The diverse functions of Src family kinases in macrophages

Abstract

Macrophages are key components of the innate immune response. These cells possess a diverse repertoire of receptors that allow them to respond to a host of external stimuli including cytokines, chemokines, and pathogen-associated molecules. Signals resulting from these stimuli activate a number of macrophage functional responses such as adhesion, migration, phagocytosis, proliferation, survival, cytokine release and production of reactive oxygen and nitrogen species. The cytoplasmic tyrosine kinase Src and its family members (SFKs) have been implicated in many intracellular signaling pathways in macrophages, initiated by a diverse set of receptors ranging from integrins to Toll-like receptors. However, it has been difficult to implicate any given member of the family in any specific pathway. SFKs appear to have overlapping and complementary functions in many pathways. Perhaps the function of these enzymes is to modulate the overall intracellular signaling network in macrophages, rather than operating as exclusive signaling switches for defined pathways. In general, SFKs may function more like rheostats, influencing the amplitude of many pathways.

Figure 1

The structure of SFKs, and approaches used to study their function. A. An outline of the structure of SFKs (without the N-terminal unique domain). Amino acid numbers refer to the SFK, Lyn. Intramolecular interactions hold SFKs in an inactive conformation; a polyproline-like sequence in the SH2-kinase linker is bound to the SH3 domain, and the tyrosine in the C-terminal tail is phosphorylated and bound to the SH2 domain. The activity of SFKs can be manipulated in several different ways both activating (green arrows) and inhibitory (red arrows). The table in B shows the percent similarities between human SFKs, comparing either the full length amino acid sequence (top-right) or the kinase domain only (bottom-left), as indicated by solid wedges on table sides. Sequences obtained from www.kinase.com were aligned using Clustal W (202). The table in C compares biochemical and cellular IC₅₀ values for SU6656 and PP2 assayed in parallel (19 and R. A. Blake, unpublished observation). The cellular assay was performed as described in (203). The asterisk (*) indicates kinases that are also inhibited at concentrations of PP2 typically used by investigators as shown in other studies (–24).

Figure 2

Use of IT AM and ITIM-based mechanisms to signal downstream of macrophage receptors. A. Macrophage integrins depend on the ITAM-containing molecules DAP12 (and to a lesser extent FcR gamma) to initiate downstream outside-in signaling reactions. In this model, SFKs phosphorylate the ITAM-containing adapters, allowing recruitment of Syk kinase by binding to the Syk SH2 domains, leading to Syk activation and downstream phosphorylation responses. Integrins recruit either DAP12 or FcR gamma by an as yet undetermined mechanism. B. Examples of other macrophage receptors using ITAM-based mechanisms to activate downstream signaling pathways. Receptors such as the Fc receptor, Dectin-2 and TREM1 associate with ITAM-containing adapter molecules through charged interactions in their transmembrane domains. Dectin-1 contains an ITAM-like sequence in its cytoplasmic tail, and PSGL-1 associates with the ITAM-containing ERM protein, moesin. C. Macrophage receptors such as the IL-4 alpha chain use ITIM-based mechanisms to down-modulate signaling pathways. Phosphorylation of the ITIM by SFKs leads to recruitment of protein and lipid phosphatases such as Shp1, Shp2 and SHIP1 that dephosphorylate signaling molecules and inhibit downstream pathways. D. Examples of other macrophage receptors that utilize ITIM-containing domains for inhibitory signaling.

Figure 3

Involvement of SFKs in TLR signaling in macrophages. Signaling downstream of TLRs 2, 4 and 9 in response to their respective ligands leads to activation of MAP kinases, production of cytokines, and actin cytoskeletal changes. Studies mainly using SFK inhibitors such as PP1, PP2 and SU6656 have shown that SFKs are involved in many of these responses, however experiments with SFK-deficient mice have produced different results (indicating no differences or even paradoxical increases in TLR type signaling) hence the controversy regarding the role of SFKs in TLR signaling. There are several ways in which SFKs may modulate TLR signaling, both positively and negatively, rather than being directly involved. CD14 binds to SFKs, which could recruit the SFKs to TLR4 during LPS stimulation and therefore be involved in proximal TLR signaling events, such as phosphorylation of substrates such as Vav and paxillin. TLR2 and 9 do not require co-receptors, but may recruit SFKs by clustering with other receptors such as integrins that are known to signal through SFKs. SFKs have also been shown to act more distal to TLRs, possibly interacting with downstream molecules such as TRAF6. Furthermore, through phosphorylation of ITAMs or ITIMs, SFKs can provide signals that down-modulate signaling through TLRs. Solid arrows indicate well-defined pathways, while dotted arrows indicate potential roles by which SFKs may modulate specific TLR pathways.