Cytoplasmic tyrosine kinases in the activating signaling pathways utilizing ITAM-containing adapters. Examples of immunoreceptors, hemi-ITAM C-type lectin receptors, and nonimmunoreceptors that utilize ITAM-signaling adapters and the cytoplasmic tyrosine kinases (indicated as shaded molecules) discussed in this article are shown. “Classical immunoreceptors” refers to those signaling molecules that are directly coupled to ITAM adapters FcRγ (group shown on the left) or DAP12 (group shown on the right) through transmembrane charged residues, shown as “+” and “−” in the figure. These immunoreceptors consist of immunoglobulin superfamily-containing proteins (such as the Fc receptors, PIR-A, or the TREMs) or the C-type lectin receptors (Dectin-2, Mincle, or MDL-1). In some cases, receptors may utilize either signaling adapter (see Hamerman et al. 2009). Examples of the C-type lectin receptors that have ITAM-like sequences as imbedded domains within their cytoplasmic tails are Dectin-1, CLEC2, and CLEC9A. The sequences with the ITAM domain of these receptors differs in that the membrane distal tyrosine resides in a YxxxL sequence (or YxxxI for mouse), as opposed to the traditional YxxL ITAM sequence found around the proximal tyrosine. As a result, the membrane distal tyrosine is dispensable for signaling, leading to the designation of these receptors as “hemi-ITAM” molecules (Kerrigan and Brown 2010). For a more complete list of FcRγ- and DAP12-associated receptors, see Lanier (2009), Graham and Brown (2009), and Kanazawa (2007). Not shown is the human FcγRIIA receptor, which is unique among the Fc receptors for having an ITAM sequence directly imbedded in its cytoplasmic tail (see Nimmerjahn and Ravetch 2008). To the right are shown examples of receptors that link to or co-opt the ITAM pathway, with the best example being the leukocyte integrins (see Abram and Lowell 2009). For all of these receptors, it remains unclear how they are coupled to the ITAM adapters; hence this association is indicated as a “∼” within the membrane region. Despite the difference in the coupling of these receptors to the initial activating pathways, the overall signaling events that follow receptor engagement by their respective ligands are quite similar (as an examples, see Gilfillan and Rivera 2009; Mocsai et al. 2010). The first step involves activation of Src-family kinases (shown as “SFK”), which are anchored in the membrane by their N-terminal acetylation sites. The SFK phosphorylate the ITAM adapters, leading to docking sites for Syk kinase, which then phosphorylates a number of substrates, including signaling scaffolding proteins such as SLP76, LAT, or NTAL, which in turn recruit other molecules (Vav family members) to initiate downstream responses. Syk function is also critical for PI3-kinase activation, which in turn leads to generation of membrane-bound PIP3 lipids that serve as membrane binding sites for the Tec-family kinases. Tec-family members contribute to scaffold protein phosphorylation. Together, these three cytoplasmic kinases contribute to the main downstream pathways of Ca²⁺ signaling, MAPK activation, and NF-κB activation. Syk and Src-family kinases also contribute to activation of the FAK/Pyk2 kinases that feed primarily into the Rho/WASP pathway of actin polymerization, leading to cytoskeletal changes in innate immune cells required for adhesion, migration, and degranulation responses.

Tyrosine kinase pathways leading to CARD9 and TLR signaling responses. The central role of the CARD9/Malt1/Bcl10 complex in activating primarily the NF-κB pathway and to a lesser extent the MAPK pathway is shown (see Colonna 2007). Engagement of C-type lectin receptors, leading to Syk activation, is coupled to the CARD9 complex through an unclear pathway, but may involve PKCs. This in turn directly leads to activation of IKK, which in turn activates downstream NF-κB pathways (see Hara and Saito 2009). The importance of this pathway in innate immune cell recognition has been revealed by the similar defect in host defense in mice lacking either the C-type lectin receptors, Syk, or CARD9 (Gross et al. 2006). The CARD9 complex may also facilitate the ability of the TLRs to engage downstream MAPK pathway activation. Upstream of the TLRs, the major cytoplasmic tyrosine kinase involved in this innate immune receptor function is Tec-family members, as revealed by poor TLR signaling function in cells derived from either mice or humans lacking various Tec kinases. It remains unclear how the Tec kinases operate in the TLR pathway, but could involve phosphorylation of signaling adapters MyD88 or Mal.

Inhibitory pathways in innate cells utilizing ITIM or “inhibitory” ITAM signaling pathways. Shown to the left are classical inhibitory receptors that contain ITIM binding sequences within their cytoplasmic tails. Like activating receptors, the inhibitory receptors can be both immunoglobulin superfamily proteins or C-type lectin receptors. For a more exhaustive list of inhibitory receptors in innate cells, see Munitz (2010). Inhibitory receptors are often engaged simultaneously with activating receptors, though they recognize distinct sets of ligands, during innate immune recognition of complex pathogens such as bacteria and yeast. The ITIM domains on these proteins are then phosphorylated by SFKs, which results in the recruitment of tyrosine or lipid phosphatase (Shp1/2 or Ship, respectively), which in turn dampen signaling by dephosphorylation of a number of substrates (the ITAMs themselves or downstream substrates in the MAPK, Ca²⁺, or Rho/WASP pathways). Ship works directly to convert PIP3 back to PIP2 and hence opposes PI3-kinase function (Sly et al. 2007). Not shown are inhibitory DOK proteins, which are cytoplasmic adapter proteins that also recruit RasGAP and Ship to down-regulate signaling responses (see Mashima et al. 2009). This inhibitory receptor pathway also functions to down-modulate other signaling responses besides just ITAM/immunoreceptor pathways, in particular G-protein-coupled pathways and TLR responses (Zhang et al. 2005; O'Neill 2008). To the right are shown examples of inhibitory receptors that function through “inhibitory” ITAM signaling. This pathway is believed to be engaged following low avidity interactions of these receptors with various ligands, which results in only weak phosphorylation of associated ITAM adapters (shown as the ∼P on FcRγ and DAP12). The partial phosphorylation of the ITAMs leads to Shp-1/2 recruitment instead of Syk recruitment, hence engaging an inhibitory response analogous to an ITIM pathway (see Kanamaru et al. 2008; Hamerman et al. 2009). This more novel mechanism of signal inhibition has mainly been revealed by studies of innate cells lacking DAP12 or FcRγ. Like the classical ITIM pathway, the low avidity inhibitory ITAM pathway also regulates TLR responses (Hamerman et al. 2005). However, when these receptors are engaged by more high avidity ligands, they can induce activating signaling responses.