Cell Activation Linker (NTAL) Regulates Triggering Receptor Expressed on Myeloid Cells (TREM)-2 Signaling and Macrophage Inflammatory Responses Independently of the Linker for Activation of T Cells

“…Cells were lysed with radioimmune precipitation assay lysis buffer (0.5% deoxycholic acid, 1% Triton X-100, 150 mM NaCl, 20 mM Tris (pH 8), 5 mM EDTA, 0.4 mM Na 3 VO 4 , aprotinin, leupeptin, and phenylmethylsulfonyl fluoride) or in some instances with laurylmaltoside buffer (1% laurylmaltoside in 20 mM Tris (pH 7.5), 100 mM NaCl, 10% glycerol, and protease and phosphatase inhibitors as above). Lysates were clarified by centrifugation and precipitated as described previously (27). Proteins were separated using 4 -12% gels (Nupage, Invitrogen), transferred onto polyvinylidene difluoride membranes (Millipore), and analyzed by Western blot.…”

“…Because DAP12-dependent signaling is known to lead to phosphorylation of the E3 ubiquitin ligase c-Cbl in NK cells (27), we next determined whether c-Cbl is downstream of TREM-2 in macrophages. Using the GPVI/DAP12 chimera system, we found that c-Cbl was rapidly tyrosine-phosphorylated ( Fig.…”

SHP-1- and Phosphotyrosine-Independent Inhibitory Signaling by a Killer Cell Ig-Like Receptor Cytoplasmic Domain in Human NK Cells

“…2DL4 possesses a number of unique structural elements, which include 1) an extracellular domain consisting of D0 and D2 Ig-like domains (a feature shared only by KIR2DL5) (23), 2) a cytoplasmic domain possessing only a single ITIM sequence, and 3) a transmembrane domain containing a charged arginine residue (24). By analogy, activating forms of KIR with truncated cytoplasmic domains that lack functional ITIMs also exist and associate noncovalently with a homodimer of the activating accessory protein DAP12 via similar basic transmembrane residue in the activating KIR and an acidic transmembrane residue in DAP12 (3,(25)(26)(27). Therefore, the presence of both an ITIM and a basic transmembrane residue suggests that 2DL4 may have both inhibitory and activating functions.…”