Src-Family and Syk Kinases in Activating and Inhibitory Pathways in Innate Immune Cells: Signaling Cross Talk

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Src-family and Syk Kinases in Activating and Inhibitory Pathways in Innate Immune Cells: Signaling Cross Talk

Clifford A. Lowell

Department of Laboratory Medicine, University of California, San Francisco, California 94143 Correspondence: [email protected]

The response of innate immune cells to growth factors, immune complexes, extracellular matrix proteins, cytokines, pathogens, cellular damage, and many other stimuli is regulated by a complex net of intracellular signal transduction pathways. The majority of these pathways are either initiated or modulated by Src-family or Syk tyrosine kinases present in innate cells. The Src-family kinases modulate the broadest range of signaling responses, including regulating immunoreceptors, C-type lectins, integrins, G-protein-coupled receptors, and many others. Src-family kinases also modulate the activity of other kinases, including the Tec-family members as well as FAK and Pyk2. Syk kinase is required for initiation of signaling involving receptors that utilize immunoreceptor tyrosine activation (ITAM) domains. This article reviews the major activating and inhibitory signaling pathways regulated by these cytoplasmic tyrosine kinases, illuminating the many examples of signaling cross talk between pathways.

nnate immune cells, including macrophages, important roles in innate cells. They are not dis- Idendritic cells, granulocytes, and mast cells, cussed in detail in this article, but are reviewed function as the ﬁrst line of defense against elsewhere in articles on the subject. pathogens. These cells use a dizzying array of There are eight members of the Src family; cell-surface receptors, which are connected to innate immune cells primarily express Hck, an equally complicated intracellular signal trans- Fgr, Lyn, and to a lesser extent, Src (Lowell duction network, to sense pathogen molecules 2004). The Syk-ZAP70 family has only two and then orchestrate the appropriate immune members and only Syk is found in innate cells. response. Among the intracellular signaling mol- Most innate cell types express the same spec- ecules that are most crucial for innate immune trum of kinases with some speciﬁc cellular dif- cells are the cytoplasmic tyrosine kinases. Two ferences. For example, mast cells express a majorkinasefamiliesthat operateintheproximal broader range of Src-family kinases than macro- intracellular signaling pathways in innate cells are phages or dendritic cells (Colgan and Hankel the Src-family kinases and the Syk-ZAP70 family. 2010). In general, Src-family and Syk kinases A third familyof kinases, the Tek family, also have tend to operate together in signaling pathways,

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with the Src-family being “upstream” or acti- of interaction and cross regulation. Together, vated ﬁrst in response to pathogen detection. these pathways impinge on downstream factors, These enzymes then communicate downstream such as MAPK kinases, which have broad effects to Tec-family members. The Tec-family kinases on gene transcription; the Rac/Rho pathway to expressed in innate cells include Btk, Bmx, and modulate cytoskeletal function; the inositol Tec (Koprulu and Ellmeier 2009; Tohyama and trisphosphate (IP3), and diacylglycerol pathway Yamamura 2009). Additionally, Src-family kin- (DAG), which regulates Ca2þ entry into cells ases activate yet another family of PTKs, the and activation of various isoforms of protein FAK/Pyk2 tyrosine kinases, which play a major kinase C (PKC). Overall, the outline of the pro- role in integrin signaling (Hauck et al. 2000). totypical immunoreceptor pathway as described Though primarily studied in activating path- here is similar in both innate and adaptive im- ways, Src-family and Syk kinases also activate mune cells (Smith-Garvin et al. 2009; Kurosaki inhibitory signaling pathways (Nimmerjahn and et al. 2010). Ravetch 2008). In many situations, inhibitory signaling often overrides the activating signal. New Concepts in Immunoreceptor Signaling: Pathways can also be initiated at different times CARD9 and Receptor Diversity or rates. Finally, to add even more complexity, activating and inhibitory pathways often inter- Recent and exciting developments in the immu- act indirectly, for example, through the produc- noreceptor paradigm include recent progress in tion of cytokines and growth factors and not delineating how this pathway is connected to through direct intracellular biochemical inter- NF-kB and the demonstration that many innate actions; Hence the term signaling “cross talk,” immune receptors utilize the “immunoreceptor which now appears commonly in the literature pathway” even though they lack ITAMs and (O’Neill 2008; Ivashkiv 2009; Page et al. 2009). therefore are not technically immunoreceptors. The adapter protein CARD9, which con- tains a caspase-recruitment domain (CARD), OVERVIEW OF ACTIVATING PATHWAYS has now been shown to be the link between a variety of ITAM-containing receptors involved Classical Immunoreceptor Pathways in recognition of fungal and probably other In the prototypical immunoreceptor pathway, pathogen structures and NF-kB (Fig. 2) (Gross engagement of the receptor leads to activation et al. 2006; Gross et al. 2009). CARD9 is closely of Src-family kinases, which in turn phosphor- related to the lymphocyte protein CARMA-1, ylate immunoreceptor tyrosine-based activa- which forms a complex with the adapter pro- tion motifs (ITAMs) present on either the teins Bcl-10 and MALT1, and thus links the T- receptor or associated subunits (Fig. 1). This and B-cell receptors to the NF-kBpathway leads to recruitment of Syk, by binding of the (Rawlings et al. 2006). CARD9 forms the same Syk SH2 protein domain to the phospho-ITAM complex in innate cells. In lymphoid cells, residues, and activation of Syk allowing it CARMA-1 is activated by PKC isoforms (PKCu to phosphorylate downstream substrates. One in T-cells and PKCb in B-cells), which phos- of the enzymes activated downstream is phosphorylate CARMA-1, resulting in a conforma- phoinositide3-kinase(PI3-kinase), which gener- tional change that allows it to interact with ates membrane-associated phosphatidylinositol IKK, leading to IkB turnover and NF-kB activa- (3,4,5)-triphosphate (PIP3). The FAK/Pyk2 tion. In myeloid cells, it remains unclear if kinases are activated directly via the Src-family CARD9 activation is directly downstream of kinases, where they contribute to downstream PKC activation (Hara and Saito 2009). Never- responses involving cell adhesion and migra- theless, it is clear that in the absence of tion. Though usually depicted as a linear signal- CARD9, receptors involved in fungal pathogen ing pathway with Src-kinases at the top and recognition (Dectin-1, Dectin-2) are unable to FAK/Pyk2 at the bottom, there are many points activate NF-kB, and more importantly, the

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Classical immunoreceptors Receptors co-opting ITAM signaling FcRγ Receptors DAP12 Receptors FcεR, FcγRs TREMs Dual Receptors Dectin-2 MDL-1 Hemi-ITAM Receptors Integrins Mincle CD200R3 Dectin-1 IL-3R DCAR Siglec-H CLEC2 PSGL-1 γ PIR-A MAIR-II CLEC9A IFN R SIRP-β RANK

FcRγ DAP12 DAP12 FcRγ

+– + – PIP3 PIP2

SLP-76SSLLLP-7P-P P Y Y P P Y Y P Y P SOS PI3-KPPI3-K P Y Y P Y Y Syk Syk Syk Syk P P LAT/NTAL Y Y P P Y Y P Y γ P Y Y Y Y SFK SFK PPLCLC Vav Tec Ks

Fak/Pyk2

CARD9 Ras DAG IP3 RasGRP RhoRho GTPase WASPW NF-kB pathway PKC 2+ MAPKs Ca Actin polymerization Cytokine secretion Cytokine secretion NFAT proliferation gene transcription Migration degranulation

Figure 1. 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 ITAMadapters FcRg (group shown on the left) or DAP12 (group shown on the right) through transmembrane charged residues, shown as “þ” and “2” 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 (Ker- rigan and Brown 2010). For a more complete list of FcRg- and DAP12-associated receptors, see Lanier (2009), Graham and Brown (2009), and Kanazawa (2007). Not shown is the human FcgRIIA receptor, which is unique among the Fc receptors for having an ITAMsequence directly imbedded in its cytoplasmic tail (see Nimmerjahn and Ravetch 2008). Tothe right are shown examples of receptorsthat link to orco-opt the ITAMpathway, 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 ITAMadapters; hence this association is indicated as a “” within the membrane region. Despite the difference in the coupling of these receptorsto 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-familykinases. Tec-familymembers contribute to scaffold protein phosphorylation. Together, these three cytoplasmic kinases contribute to the main downstream pathways of Ca2þ signaling, MAPK activation, and NF-kB activation. Syk and Src-family kinases also contribute to activation of the FAK/Pyk2 kinases that feed primarily into the Rho/WASPpathway of actin polymerization, leading to cytoskeletal changes in innate immune cells required for adhesion, migration, and degranulation responses.

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C.A. Lowell

TLRs C-type lectin receptors

FcRγ DAP12

+ – PIP3 PIP2

PI3-K P Y Y P P Y Y P Y P

P Y Y P P Y Y P Y P MyD88 Tec Ks Tec

Syk Syk IRAKs

CARD9

Malt1 MAPKs NF-κB pathway Bcl10

Cytokine secretion Cytokine secretion

Figure 2. Tyrosine kinase pathways leading to CARD9 and TLR signaling responses. The central role of the CARD9/Malt1/Bcl10 complex in activating primarily the NF-kB 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 leadsto activation of IKK, which in turn activates downstream NF-kB 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 theC-typelectinreceptors,Syk,orCARD9(Grossetal.2006).The CARD9complexmayalsofacilitatetheabilityof the TLRsto engage downstream MAPK pathwayactivation. Upstream of the TLRs, the majorcytoplasmic 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 Teckinases. It remains unclear how the Tec kinases operate in the TLR pathway, but could involve phosphorylation of signaling adapters MyD88 or Mal.

entire repertoire of ITAM-containing receptors protein (Fig. 1). Surprisingly, a number of non- in myeloid cells are uncoupled from NF-kB immunoreceptor pathways, such as neutrophil (Robinson et al. 2006; Yamasaki et al. 2008). integrin signaling or IL-3 responses in baso- This results in profound defects in cytokine phils, are lost in innate cells derived from mice responses, which in vivo translates to poor re- lacking DAP12 or FcRg (Mocsai et al. 2006; sponses to pathogens, speciﬁcally fungi such as Hida et al. 2009). Examples of receptors that co- Candida albicans,bacteriasuchasListeria mono- opt the classical immunoreceptor pathway are cytogenes,andM.tuberculosis (Ruland 2008; Dor- shown in Figure 1. The mechanisms by which hoi et al. 2010). these nonimmunoreceptors couple to DAP12 Immunoreceptor signaling has also recently and/or FcRg to initiate signaling remain un- been shown to play a role downstream of innate clear. receptors that lack ITAM sequences. This con- cept evolved from studies of innate cells lacking OVERVIEW OF INHIBITORY PATHWAYS the ITAM-signaling adapters DAP12 and the Classical Inhibitory Pathways Fc1RIg chain (referred to as FcRg) (Nimmer- jahn and Ravetch 2008; Lanier 2009). Typically, Most inhibitory signals are mediated in a fashion DAP12 and FcRg are coupled to immunore- very similar to the immunoreceptor pathway, ceptors through charged amino acid interac- except that inhibitory pathways are initiated tions within the transmembrane regions of each through phosphorylation of immunoreceptor

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Cytoplasmic Tyrosine Kinases in Innate Immune Cells

tyrosine-based inhibitory motifs (ITIMs) de- phosphatases, such as SHP-1 (Src homology 2 ﬁned by the sequence amino acids I/V/L/ domain containing protein tyrosine phospha- SxYxxL/V (Fig. 3) (Munitz 2010). The Src- tase-1/2) or SHIP-1 (SH-2-containing inositol family kinase Lyn is primarily responsible for phosphatase), which then down-modulate sig- ITIM phosphorylation. Phospho-ITIM domains naling responses by dephosphorylating down- serve as docking sites for several types of stream substrates. Like the activating pathways,

ITIM Receptors FcγRIIB ILTs PIR-B α Sirp- DAP12 Receptors Classical inhibitory receptors Siglecs FcRγ, FcRα Low avidity ITAM TREM-2 BDCA-2 inhibitory receptors

– + PIP3 PIP3 – +

SLP-76 P Y Y P Y P SOS PI3-K Y Y Syk Shp1 Shp1 P P SFK LAT/NTAL P SFK Y Y Y Y P Y P Shp2 PLCγ Vav Shp2 Tec Ks

Y P Ship1 Y P Fak/Pyk2

DAG IP Ras RasGRP 3 Rho GTPase WASP PKC Ca2+ MAPKs Actin polymerization

Cytokine secretion NFAT gene transcription proliferation Migration degranulation

Figure 3. 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, Ca2þ, 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). Tothe 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 FcRg 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 FcRg. Like the classical ITIM pathway, the low avidity inhibitory ITAM pathway also regulates TLR responses (Hamer- man et al. 2005). However, when these receptors are engaged by more high avidity ligands, they can induce activating signaling responses.

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this inhibitory pathway was first worked out in in an SHP-1-dependent fashion, resulting in the lymphocytes and NK cells, mainly through the attenuation of tissue injury in an Ab-induced study of the inhibitory Fc receptor, FcgRIIb glomerulonephritis model (Kanamaru et al. (Ravetch and Lanier 2000). The number of 2008). This has led to the model whereby low newly recognized ITIM-containing inhibitory affinity or partial (i.e., monomeric) engage- receptors in innate immune cells has grown to ment of activating receptors results in only include a number of Ig superfamily molecules modest ITAM phosphorylation events, leading (PIR-B, Sirp-1a), sialic binding proteins, to the recruitment of phosphatases (SHP-1 in referred to as Siglecs, and a few C-type lectin particular) instead of Syk kinase (Pinheiro da receptors (Robinson et al. 2006; Crocker et al. Silva et al. 2008), resulting in an overall dimi- 2007). In many cases, the ligands for these nution of cellular response (Fig. 3). inhibitory receptors are unknown. In a broad The activation of ITAM signaling pathways sense, the inhibitory receptors function by lim- also functions to inhibit other signaling re- iting innate immune cell functions, including sponses, such as TLR signaling (Hamerman proliferation, cytokine responses, and cell ad- et al. 2009). In particular, both macrophages hesion. Since inhibitory pathways are often and dendritic cells derived from DAP12 and engaged at the same time as the activat- FcRg-deficient mice display increased respon- ing pathways, the resulting cellular response ses to various TLR ligands, such as CpG, endo- depends on a balance between the activating toxin (LPS), or yeast zymosan (Hamerman signal and the inhibitory signal. Mutagenesis et al. 2005; Chu et al. 2008). In macrophages, studies suggest that inhibitory signaling is usu- this inhibitory signaling may be mediated ally dominant; for example, innate immune through TREM-2, which is a DAP12 coupled cells from Lyn kinase-deficient mice display receptor thought to be involved in recognition hyperproliferative responses to cytokines and a of various bacterial structures and potentially hyperadhesive phenotype that correlates with apoptoticcells (Hamerman et al. 2006; Takahashi the lack of phosphatase activation in these cells et al. 2007; Hsieh et al. 2009; N’Diaye et al. 2009). (reviewed by Scapini et al. 2009). Additional FcRg and DAP12-associated receptors on both mouse and human plasmacytoid DCs have also been found to inhibit interferon Inhibitory ITAMs production following TLR stimulation (Blasius There is increasing evidence that ITAM-coupled et al. 2004; Fuchs et al. 2005; Swiecki and Co- activating receptors also contribute to inhibi- lonna 2007). tory signaling (Fig. 3). This was first demon- The molecular mechanisms by which ITAM- strated in mast cell experiments that compared based pathways cross-inhibit TLR responses engagement of IgE-Fc1R receptors by low remain unclear (reviewed by Ivashkiv 2008). versus high affinity haptens (Torigoe et al. 1998). In addition to phosphatase recruitment, activa- The low affinity haptens suppressed the signal- tion of PI3-kinase-mediated pathways that both ing responses. A similar observation was made suppress NF-kB activation and alter recruit- by Pasquier et al., who found that a Fab frag- ment of MyD88 and Mal adaptors to TLRs ment of an anti-FcaRI mAb inhibited IgG- have been proposed (Kagan and Medzhitov mediated phagocytosis in a transfected macro- 2006). Activation of calcineurin by ITAM sig- phage cell line, suggesting that low affinity or naling, leading to dephosphorylation of TLR sig- partial activation of one ITAM-containing naling pathway molecules, may also be involved receptor can inhibit the function of another (Kang et al. 2007). Recently, Han et al. have pro- ITAM-containing immunoreceptor (Pasquier vided an elegant model for ITAM-mediated et al. 2005). Subsequently, this same group down regulation of TLR responses (Han et al. demonstrated that monovalent engagement 2010). Building on the observation that Mac-1- of FcaRI inhibits chemotaxis to MCP-1 and deficient mice (lacking the integrin amprotein TNF,as well as downstream signal transduction, that dimerizes with the b2 chain to form Mac-1)

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demonstrate increased inflammatory responses (Gringhuis et al. 2007). More recently, IL-10 to dextransodium sulfate-induced colitis(Abdel- production by neutrophils, following activation baqi et al. 2006), this group and others (Wang through C-type lectin receptors or other ITAM- et al. 2010) found that strong ligation of b2 integ- Syk pathways, has been found to have an im- rins in macrophages and dendritic cells induces munomodulatory effect on subsequent inflam- resistance to subsequent stimulation by various matory responses (Zhang et al. 2009). Hence, in TLRligands. Since signalingthrough b2integrins states of chronic mycobacterial infection, engage- proceeds in a DAP12/FcRg to Syk pathway, Han ment of the C-type lectin/Syk pathway leads et al. (2010) investigated potential intracellular to enough neutrophil-derived IL-10 to limit re- Syk substrates. They found that Syk phosphory- sponses of macrophages and DCs. Thus, neu- lates the TLR adapter proteins MyD88 and trophil depletion can sometimes increase the TRIF on specific tyrosine residues (Y277 and immunopathology associated with infection. Y375, respectively), and that phosphorylation of Similarly, high affinity engagement of b2 integ- these residues results in recruitment of the E3 rins on human macrophages can lead to suffi- ubiquitin ligase Cbl-b, which targets both the cient production of IL-10 to down-modulate adapter proteins and Syk kinase itself for ubiqui- subsequent TLR stimulation (Wang et al. 2010). tin-mediated proteolysis, thus attenuating TLR In all of these cases, the activating stimulation responses (reviewed by Means and Luster 2010). (either throughtheC-typelectinsor theb2integ- This unique pathway provides a direct intracellu- rins) needsto be presentfor hoursto daystoallow lar mechanism for signaling cross talk between for sufficient production of IL-10 and other ITAM and TLR pathways. It is likely that under inhibitors. For integrin signaling, this indirect physiologic situations of high affinity integrin pathway is probably additive with the direct bio- ligation, such as during diapedisisthroughvascu- chemical mechanism of inhibitory cross talk via lar endothelium, signaling through this pathway MyD88/TRIFphosphorylation,but woulddom- limits innate immune cell activation and reduces inateduringprolongedperiodsofintegrinactiva- inadvertent immunopathology. tion potentially to limit chronic inflammation.

Indirect Down-modulation of Signaling: SRC-FAMILY AND SYK TYROSINE KINASES Pathway Cross Talk AND INNATE SIGNALING While most studies have focused on intracellu- The history of tyrosine kinase involvement in lar mechanisms for inhibitory signaling, there innate immune responses to pathogen mole- is growing recognition that immunoreceptor cules (LPS, bacterial cell wall components, and activation can also be limited by post-transcrip- foreign DNA) is long and confusing. Many of tional events. Following engagement of ITAM these studies pre-date the identification of the pathways in macrophages or dendritic cells, Toll-like receptors (TLRs) as the major recep- production of inhibitory cytokines such IL-10 tors involved in recognition of pathogens. These and TGF-b can lead to subsequent down- studies were based on the findings that LPS modulation of signaling, both for the ITAM treatment of innate immune cells increases pathway itself and for other pathways, such as overall protein tyrosine phosphorylation and the TLRs. For example, engagement of the various tyrosine kinase inhibitors, particularly C-type lectin receptor DC-SIGN by ManLAM, those that broadly target Src-family kinases, a cell-wall component present in Mycobacte- and could block cellular responses to LPS (Orli- rium tuberculosis, down-modulates subsequent cek et al. 1999; Smolinska et al. 2008). The stimulation of DCs by LPS (Geijtenbeek et al. hypothesis that TLRs could activate Src-family 2003). The mechanism involves acetylation of kinases were refuted by the finding that macro- the p65 subunit of NF-kB, leading to prolonged phages lacking Hck, Fgr, and Lyn displayed and increased production of IL-10, which in normal (or even enhanced) responses to LPS turn inhibits LPS and other TLR responses treatment (Meng and Lowell 1997). Indeed,

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recent studies have suggested that Lyn kinase immunoreceptors (Fc receptors, NK activating functions as a negative regulator of TLR re- receptors, and pathogen recognition molecules sponses, based on the finding that Lyn-deficient such as TREM family members), where various macrophages show increased cytokine responses Src-family members are known to phosphory- to TLR4 and TLR2 stimulation (Keck et al. late residues on the receptor associated ITAM 2010). The mechanism by which Lyn functions adapter to initiate downstream signaling re- as an inhibitor of TLR responses is still unclear sponses. As stated above, the major new finding (Kagan and Medzhitov 2006). in this area has been the demonstration that many innate immune receptors that are not classical immunoreceptors in fact utilize this FUNCTIONS OF SPECIFIC KINASE FAMILIES same ITAM-mediated mechanism for intracellular signaling, and hence loss of Src-family Src-family Kinases activity directly affects these pathways as well. The Src-family kinases make up the largest fam- The best example is the integrin pathway— ily of cytoplasmic tyrosine kinases expressed in loss of Src-family kinase activity (or removal innate cells. Of the nine Src-family kinases, of the ITAM adapters FcRg and DAP12, the most are present in one type of innate cell or substrates of the kinases) results in a complete another, with Hck, Fgr, and Lyn kinase being deficiency of b1, b2, and b3 integrin function most heavily expressed in monocytes, macro- in innate cells (Abram and Lowell 2009). In all phages, granulocytes, and DCs. These kinases of these pathways, it is uncertain how the Src- have been implicated as primary signaling mole- family kinases are activated following ligand cules downstream of a host of immune cell re- binding by the receptor. Dephosphorylation of ceptors, including immunoreceptors, cytokine the regulatory C-terminal tyrosine in Src-family receptors, integrins, and various pathogen recep- kinases, which activates these enzymes, occurs tors (TREMs and Dectins, as examples). Inhi- through the action of either receptor tyrosine bitor studies and use of knockout mice have phosphatases such as CD45 or CD148, or demonstrated acritical role for Src-family kinases potentially other cytoplasmic tyrosine phos- in a variety of host defense and inflammatory phatases (LMW-PTP and Lyp/PEP, as exam- conditions (Okutani et al. 2006; Abram and ples) (Hermiston et al. 2009; Saunders and Lowell 2008; Ingley 2008). In some cases, these Johnson 2010; Zambuzzi et al. 2010); however, kinases play clear and direct roles in innate whether these phosphatases are directly immune signaling, in both activating and inhib- recruited to the signaling receptors remains itory pathways (Figs. 1 and 3), but in other cases unclear. their role may be more indirect (for example, by modulating cytokine responses that impact Src-family Kinases in Other Innate TLR pathways). The Src-family kinases are also Pathways responsible for phosphorylation and direct activation of other cytoplasmic tyrosine kinases, in Src-family kinases also play important roles in particular Tec-family and FAK/Pyk2 and less pathways where ITAM/immunoreceptor mole- directly Syk; hence Src-family members are cules are not involved. A number of studies often at the top of most signaling pathways in suggest that Src-family kinases work with Jak innate cells. kinases in supporting cytokine responses, either by phosphorylation of receptor subunits or potentially phosphorylating Stat molecules Src-family Kinases in Immunoreceptor (Reddy et al. 2000; Hayakawa and Naoe 2006). Pathways For the classical growth factor receptors, such The most well-understood function of these as the G-CSF or the GM-CSF receptor, kinases is in the classical immunoreceptor acti- Src-kinases have been found to be physically vating (ITAM)pathway (Fig. 1) utilized by many associated with the receptor through SH3/

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Cytoplasmic Tyrosine Kinases in Innate Immune Cells

receptor interactions involving membrane pro- Src-family Kinases and GPCRs ximal regions (Sampson et al. 2007; Peru- Several Src-family kinases have been implicated gini et al. 2010). In other cytokine responses, in regulating chemokine or chemoattractant Src-family kinases are thought to act more receptor signaling, which are mediated by G- downstream of the receptor, often through protein-coupled receptors (GPCRs) (Luttrell interactions with TRAF signaling molecules. and Luttrell 2004). Lyn kinase is activated in Such is the case in the RANK/RANKL signaling macrophages downstream of both CXCR4 and response, where interaction of Src and TRAF6 CCR5, and is thought to couple these GPCRs lead to enhancement of downstream responses to the MAPK and PI3-kinase pathways (Ptasz- (particularly activation of PI3-kinase activity nik et al. 2002; Tomkowicz et al. 2006; Cheung and downstream Akt function) (Leibbrandt et al. 2009). Inhibitor studies have also placed and Penninger 2008). Formation of TRAF6/ Src-family kinases downstream of monocyte- Src signaling complexes has also been reported chemoattractant protein (MCP-1) signaling via downstream of IL-1 and TNFa signaling path- CCR2 (Arefieva et al. 2005) and IL-8 signaling ways, as well as in CD40/CD40L interactions, via CXCR1 (Sai et al. 2008). Similarly, neu- again with the implication that this complex is trophils derived from hck2/2fgr2/2 double involved in stimulating downstream PI3-kinase mutant mice show significant functional defects signaling (Mukundan et al. 2005; Wang et al. following formyl peptide (fMLF) stimulation, 2006). which involves signaling through GPCR cou- Src-family kinases have also been shown to pled formyl peptide receptors (Fumagalli et al. be involved in IL-6 signaling pathways, through 2007). But even in the GPCR signaling pathway, a direct interaction with the IL-6 receptor sig- the same paradigm of Src-kinases acting both in naling protein gp130 (Hallek et al. 1997; Haush- a positive and inhibitory fashion seems to be err et al. 2007). This signaling function of established. While Hck/Fgr-deficient neutro- Src-family kinases may be particularly impor- phils have reduced responses to foryml peptide tant in myeloma cells, where IL-6 serves as a stimulation, they manifest hyper-responsive growth factor for these cells. A number of stud- signaling to chemokines that signal through ies have shown involvement of Src-family kin- CCR1 and CXCR2 (Zhang et al. 2005). This in- ases downstream of the M-CSF receptor in hibitory function in the CCR1/CXCR2 path- macrophages and myeloid progenitors, with ways involves phosphorylation of the ITIM- their function being to couple receptor activa- containing inhibitory receptor PIR-B, which tion to downstream PI3-kinase pathways (Lee in turn is involved in recruiting tyrosine phos- and States 2000; Bourgin-Hierle et al. 2008). phatases that modulate downstream responses As in the case of the IL-3 receptor, the M-CSF from the receptors. receptor pathway may also co-opt ITAM- containing adapters to activate downstream functions, since DAP12 has been recently found Src-family Kinases and Selectin Signaling to be required for optimal M-CSF receptor signaling (Zou et al. 2008). Finally, as in the immu- Src-family kinases have been implicated in the noreceptor pathways, not all the signaling innate immune cell response to selectin engage- functions of Src kinases in cytokine pathways ment, primarily recognition of E-selectin by are activating, since the deficiency of Lyn kinase leukocyte PSGL-1 and CD44 counter-receptors. actually leads to enhanced responses to some Engagement of these receptors leads to activa- cytokines, such as G-CSF, potentially through tion of tyrosine phosphorylation that is inhib- reduced recruitment of phosphatases (Mermel ited by PP2 and other relative Src-family- et al. 2006). The mechanism through which kinase-specific inhibitors (Hidari et al. 1997; Lyn kinase functions as an inhibitor, while other Kumar et al. 2001). The Fgr kinase seems Src-family members function as activators of dominant in this signaling response (Zarbock cytokine signaling, remains unclear. et al. 2008), although there is clearly some

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redundancy with other Src-family members part, through DAP12 (Koga et al. 2004), the (Yago et al. 2010). However, like the integrin effect of Tec/Btk deficiency may reflect im- pathway, it appears that selectin recognition paired activation of downstream actin polymer- also depends on ITAM adapters, since this sig- ization responses. naling is reduced in DAP12-deficient neutrophils (Zarbock et al. 2008). Src-family Kinase Connections to FAK/Pyk2 in Integrin Pathways Src-family Kinases and Membrane Based on studies in nonimmune cells, Src- Bound Receptors family kinases can directly modulate the activity A number of GPI-linked proteins in innate cells of FAK/Pyk2 kinases (Fig. 1). FAK is widely are known to signal through Src-family kinases, expressed in most cells of the body, while Pyk2 including TLR4-associated CD14 and the uro- is expressed mainly in the nervous system, T- kinase plasminogen activator receptor (uPA-R) cells, and various innate cells. FAK kinase has (Stefanova et al. 1993; D’Alessio and Blasi been very extensively studied in fibroblasts and 2009). In innate immune cells (mainly macro- various tumor cell types, where it plays an phages), engagement of these receptors leads important role in cell adhesion signaling down- to increased tyrosine phosphorylation and sub- stream of integrin activation (Tomar and Sch- sequent signaling events that affect adhesion and laepfer 2009). More limited studies of Pyk2 migration. It is likely that clustering of these suggest a similar function. Both of these kinases receptors leads to aggregation of lipid raft mem- are substrates for Src-family kinases following brane structures, through the GPI linkages in the integrin ligation. Phosphorylation of FAK or receptors, which in turn brings the kinases Pyk2 by Src-family members leads to protein together, since they too are located in the raft unfolding and activation of their enzymatic structures. activity. In innate cells, FAK and Pyk2 are found in podosomes, which are the main contact sites in leukocytes (Calle et al. 2006). Macrophages Src-family Kinase Connections to Tec Kinases lacking FAK exhibit elevated protrusive activity, Besides their function in TLR pathways, Tec- altered adhesion dynamics, impaired chemo- family kinases also play important roles in taxis, and elevated basal Rac1 activity, leading downstream immunoreceptor signaling, where to a marked inability to form stable lamellipo- their activation is mediated both through PIP3 dia necessary for directional locomotion (Oki- generation by PI3-kinase and by direct phos- gaki et al. 2003; Owen et al. 2007a). These phorylation by Src-family kinases. Monocytes defects point to an alteration in integrin out- from patients with Bruton’s agammaglobuline- side-in signaling, consistent with the role of mia show reduced uptake of both Ig and com- this kinase in fibroblasts and tumor cells. As a plement opsonized particles (Amoras et al. result of this defect, recruitment of FAK- 2003; Jongstra-Bilen et al. 2008). Studies in deficient monocytes to sites of inflammation macrophage cell lines suggest that Btk and Tec is impaired. A similar phenotype is observed communicate to the actin polymerization path- in macrophages derived from Pyk2-deficient way, since these kinases localize to phagocytic animals (Okigaki et al. 2003). The involvement cups near sites of actin polymerization. Btk of FAK and Pyk2 in macrophage integrin signal- and Tec also play a significant role in macro- ing may also affect bacterial phagocytosis by phage/osteoclast RANK/RANKL signaling. Re- these receptors, since siRNA-mediated knock- markably, like Src or Src/Hck-deficient mice, down of FAK and/or Pyk2 can reduce uptake the double mutant Tec 2/2Btk2/2 mice have of various Yersina strains of bacteria (Owen severe osteopetrosis due to impaired osteoclast et al. 2007b). FAK deficiency also reduces neu- maturation (Shinohara et al. 2008). Since it is trophil adhesion, migration, and antibacterial known that RANK signaling is mediated, in uptake, again potentially through defects in

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Cytoplasmic Tyrosine Kinases in Innate Immune Cells

integrin-mediated signaling events (Kasorn This is particularly true for the C-type lectin et al. 2009). Inhibition of Pyk2 function in neu- receptors in innate cells. trophils, through use of inhibitory peptides transduced into the cells, reduces cell adhesion Syk and ITAM Pathways and spreading following integrin activation, again suggesting a similar role for this kinase Syk kinase plays a critical role in all innate in neutrophil integrin signaling (Han et al. immune cell signaling pathways involving 2003). Pyk2 has also been implicated in the ITAM or ITAM-like signaling adapters. This integrin signaling responses leading to cytokine includes signaling from classical immunore- (in particular IL-10) production (Wang et al. ceptors (FcRs, TREMs, Dectin-2, and others), 2010). Despite these initial studies, the specific nonimmunoreceptors that co-opt the ITAM individual functions of FAK versus Pyk2 in adapters for signaling (integrins, selectins, innate immune integrin signaling remains to IL-3 receptor), and those C-type lectin recep- be defined. tors that have hemi-ITAM sequences embedded in their C-terminal tails (Dectin-1) (Fig. 1). Since many of the C-type lectin and other Syk Kinase immunoreceptor-like pathways converge on The repertoire of signaling pathways that Syk Syk, loss of Syk activity produces a more pro- has been implicated in (reviewed by Mocsai found block than loss of any specific receptor et al. 2010) is more restricted than Src-family (LeibundGut-Landmann et al. 2007). These kinases. Syk kinase is activated by engagement same C-type lectin receptors also are involved of its two SH2 domains by phospho-ITAM in innate immune recognition of mycobacterial domains; hence it functions only in ITAM-like and viral pathogen molecules, all of which are pathways. This includes, of course, the novel affected by Syk blockade in either macrophages “inhibitory ITAM” pathway described above. or dendritic cells (Chen et al. 2008; Werning- It is likely that Syk is only involved in the haus et al. 2009). subset of signaling pathways that also depend on Src-family kinases. However, this doesn’t Syk and the Inflammasome mean that blockade of Syk kinases always produces the same phenotype in a given immuno- In addition to signaling downstream through receptor signaling response that blockade of CARD9 to NF-kB, Syk-dependent signaling Src-family members does. For example, the events have been linked to activation of intracel- defect in macrophage phagocytosis in Src-fam- lular pattern recognition receptors, which in ily-kinase-deficient cells is different than in Syk- turn activate the inflammasome complex lead- deficient cells. Lack of Src-family kinases pro- ing to IL-1b production. For example, activa- duces a moderate to severe defect in FcR-medi- tion of the Nod-like receptor protein NLRP3 ated particle uptake due to a reduction in initial requires upstream Syk activity during innate actin polymerization at the phagocytic cup (Fit- immune cell responses to fungal molecules zer-Attas et al. 2000). However, Syk-deficient (Gross et al. 2009). Similarly, Syk-deficient den- cells show a complete block in FcR-mediated dritic cells fail to respond to monosodium urate particle uptake due to a block in fusion of the crystals (present in the joints of gout patients), a arms of the phagocytic cup, a step subsequent pathway also known to require NLRP3 (Ng et al. to actin polymerization events (Crowley et al. 2008; Martinon 2010). It remains unclear how 1997). Hence, it is likely that the more proximal Syk activation is coupled to the NLRP3/inflam- Src-family kinases signal to many other path- masome complex. ways besides just ITAM-mediated Syk activa- In contrast to Src-family kinases, it is likely tion. Finally, it should be noted that Syk has that Syk does not signal in cytokine or GPCR- been implicated in some pathways in which linked pathways. Lack of Syk has no impact on the role of Src-family kinases are unknown. neutrophil, macrophage, or mast cell recognition

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of various cytokine growth factors or GPCR This may be useful for when treating cancer, agonists, such as formyl peptides, ATP,or other which is where most of the anti-Src kinase drugs agents (Mocsai et al. 2003). have been used (Kim et al. 2009), but is likely problematic for chronic treatment of immune- mediated disease. Moreover, distinguishing CONCLUSIONS between individual Src-family members may The roles of cytoplasmic tyrosine kinases in be chemically impossible to achieve. Neverthe- innate immune responses are complex, ranging less, given our ever-expanding understanding from direct signaling involvement in very of kinase-mediated signaling pathways in innate defined pathways (such as Syk in C-type lectin cells, it is quite likely that in the very near receptor signaling) to more diffuse interactions future we will see highly active drugs that block (Src-family kinases somehow regulating GPCR these pathways, resulting potentially in clinical responses) to indirect secondary effects (Src- benefit. family and Syk function in immunoreceptor pathways leading to IL-10 production that feeds back on TLR-mediated responses). It is clear ACKNOWLEDGMENTS that Src-family members have the broadest CAL is supported by NIH grants AI065495 and effects on overall signaling, while Syk has AI068150. We thank Clare Abram, Patrizia Sca- more defined roles. This diffuse, versus very pini, and Chrystelle Lamagna for critical read- defined, functional role is mirrored in the fact ing of the manuscript. that deficiencies in single Src-family kinases tend to produce limited signaling defects in innate cells, while loss of Syk has very defined REFERENCES broad functional effects. Because of redun- Abdelbaqi M, Chidlow JH, Matthews KM, Pavlick KP, Bar- dancy, studying the role of Src-kinases in any low SC, Linscott AJ, Grisham MB, Fowler MR, Kevil pathway often requires blocking multiple family CG. 2006. Regulation of dextran sodium sulfate induced members, after which one often finds that many colitis by leukocyte beta 2 integrins. Lab Invest 86: 380– signaling responses are affected. This overall 390. Abram CL, Lowell CA. 2008. The diverse functions of Src broad function for Src-family members is anal- family kinases in macrophages. Front Biosci 13: 4426– ogous to how a rheostat controls lighting: It 4450. dials up and dials down responses in a graded Abram CL, Lowell CA. 2009. The ins and outs of leukocyte fashion. In contrast, Syk acts more like a signal- integrin signaling. Annu Rev Immunol 27: 339–362. Amoras AL, Kanegane H, Miyawaki T, Vilela MM. 2003. ing switch: It is critically required in an absolute Defective Fc-, CR1- and CR3-mediated monocyte phag- way in a limited number of pathways. ocytosis and chemotaxis in common variable immuno- When viewed in this fashion, it becomes deficiency and X-linked agammaglobulinemia patients. obvious that if we are to design therapeutics J Investig Allergol Clin Immunol 13: 181–188. Arefieva TI, Kukhtina NB, Antonova OA, Krasnikova TL. that target these kinases for use in inflammatory 2005. MCP-1-stimulated chemotaxis of monocytic and or autoimmune disease, we are better off focus- endothelial cells is dependent on activation of different ing on the switch kinases (Syk) rather than the signaling cascades. Cytokine 31: 439–446. rheostat kinases (Src-family), since targeting Blasius A, Vermi W, Krug A, Facchetti F, Cella M, Colonna M. 2004. A cell-surface molecule selectively expressed the former will produce defined effects. Indeed, on murine natural interferon-producing cells that blocks a number of companies are close to producing/ secretion of interferon-alpha. Blood 103: 4201–4206. releasing highly active Syk kinase inhibitors that Bourgin-Hierle C, Gobert-Gosse S, Therier J, Grasset MF, Mouchiroud G. 2008. Src-family kinases play an essential have strong potential for treatment of immune- role in differentiation signaling downstream of macro- mediated disease (Cohen and Fleischmann phage colony-stimulating factor receptors mediating 2010; Colonna et al. 2010). In contrast, most persistent phosphorylation of phospholipase C-gamma2 and MAP kinases ERK1 and ERK2. Leukemia 22: 161– of the Src-family inhibitors produced and used 169. clinically so far are rather broadly acting, and Calle Y,Burns S, Thrasher AJ, Jones GE. 2006. The leukocyte hit enzymes besides just Src-family members. podosome. Eur J Cell Biol 85: 151–157.

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Cytoplasmic Tyrosine Kinases in Innate Immune Cells

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C.A. Lowell

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Src-family and Syk Kinases in Activating and Inhibitory Pathways in Innate Immune Cells: Signaling Cross Talk

Clifford A. Lowell

Cold Spring Harb Perspect Biol published online November 10, 2010

Subject Collection Immunoreceptor Signaling

The Coordination of T-cell Function by Perspectives for Computer Modeling in the Study Serine/Threonine Kinases of T Cell Activation David Finlay and Doreen Cantrell Jesse Coward, Ronald N. Germain and Grégoire Altan-Bonnet ITAM-mediated Signaling by the T-Cell Antigen Structural Biology of the T-cell Receptor: Insights Receptor into Receptor Assembly, Ligand Recognition, and Paul E. Love and Sandra M. Hayes Initiation of Signaling Kai W. Wucherpfennig, Etienne Gagnon, Melissa J. Call, et al. Coordination of Receptor Signaling in Multiple Src-family and Syk Kinases in Activating and Hematopoietic Cell Lineages by the Adaptor Inhibitory Pathways in Innate Immune Cells: Protein SLP-76 Signaling Cross Talk Martha S. Jordan and Gary A. Koretzky Clifford A. Lowell The Cytoskeleton Coordinates the Early Events of The LAT Story: A Tale of Cooperativity, B-cell Activation Coordination, and Choreography Naomi E. Harwood and Facundo D. Batista Lakshmi Balagopalan, Nathan P. Coussens, Eilon Sherman, et al. An Enigmatic Tail of CD28 Signaling Antigen Receptor Signaling to NF-κB via Jonathan S. Boomer and Jonathan M. Green CARMA1, BCL10, and MALT1 Margot Thome, Jean Enno Charton, Christiane Pelzer, et al. Mediation of T-Cell Activation by Actin It's All About Change: The Antigen-driven Meshworks Initiation of B-Cell Receptor Signaling Peter Beemiller and Matthew F. Krummel Wanli Liu, Hae Won Sohn, Pavel Tolar, et al. T-Cell Signaling Regulated by the Tec Family ZAP-70: An Essential Kinase in T-cell Signaling Kinase, Itk Haopeng Wang, Theresa A. Kadlecek, Byron B. Amy H. Andreotti, Pamela L. Schwartzberg, Raji E. Au-Yeung, et al. Joseph, et al. For additional articles in this collection, see http://cshperspectives.cshlp.org/cgi/collection/

Lipid Signaling in T-Cell Development and Understanding the Structure and Function of the Function Immunological Synapse Yina H. Huang and Karsten Sauer Michael L. Dustin, Arup K. Chakraborty and Andrey S. Shaw

For additional articles in this collection, see http://cshperspectives.cshlp.org/cgi/collection/