The SYK Tyrosine Kinase: a Crucial Player in Diverse Biological Functions

REVIEWS

The SYK tyrosine kinase: a crucial player in diverse biological functions

Attila Mócsai*, Jürgen Ruland‡ and Victor L. J. Tybulewicz§ Abstract | Spleen tyrosine kinase (SYK) is known to have a crucial role in adaptive immune receptor signalling. However, recent reports indicate that SYK also mediates other, unexpectedly diverse biological functions, including cellular adhesion, innate immune recognition, osteoclast maturation, platelet activation and vascular development. SYK is activated by C‑type lectins and integrins, and activates new targets, including the CARD9–BCL‑10–MALT1 pathway and the NLRP3 inflammasome. Studies using Drosophila melanogaster suggest that there is an evolutionarily ancient origin of SYK‑mediated signalling. Moreover, SYK has a crucial role in autoimmune diseases and haematological malignancies. This Review summarizes our current understanding of the diverse functions of SYK and how this is being translated for therapeutic purposes.

Our understanding of signal transduction in cells of the was due to a defect in lymphatic vascular development3 immune system was greatly advanced during the 1990s provided evidence of a role for SYK beyond the adap‑ when it was shown that B cell receptors (BCRs), T cell tive immune response. The discovery of ITAM‑based receptors (TCRs) and Fc receptors (FcRs) (collectively signalling in Drosophila melanogaster 4 also indicated termed classical immunoreceptors) signal by a conceptu‑ a role for such pathways beyond adaptive immunity ally similar mechanism. All these receptors associate with (insects do not have an adaptive immune system). transmembrane proteins that have cytoplasmic domains Additional studies have revealed that SYK is required containing immunoreceptor tyrosine-based activation motifs for several functions of innate immune cells and for cer‑ (ITAMs)1, which are short peptide sequences with two tain non‑immune functions, such as bone resorption by tyrosine residues that are 6–12 amino acids apart. ITAM osteoclasts5. SYK has also been shown to mediate sig‑ tyrosine residues are rapidly phosphorylated following nalling by classes of receptors, including integrins6 and 7 *Department of Physiology, receptor engagement, leading to the recruitment and C-type lectins , that do not contain conventional ITAMs. Semmelweis University School activation of spleen tyrosine kinase (SYK) or the related Collectively, these studies have dramatically changed of Medicine, 1094 Budapest, protein ζ‑chain‑associated protein kinase of 70 kDa our view of the SYK tyrosine kinase. This Review sum‑ Hungary. ‡Institut für Molekulare (ZAP70). This signalling model soon entered immu‑ marizes our current understanding of the diverse roles Immunologie, III Medizinische nology textbooks and became the central paradigm of of SYK, in both immune and non‑immune biological Klinik, Klinikum rechts der immune cell signalling. responses, and describes how this knowledge is being Isar, Technische Universität SYK is a 72 kDa non‑receptor tyrosine kinase that translated for the therapy of human diseases. München, 81675 Munich, contains two SRC homology 2 (SH2) domains and and Laboratory of Signalling (BOX 1) in the Immune System, a kinase domain and is most highly expressed ITAM- and hemITAM-based signalling pathways Helmholtz Zentrum München by haematopoietic cells. Mammals also express a SYK Classical immunoreceptors (BCRs, TCRs and vari‑ (German Research Center homologue, ZAP70, which is mostly restricted to T cells ous FcRs) mediate, either directly or indirectly, the for Environmental Health), and natural killer (NK) cells. SYK‑related kinases are also adaptive recognition of self and foreign antigens. 85764 Neuherberg, Germany. found in invertebrates. Further information about SYK Immunoreceptor‑associated transmembrane adaptors §Division of Immune Cell and SYK‑related molecules can be found in BOX 1. or, in the case of FcγRIIA, the ligand binding receptor Biology, MRC National The SYK signalling pathway was initially thought to chain itself contain one or more ITAMs (FIG. 1a), and Institute for Medical Research, be restricted to classical immunoreceptors of the adap‑ receptor ligation leads to the phosphorylation of these London NW7 1AA, UK. tive immune response. However, later studies showing ITAMs, primarily by members of the SRC tyrosine Correspondence to A.M. glycoprotein VI e‑mail: [email protected] that (GPVI), a collagen receptor expressed kinase family. Through interaction with their tandem 2 doi:10.1038/nri2765 by platelets, also signals by a similar mechanism and that SH2 domains, dual‑phosphorylated ITAMs recruit SYK, Published online 14 May 2010 the petechiated appearance of SYK‑deficient embryos in the case of the BCR and FcR, or ZAP70, in the case of

Box 1 | Facts and figures on SYK and SYK-related molecules Spleen tyrosine kinase (SYK) contains two tandem SRC homology 2 (SH2) domains and a carboxy‑terminal tyrosine Immunoreceptor tyrosine- kinase domain (see the figure). These domains are linked by two linker regions: interdomain A between the two SH2 based activation motif domains and interdomain B between the C‑terminal SH2 domain and the kinase domain. An alternatively spliced form (ITAM). A short peptide motif of SYK (known as SYK‑B) lacks 23 amino acids of interdomain B, including a nuclear localization signal9,185. containing two tyrosine SYK is highly expressed by all haematopoietic lineage cells. It was initially thought to be a haematopoietic residues that is found in the cell‑specific kinase; however, later studies showed its expression in other tissues as well145. Although the expression cytoplasmic tail of several of SYK is tightly regulated30, the mechanism of this regulation, and that leading to the generation of the SYK‑B signalling adaptor proteins and is necessary to recruit proteins isoform, is not well understood. that are involved in triggering Mammals also express the SYK‑related molecule ζ‑chain‑associated protein kinase of 70 kDa (ZAP70), the activating signalling proteins. expression of which is mostly confined to the T and natural killer (NK) cell lineages. Readers interested in further The consensus sequence is details of ZAP70 biology are referred to an excellent recent review36.

Tyr-X-X-(Leu/Ile)-X6–12-Tyr-X-X- SYK family kinases have an evolutionarily ancient origin but (Leu/Ile), where X denotes any they have appeared and disappeared during the evolution Interdomain A Interdomain B amino acid. of animals186. Drosophila melanogaster express a single SYK SYK‑related molecule, SH2 domain ankyrin repeat kinase SH2 SH2 Kinase Glycoprotein VI (629 amino acids) (SHARK)4, which contains ankyrin‑like (ANK) repeats (GPVI). One of several Alternative splicing collagen receptors expressed between its two SH2 domains. Interestingly, hydras and SYK-B sponges express two SYK‑related kinases, one (known as SH2 SH2 Kinase on platelets. GPVI is a (606 amino acids) transmembrane glycoprotein HTK98) is similar to vertebrate SYK and the other (known that is closely related to Fcα as HTK16) is more closely related to D. melanogaster ZAP70 receptors (FcαRs). It associates SHARK186,187. By contrast, the Caenorhabditis elegans genome SH2 SH2 Kinase (618 amino acids) with, and signals through, the does not contain any SYK‑related tyrosine kinases188. ITAM-containing adaptor FcR The Syk gene has been disrupted in mice by two Hydra HTK98 γ-chain. 23,24 SH2 SH2 Kinase independent groups . The major phenotypes displayed (624 amino acids) by SYK‑deficient mice are perinatal lethality, a petechiated Petechia ANK in utero appearance and the lack of mature B cells. A small red spot on the skin Drosophila SHARK SH2 SH2 Kinase that results from localized Two groups have also reported mutations allowing the (939 amino acids) 189,190 bleeding owing to broken Cre‑mediated conditional deletion of Syk . To the blood vessels. A large number best of our knowledge, no human patients or animal Hydra HTK16 SH2 SH2 Kinase of petechiae usually indicates strains with a spontaneous germline mutation in SYK (757 amino acids) a platelet defect. Note that, have been described. in contrast to the original assumptions, the red spots of SYK-deficient embryos are not Nature Reviews | Immunology bona fide petechiae as they are the TCR, and this triggers kinase activation and down‑ ligation of classical immunoreceptors also trig‑ caused by a vascular stream signalling. It should be noted that ZAP70 is more gers pro‑inflammatory transcriptional programmes, developmental defect. dependent on the SRC family kinases than SYK, prob‑ through activation of nuclear factor‑κB (NF‑κB).

Osteoclasts ably because SYK can phosphorylate the ITAM tyrosine Recent studies have revealed that ITAM‑based sig‑ 8,9 Large multinucleated cells residues itself . nalling events lead to caspase‑recruitment domain that arise from macrophage (CARD)‑mediated heterotypic aggregation of CARD9 precursors and are specialized Signalling downstream of SYK and ZAP70. Of the sev‑ or CARMA1 (also known as CARD11) with the adap‑ for bone resorption. Once eral intermediate molecules implicated in relaying SYK‑ tor protein B cell lymphoma 10 (BCl‑10) and the firmly adhered to the bone paracaspase surface, osteoclasts release or ZAP70‑mediated downstream signalling, VAV family associated mucosa‑associated lymphoid 11–16 hypochlorous acid and members, phospholipase Cγ (PlCγ) isoforms, the regu‑ tissue lymphoma translocation protein 1 (MAlT1) , hydrolytic enzymes to latory subunits of phosphoinositide 3‑kinases (PI3Ks) triggering activation of the NF‑κB pathway. whereas digest the bone tissue. and the SH2 domain‑containing leukocyte protein (SlP) CARMA1 relays antigen receptor‑mediated activation

C-type lectins family members SlP76 (also known as lCP2) and SlP65 of the BCl‑10–MAlT1 complex in B cells, T cells, Carbohydrate-recognizing (also known as BlNK) can directly associate with SYK NK cells and mast cells, CARD9 is responsible for proteins (lectins) that require (FIG. 1b) and/or ZAP70. These molecules probably partici‑ BCl‑10–MAlT1 complex activation in macrophages Ca2+ ions for their proper pate in forming receptor‑proximal signalling complexes and dendritic cells (DCs)11–17. The role of this pathway function. A characteristic and trigger downstream processes including Ca2+ and pro‑ in fungal recognition will be discussed below; further feature of their structure is a carbohydrate recognition tein kinase C (PKC) signalling, RAS homologue (RHO) details of CARMA1 and CARD9 signalling can be 18 domain. C-type lectins include family‑ and protein tyrosine kinase 2 (PYK2)‑mediated found in an excellent recent review . soluble molecules such as cytoskeletal rearrangement, reactive oxygen species (ROS) the complement-activating production and phagocytosis, PI3K‑mediated TeC family Negative regulation of SYK function. The action of mannose binding lectin, as well as cell surface receptors and AKT signalling pathways, as well as transcriptional SYK is often counteracted by phosphatases such as SH2 such as various CLEC proteins. regulation through the RAS–extracellular signal‑regulated domain‑containing protein tyrosine phosphatase 1 kinase (eRK) and Ca2+–nuclear factor of activated T cells (SHP1; also known as PTPN6), and it is the balance of Paracaspase (NFAT) pathways (FIG. 1b). Although the transmembrane SYK and SHP1 activities that determines the ultimate A group of proteolytic enzymes adaptor protein linker for activation of T cells (lAT) is an signalling output19. The expression of SYK is also nega‑ distinct from, but closely E3 ubiquitin ligase related to, the caspases. The important component of TCR and FcεRI signalling, it is at tively regulated by the Casitas B‑lineage most important mammalian present unclear whether any lAT‑related molecules have lymphoma (CBl), leading to ubiquitylation and paracaspase is MALT1. similar roles in other lineages10. degradation of SYK20–22.

a ITAM in adaptor chain ITAM in receptor tail HemITAM in receptor tail For example, BCR, FcγRIIA CLEC2, CLEC7A or CLEC9A TCR, FcεRI and CLEC4E

Receptor Transmembrane Plasma adaptor membrane

ITAM or ITAM hemITAM hemITAM C-terminus P P P P P P N-terminus SH2 SH2

SYK or ZAP70 Kinase SYK or ZAP70 SYKSYK b

ITAM

P P

SRC family kinases

SYK

Direct binding SLP76 or VAV PLCγ PI3K partners of SYK SLP65

E3 ubiquitin ligase CARD–BCL-10– p38 JNK RHO family Ca2+ DAG TEC family LAT An enzyme that is required MALT1 complex to attach the molecular tag Other signalling ubiquitin to proteins. intermediates NLRP3 PYK2 NFAT PKC RASGRP ERK AKT NF-κB Depending on the position and inflammasome number of ubiquitin molecules that are attached, the ubiquitin Cytoskeletal ROS Cytokine tag can target proteins for Cellular responses Differentiation Proliferation Survival changes production release degradation by the proteasome, sort them to specific subcellular Figure 1 | general mechanism of SyK-mediated signalling. a | Recruitment of spleen tyrosine kinase (SYK) or compartments or modify ζ‑chain‑associated protein kinase of 70 kDa (ZAP70) to plasma membrane receptors occurs through binding of the tandem their biological activity. SH2 domains of SYK or ZAP70 to two phosphorylated tyrosine residues in the receptor complex. NaThetur twoe Re phosphorylatedviews | Immunolog y tyrosine residues are either in a single immunoreceptor tyrosine‑based activation motif (ITAM) or in two hemITAMs on two Pro-B cells separate receptor peptide chains. The ITAMs are either present in receptor‑associated transmembrane adaptors or in the Cells in the earliest stage of cytoplasmic tail of the receptor chain itself. b | The general scheme of signal transduction through SYK is shown. Signal B cell development in the bone transduction is mostly initiated by phosphorylation of ITAM tyrosine residues by SRC family kinases. Recruitment of SYK to dually marrow. They are characterized by incomplete immunoglobulin phosphorylated ITAMs triggers the activation of SYK and its direct binding to members of the VAV and phospholipase Cγ (PLCγ) heavy-chain rearrangements families, the p85α subunit of phosphoinositide 3‑kinase (PI3K), as well as SH2 domain‑containing leukocyte protein 76 and are defined as (SLP76) and SLP65. These direct binding partners activate downstream signalling components, which eventually trigger CD19+cytoplasmic IgM– or various cellular responses. The SYK‑mediated signalling pathways are also regulated by several feedback mechanisms, sometimes as B220+CD43+ such as the phosphorylation of ITAM tyrosine residues by SYK or the regulation of direct SYK binding partners by further (by the Hardy classification downstream molecules. SYK activation by hemITAM‑containing receptors probably proceeds through similar scheme). mechanisms. BCR, B cell receptor; BCL‑10, B cell lymphoma 10; CARD, caspase‑recruitment domain; CLEC, C‑type lectin; DAG, diacylglycerol; ERK, extracellular signal‑regulated kinase; FcR, Fc receptor; JNK, Janus kinase; LAT, linker for activation of Pre-B cells T cells; MALT1, mucosa‑associated lymphoid tissue lymphoma translocation protein 1; NF‑ B, nuclear factor‑ B; NFAT, nuclear Cells in a stage of B cell κ κ development in the bone factor of activated T cells; NLRP3, NLR family, pyrin domain‑containing 3; PKC, protein kinase C; PYK2, protein tyrosine kinase 2; marrow that are characterized RASGRP, RAS guanyl‑releasing protein; RHO, RAS homologue; ROS, reactive oxygen species; TCR, T cell receptor. by complete immunoglobulin heavy-chain rearrangement in the absence of immunoglobulin The role of SYK and ZAP70 in leukocyte function. As defects in T cell development25–27. More detailed studies light-chain rearrangement. B cell and T cell development require signal transduc‑ have revealed that SYK has an important, although not They express the pre-B cell pro-B cells receptor, which comprises a tion by properly rearranged antigen receptors, SYK defi‑ indispensable, role in the transition of into 23,24 pseudo light chain and a ciency leads to complete absence of mature B cells , pre-B cells, and is essential for immature B cells to pass heavy chain. and ZAP70‑deficient humans and mice have severe a recently described RAC‑dependent checkpoint that

controls their entry into the splenic white pulp (termed Structural basis of SYK function transitional type 0 stage)23,28. Signalling by the BCR and Three different SYK activity states. Immunoreceptor TCR was originally thought to be specifically mediated signalling through SYK requires the kinase activ‑ by SYK and ZAP70, respectively. unexpectedly, however, ity of SYK, as well as both SH2 domains. The kinase ZAP70 was recently found to be expressed throughout domain of SYK is inactive in the resting state of the B cell development and to participate in pre-BCR signal‑ protein but can be activated by binding of both SH2 ling, and therefore also in allelic exclusion and transi‑ domains to dually phosphorylated ITAMs (FIG. 2b). Transitional type 0 stage tion from the pro‑B cell stage to the pre‑B cell stage29. Phosphorylation of tyrosine residues in the linker Transitional B cells are recent emigrants from the bone Similarly, SYK was shown to have an important role in regions (interdomain A or B) also results in kinase marrow that are in the process pre-TCR signalling, which occurs during the transition activation even in the absence of phosphorylated of maturing into follicular and from the double negative 3 (DN3) to the DN4 stage ITAM binding (see below). This observation led to marginal zone B cells. They can of early thymocyte development30. Hence, SYK and the proposal that SYK functions as an ‘OR’ logic gate be found in the spleen and ZAP70 have overlapping functions in early lymphocyte switch47 (FIG. 2b). The physiological relevance of this typically have a half-life of a few days. Transitional B cells development. activation is discussed below. have been subdivided into SYK is also required for FcεRI signalling in mast subsets type 0 to type 3 based cells31 and FcγR signalling in neutrophils and macro‑ Autoinhibition of the kinase domain. Although no high on expression of IgM, IgD, phages32,33, whereas FcγR‑mediated antibody-dependent resolution three‑dimensional structure of the entire SYK CD93 and CD23. Type 0 B cells cell-mediated cytotoxicity are the earliest migrants from (ADCC) reactions are defective protein is available, the structure for the related protein the bone marrow and are in NK cells that lack both SYK and ZAP70 (REF. 34). The ZAP70 indicates that the kinase adopts an autoinhibited located in the red pulp of the reader is referred to excellent reviews on further aspects conformation in the absence of phosphorylated ITAM spleen. of these signal transduction processes9,35–38. binding48,49. In this structure, binding between inter‑ domain A, interdomain B and parts of the kinase domain Pre-BCR (Pre-B cell receptor). A ITAM-mediated ‘tonic’ signalling. ITAM‑mediated hold the catalytic centre in an inactive conformation. receptor that is formed at the signalling was initially thought to occur only following engagement of the SH2 domains by a phosphorylated surface of pre-B cells by ligand‑induced receptor cross‑linking. However, recent ITAM disassembles this tripartite binding, allowing the the pairing of rearranged studies revealed that the disruption of BCR signalling kinase domain to adopt an active conformation. immunoglobulin heavy chains with surrogate light chains and by inactivation of the ITAM‑bearing Igα chain leads Several studies indicate that, analogous to ZAP70, 39 (FIG. 2b) the heterodimer of Igα and Igβ. to a rapid loss of resting mature B cells . These studies SYK is also regulated by similar autoinhibition . Signalling by the pre-BCR indicated that the maintenance of mature B cells requires A low resolution three‑dimensional structure of SYK occurs in the absence of known ITAM‑dependent constitutive (‘tonic’) signalling, seem‑ shows that SYK has a compact structure, similar to auto‑ ligands and is a crucial event in ingly in the absence of a BCR ligand. It has yet to be tested inhibited ZAP70 (REF. 50). Deletion of both SH2 domains, B cell development. whether this tonic signalling requires SYK, although or of interdomain A alone51,52, as well as binding of a 52 Pre-TCR the effect of SYK inhibitors on BCR‑expressing B cell phosphorylated ITAM to the SH2 domains , results in (Pre-T cell receptor). A cell lymphomas suggests that this is the case (see below). SYK activation, consistent with it being relieved from an surface receptor complex Recent studies have also revealed antigen‑independent autoinhibited conformation. consisting of TCRβ, pre-TCRα 40,41 and CD3 that is expressed by signal transduction by FcεRI on mast cells . As these immature CD4–CD8– responses require receptor aggregation and SYK, they Interaction of SYK with phosphorylated ITAMs. thymocytes. Signalling through seem to conform to conventional ITAM–SYK‑mediated Binding of the SH2 domains of SYK to phosphorylated this complex is essential for signalling. ITAMs is a crucial step in SYK activation and down‑ + + maturation to the CD4 CD8 stream signalling. A high‑resolution three‑dimensional stage. Signalling by hemITAM-containing C-type lectins. It has structure of the SH2 domains of SYK in complex with Antibody-dependent long been thought that the two phosphorylated tyrosine the doubly phosphorylated ITAM of CD3ε showed cell-mediated cytotoxicity residues have to reside in a single peptide chain (as in that the amino‑terminal SH2 domain binds to the (ADCC). A cytotoxic a classical ITAM) to recruit and activate SYK. However, a carboxy‑terminal phosphorylated tyrosine of the ITAM mechanism by which an 53 (FIG. 1a) antibody-coated target cell is new family of C‑type lectins, including the fungal rec‑ and vice versa . The structure shows substan‑ 53,54 directly killed by a leukocyte ognition receptor CleC7A (also known as dectin 1) or tial flexibility , which might account for the ability of that expresses Fc receptors, the platelet receptor CleC2 (also known as CleC1B) SYK, in contrast to ZAP70, to bind to phosphorylated such as a natural killer cell, can activate SYK even though these receptors only have ITAMs with a broad range of spacing between the macrophage or neutrophil. a single Tyr‑X‑X‑leu motif (termed ‘hemITAM’) in their phosphory lated tyrosine residues (including the long‑ 42,43 55 HemITAM cytoplasmic tail . Further studies provide support spaced FcγRIIA ITAM ) and to bridge two hemITAMs 43–45 A peptide motif in the for the role of the tyrosine residue in the Tyr‑X‑X‑leu in C‑type lectins (FIG. 1a). cytoplasmic tail of various motif, as well as both SYK SH2 domains, in signal trans‑ C-type lectins, characterized by duction43,44, which suggests that hemITAM‑containing The role of phosphorylated tyrosines in SYK. There are the presence of a single Tyr-X-X-Leu sequence (in C‑type lectins activate SYK by dimerization followed by ten tyrosine residues in SYK that are sites of autophos‑ 56 contrast to the two such engagement of the two SH2 domains of SYK by phos‑ phorylation (FIG. 2a), most of which have been shown to sequences present in phorylated hemITAMs on two separate receptor chains participate in SYK‑mediated signal transduction. Amino consensus ITAMs). (FIG. 1a). evidence for this stoichiometry has recently acids are numbered as in mouse SYK. Phosphorylation of the been provided for the CleC2–SYK interaction45. The BCR activation triggers phosphorylation of Tyr130 tyrosine residue in the 56 hemITAMs leads to binding to functional role of SYK in signalling by C‑type lectins is in interdomain A , leading to the dissociation of SYK 57,58 the SYK SH2 domains in a 2/1 discussed below; other aspects of these receptors can be from the phosphorylated ITAMs of the BCR complex stoichiometry. found in excellent recent reviews7,46. (FIG. 2c). Mutation of Tyr130 to the phosphomimetic

a VAV1 glutamate also decreases ITAM binding and results in SLP65 CBL p85α increased kinase activity, further supporting an inhibitory p85α PLCγ role for interdomain A57. 20 24 25 r519 r5 r6 Phosphorylated Tyr317 is a binding site for the e3 2 Ty Ty Ty Tyr6 r130 r346 ubiquitin ligase CBl (FIG. 2a), which is involved in the Ty Tyr290 Tyr317 Tyr34 Ty Tyr358 SYK SH2 SH2 Kinase ubiquitylation and degradation of SYK20,21. Accordingly, mutation of this residue results in enhanced FcεRI‑ and Interdomain A Interdomain B BCR‑mediated signalling59,60. Phosphorylated Tyr317 also binds to the C‑terminal SH2 domain of the PI3K regula‑ b Plasma membrane tory subunit p85α, whereas the N‑terminal SH2 domain of p85α binds phosphorylated Tyr342 and phosphorylated ITAM Tyr346 (REF. 61) (FIGS 2a,c). Mutation of all three tyro‑ Interdomain A P P sine residues abrogates FcεRI‑induced AKT activation, SH2 SH2 62 Tyr317 P P consistent with an inability to activate PI3K . Interdomain B Tyr342 P Tyr130 In the ZAP70 structure, Tyr315 and Tyr319 in inter‑ Kinase Tyr346 P domain B and Tyr597 and Tyr598 in the kinase domain stabilize the autoinhibited conformation48. Mutation of Resting Binding to Phosphorylation these residues in ZAP70 or of the analogous residues state phosphorylated of linker tyrosines ITAM in SYK (Tyr342, Tyr346, Tyr624 and Tyr625) results in kinase activation, further indicating that SYK adopts an autoinhibited conformation63–65. All four residues Kinase inactive Kinase active Kinase active 56 (autoinhibited) are phosphorylated following BCR stimulation , prob‑ Logic ‘OR’ ably contributing to kinase activation by destabilizing relationship the autoinhibited conformation of SYK (FIG. 2b), as well as providing docking sites for other signalling mol‑ c ecules that stabilize the active conformation and initiate downstream signalling (FIG. 2c). P ITAM ITAM ITAM P Phosphorylated Tyr342 binds to the SH2 domain of P P P P P P VAV1 and both phosphorylated Tyr342 and phospho‑ PLCγ P P rylated Tyr346 bind to the C‑terminal SH2 domain of SRC family P kinases SYK PlCγ1 (REFS 66,67) (FIGS 2a,c). Interestingly, binding of PlCγ1 is mediated by two phosphorylated tyrosine 68 P P residues binding to one SH2 domain . Mutation of P PI3K P these residues inhibits FcεRI signalling in mast cells62,69 P P P P P VAV P P and phenocopies the cytoskeletal changes observed in P 70 Inactive SYK P P SYK‑deficient neutrophils . SLP65 when phosphorylated, Tyr624 binds to the SH2 domain of SlP65 (REF. 71) (FIGS 2a,c). This binding acti‑ Active SYK ITAM vates the kinase, which is consistent with the putative phosphorylation Autophosphorylation ↓ ↓ role of Tyr624 in stabilizing the autoinhibitory confor‑ Amplification of Sustained activation mation. This SlP65–SYK association is also important initial activation for BCR signalling and B cell development71. Although no direct interaction between Tyr624 and the SlP65‑ Figure 2 | Structural basis of SyK activation. a | The domain structure of spleen related adaptor SlP76 has been reported, such a direct tyrosine kinase (SYK) with tyrosine residues shown to be sites of autophosphorylation. Proteins shown to bind to phosphorylated tyrosines are indicated above the tyrosine functional association would not be surprising given the 3,23,24,33,72,73 residues (p85 represents the 85 kDa phosphoinositide 3‑kinase (PI3K) regulatory strikingly similar phenotypes of SYK‑deficient α Nature Reviews | Immunology 3,74,75 subunit). b | There are three different states of SYK activation. SYK is autoinhibited in its and SlP76‑deficient mice . resting state owing to the binding of interdomain A and interdomain B to the kinase domain, in particular, the carboxy‑terminal end. This autoinhibited conformation Integration of SYK activation. The most likely physiologi‑ can be suspended by binding of the two SH2 domains to dually phosphorylated cal relevance of the dual activation of SYK either by ITAM immunoreceptor tyrosine‑based activation motifs (ITAMs) or by phosphorylation of binding or by phosphorylation of tyrosine residues in the tyrosine residues in interdomain A or B. There is a logic ‘OR’ relationship between those linker regions is that initial ITAM binding triggers rapid two activation mechanisms. c | Prior to activation, SYK is held in an autoinhibited activation of SYK, whereas subsequent phosphorylation conformation by interactions between interdomains A and B and the kinase domain. leads to prolonged activation and downstream signalling Initial phosphorylation of ITAM tyrosine residues by SRC family kinases provides docking sites for the SYK SH2 domains inducing conformational changes and kinase activation. even in the absence of binding to phosphorylated ITAMs SYK activation results in autophosphorylation on several residues, leading to release (FIG. 2c). Importantly, SYK can catalyse the autophosphory‑ from the ITAM and sustained SYK activation. SYK then recruits direct binding partners to lation of its linker region tyrosine residues, leading to its phosphorylated tyrosine residues, triggering downstream signalling. Phosphorylation sustained SYK activation after transient ITAM phosphory‑ of ITAM tyrosines by SYK provides a positive feedback loop. Amino acid numbers in this lation. In addition, SYK itself can phosphorylate ITAMs, figure correspond to the sequence of mouse SYK. CBL, Casitas B‑lineage lymphoma; suggesting the existence of a positive feedback loop PLCγ, phospholipase Cγ; SLP65, SH2 domain‑containing leukocyte protein of 65 kDa. during initial ITAM‑mediated SYK activation52 (FIG. 2c).

The role of SYK in cell adhesion a role for SYK in signal transduction by P‑selectin glyco‑ Integrin signal transduction. Integrins are a family protein ligand 1 (PSGl1), the major selectin receptor of heterodimeric transmembrane receptors that par‑ on leukocytes. SYK is activated by, and associates with, ticipate in leukocyte adhesion and migration (through PSGl1 in leukocytes89,90. Abrogation of SYK activity lymphocyte function‑associated antigen 1 (lFA1; also inhibits PSGl1‑mediated transcriptional regulation89, known as αlβ2 integrin) and macrophage receptor 1 whereas SYK‑deficient neutrophils show a defect in (MAC1; also known as αMβ2 integrin)), platelet acti‑ slow rolling, suggesting that SYK is involved in PSGl1‑ vation (through αIIbβ3 integrin) and osteoclast devel‑ induced inside-out signalling that activates lFA1 (REF. 90) opment and function (through αVβ3 integrin). Given (FIG. 3d). Both studies concluded that PSGl1‑induced their structural and functional differences, integrins and SYK activation proceeds through an ITAM‑mediated classical immunoreceptors were long thought to signal pathway89,91. However, one report suggested that SYK is by conceptually different mechanisms. activated through the ITAM‑like motif‑containing ezrin, evidence for the use of similar signalling mechanisms radixin and moesin (eRM) family proteins89, whereas the by integrins and immunoreceptors came from studies other study suggested that phosphorylation of the ITAM‑ showing defective integrin‑mediated signalling in SYK‑ bearing adaptors DAP12 and FcRγ by the SRC family Phosphomimetic deficient neutrophils72, monocytes, macrophages76,77, kinase FGR is involved91 (FIG. 3d). An amino acid of which the platelets73 and osteoclasts78. Further in vivo studies conformation resembles that of The role of SYK in innate pathogen recognition a phosphate residue, or that showed that SYK is necessary for firm leukocyte adhe‑ 79 of the phosphorylated form of sion to the inflamed endothelium and the development The innate immune system uses germline‑encoded pat‑ another amino acid. Owing to of a MAC1‑dependent vasculopathy reaction80. tern recognition receptors (PRRs) to detect pathogen‑ their negative charge, the most The mechanism of SYK activation by integrins has associated molecular patterns (PAMPs) and activate important phosphomimetic long been debated, mainly because initial studies in heter- immune responses. Recently, SYK has been found to be amino acids are glutamate and ologous expression systems (FIG. 4a) aspartate. indicated that αIIbβ3 integrin a key component of these pathways . activates SYK by an ITAM‑independent mechanism Vasculopathy through direct association between the cytoplasmic tail of Recognition of fungi. The first insights into SYK‑ A disorder of blood vessels, the integrin β‑chain and the non‑ITAM‑binding surfaces dependent PRR signalling came from studies of the C‑type such as inflammation of the 81–83 (FIG. 3a) vessel wall. of the N‑terminal SH2 domain of SYK . lectin CleC7A, an important mammalian PRR for fun‑ More recent analyses of primary cells (rather than gal β‑glucans43 with a hemITAM in its intra cellular tail Heterologous expression heterologous expression systems) came to a differ‑ (FIG. 4a). Agonist binding to CleC7A triggers hemITAM systems ent conclusion. Structure–function studies indicated a phosphorylation, resulting in direct recruitment of SYK An experimental approach crucial role of phosphorylated tyrosine binding by the to CleC7A. As with other hemITAM‑containing C‑type that relies on forced expression of a protein in cells (usually SH2 domains of SYK for integrin‑mediated functions in lectins, CleC7A‑mediated SYK activation requires a 77,84 long-propagated cell lines). neutrophils, macrophages and platelets . The ITAM‑ single tyrosine residue in a Tyr‑X‑X‑leu motif. It is rea‑ The approach is powerful but containing proteins that link β2 integrin signalling to sonable to assume that two hemITAM‑phosphorylated can lead to artefactual results SYK activation in neutrophils and macrophages have CleC7A molecules engage the tandem SH2 domains owing to the expression of 7 proteins in a ‘unnatural’ been identified as the transmembrane adaptor molecules of a single SYK molecule in a 2/1 stoichiometry , as has environment. DAP12 (also known as TYROBP) and the FcR γ‑chain recently been shown for the CleC7A‑related receptor (FcRγ)77 (FIG. 3b). later studies indicated that integrin CleC2 in platelets45. Slow rolling signalling in osteoclasts85, DCs86, microglia87 and plate‑ SYK‑mediated signalling following CleC7A ligation The reduction of leukocyte lets88 also require ITAM‑containing transmembrane triggers ROS production92,93 and phagocytosis of yeast rolling velocity in an 43 inflammatory environment. adaptors. However, the coupling between integrins particles by DCs . Moreover, SYK couples PAMP rec‑ Under normal conditions, and the ITAM‑containing adaptor molecules is poorly ognition to de novo gene transcription and induces leukocytes roll on the surface understood, despite preliminary studies77 suggesting the chemokine and cytokine production through several of endothelial cells at a speed involvement of as yet unidentified DAP12‑associated downstream pathways, including the eRK signal‑ of ~40 m per second, mainly μ (FIG. 3b) determined by the low level molecules . ling cascade and activation of transcription factors 81,84 94 11,43,93,95–97 expression of endothelial Importantly, the ITAM‑independent and ITAM‑ such as NFAT and NF‑κB . Although the P-selectins. At sites of mediated77,84–88 mechanisms of integrin–SYK coupling are precise mechanisms of SYK‑mediated phagocytosis inflammation, E-selectins are not mutually exclusive. Indeed, the most likely scenario and ROS production are not well understood, the expressed at high densities on is that the two mechanisms jointly regulate the activity CleC7A–SYK‑induced NF‑κB response is transduced the endothelial surface, leading 6,85 (FIG. 3c) to slowing down of the rolling of SYK , allowing the fine control of adhesive through CARD9, which engages BCl‑10 and MAlT1 cells to ~5 μm per second. interactions of haematopoietic lineage cells. It should also (REFS 11,98,99) (FIG. 4b). Consistent with an essential be noted that whereas SYK is crucial for several integrin‑ role of the CleC7A–SYK–CARD9 axis for anti fungal Inside-out signalling mediated functions, it makes a limited contribution to host defence, CARD9‑deficient mice and humans are The process by which 70,72 11,100 intracellular signalling β2 integrin‑mediated migration of neutrophils . highly susceptible to fungal infections . At present, mechanisms result in the it is unclear how SYK activates CARD9, but this mecha‑ activation of a cell surface Leukocyte selectin functions. Selectins are transmem‑ nism probably differs from the PKC‑mediated activa‑ receptor, such as integrins. brane glycoproteins that are involved in leukocyte roll‑ tion of CARMA1 in lymphocytes as the crucial PKC By contrast, outside-in ing on the endothelium. endothelial P‑selectins mediate phosphory lation sites of CARMA1 are not conserved signalling is the process by slow rolling (REF. 18) which ligation of a cell surface steady‑state leukocyte rolling, whereas in an in CARD9 . Moreover, the specific role of SYK‑ receptor activates signalling inflammatory environment is triggered by endothelial triggered eRK or NFAT activation in antifungal immunity pathways inside the cell. e‑selectins. Recent evidence from two studies89,90 indicates has not yet been defined.

T helper 17 (T 17) cell Although the CleC7A–SYK–CARD9 pathway is and CleC4e associate with and signal through the H 103,105 A subset of CD4+ T helper cells a PRR pathway that can couple innate immunity to ITAM‑containing adaptor protein FcRγ (FIGS 4a,b). that produce IL-17 and that adaptive immunity, it induces a distinct pattern of DC Both CleC6A–SYK and CleC4e–SYK signalling are thought to be important in cytokines with particularly robust interleukin‑2 (Il‑2), induce CARD9‑ and NF‑κB‑mediated pro‑inflamma‑ inflammatory and autoimmune 96 diseases, as well as host Il‑10 and Il‑23 production , leading to the initiation of tory responses, which promote adaptive immunity and T helper 17 (T 17) cell 96,101 102,106,107 defence against certain potent H responses . robust TH17 cell responses . infectious pathogens. Their SYK‑deficient DCs have a greater block in Il‑2 or generation involves IL-23 and Il‑10 synthesis following yeast stimulation than those Inflammasome activation. Il‑1β is a SYK‑dependent IL-21, as well as the lacking CleC7A, suggesting redundancy at the recep‑ cytokine with an essential role in antifungal immunity93. transcription factors ROR t γ 96 (retinoic acid receptor-related tor level . Indeed, two additional SYK‑coupled PRRs for Production of mature Il‑1β requires NF‑κB‑mediated orphan receptor-γt) and STAT3 fungi, CleC6A (also known as dectin 2) and CleC4e transcription of pro‑Il‑1β and proteolytic processing of (signal transducer and activator (also known as MINCle), were recently identified102–105. pro‑Il‑1β by caspase 1‑containing multiprotein complexes of transcription 3). Instead of containing a cytoplasmic hemITAM, CleC6A termed inflammasomes108. live yeast cells specifically activate Inflammasome A molecular complex of several a ITAM-independent signalling model b ITAM-mediated signalling model c Combined signalling model proteins that following Integrin ligand assembly leads to caspase 1- mediated cleavage of ITAM-associated precursor proteins for IL-1β Integrin Integrin receptor (?) and IL-18, thereby producing β-chain α-chain the mature active cytokines. Plasma DAP12 membrane or FcRγ

SRC family kinases ITAM P P P P

SRC family kinases SYK SYKSYK

Downstream Downstream Downstream signalling signalling signalling

d ITAM-associated receptor (?) Inactive LFA1 PSGL1

DAP12 Active LFA1 or FcRγ

FGR ERM family proteins P P P P

Integrin inside-out SYK SYK activation

Transcriptional regulation Figure 3 | The role of SyK in integrin and PSgl1 signalling. a | In the immunoreceptor tyrosine‑based activation motif (ITAM)‑independent model, integrins activate spleen tyrosine kinase (SYK) without phosphorylated tyrosine binding to the SYK SH2 domains. The interaction is mediated by the intracellular domain of the integrinNa βtur‑chaine Revie andws the| Immunolog y non‑phosphorylated tyrosine‑binding surface of the amino‑terminal SH2 domain of SYK. b | In the ITAM‑mediated model, integrin ligation triggers SRC family kinase‑mediated phosphorylation of ITAM‑bearing transmembrane adaptor molecules (DAP12 and Fc receptor γ‑chain (FcRγ)), leading to the recruitment of SYK through its tandem SH2 domains. This signalling probably involves DAP12‑ and/or FcRγ‑associated transmembrane receptors. c | In the combined model, the non‑phosphorylated tyrosine‑binding surface of the SYK SH2 domains bind to the carboxy‑terminal end of the integrin β‑chain, whereas the two SH2 domains bind to the phosphorylated ITAM tyrosine residues. d | Ligation of leukocyte P‑selectin glycoprotein ligand 1 (PSGL1) triggers phosphorylation of the ITAM tyrosine residues of ITAM‑like motif‑containing ezrin, radixin and moesin (ERM) family proteins, which recruit SYK through its tandem SH2 domains. A supposedly parallel mechanism under flow conditions leads to phosphorylation of the ITAM tyrosine residues of DAP12 and FcRγ by the tyrosine kinase FGR, leading to the recruitment of SYK through its tandem SH2 domains. SYK activation triggers transcriptional changes and inside‑out activation of lymphocyte function‑associated antigen 1 (LFA1).

Zymosan the NlR family, pyrin domain‑containing 3 (NlRP3) The NlPR3 inflammasome is activated in response 93 108 A protein–carbohydrate inflammasome . SYK controls both pro‑Il‑1β synthesis to various distinct danger signals . ROS production, complex prepared from yeast and NlRP3 activation in response to fungal infection93. K+ efflux and, sometimes, release of lysosomal proteases wall extract. Its polysaccharide whereas pro‑Il‑1β synthesis is regulated through the into the cytoplasm are upstream mechanisms that trig‑ components ( -glucans) β SYK–CARD9 pathway, NlRP3 activation occurs through ger NlRP3 activation in several scenarios108. NlRP3 contain d-glucose monomers that are linked by β-type a SYK‑dependent but mostly CARD9‑independent activation by fungi requires SYK‑triggered ROS gen‑ 93 + 93 glycosidic bonds. β-glucans are mechanism (FIG. 4b). As NlRP3 activation by malarial eration and K efflux (FIG. 4b). However, because SYK particularly strong activators haemozoin also requires SYK109, the role of SYK in NlRP3 activation by zymosan or non‑viable yeast is not suf‑ of antifungal host defence. activation is not restricted to antifungal immunity. ficient for inflammasome activation93,110, other as yet a Fungal pathogens Dengue virus Necrosis (mammals) Necrosis (Drosophila) and M. tuberculosis TDM β-glucan SAP130 CLEC4E CLEC7A CLEC6A CLEC5A CLEC9A CLEC4E Draper

Plasma FcRγ DAP12 FcRγ membrane

SRC42A

P P P P P P P P P P P P

SYK SYK SYK SYK SYK SHARK Figure 4 | SyK-dependent innate pathogen and damage recognition pathways. a | Innate immune receptors coupled to spleen tyrosine kinase (SYK) or SYK‑related molecules b Fungal pathogens sense foreign pathogens and tissue damage. Fungal pathogens and Mycobacterium tuberculosis engage C‑type CLEC6A lectin 7A (CLEC7A; also known as dectin 1), CLEC6A (also CLEC7A CLEC4E known as dectin 2) and CLEC4E (also known as MINCLE); Others? Others? dengue virus activates CLEC5A; necrotic cells activate the Plasma mammalian receptors CLEC9A and CLEC4E and the FcRγ membrane Drosophila melanogaster receptor Draper. The ligand‑binding chains of those receptors have an immunoreceptor DC (or tyrosine‑based activation motif (ITAM) or an ITAM‑like macrophage?) P P P P K+ motif with a single phosphorylatable tyrosine residue (hemITAM), or associate with the ITAM‑bearing transmembrane adaptors Fc receptor γ‑chain (FcRγ) or DAP12. All of these receptors activate SYK family kinases (SYK or SH2 domain ankyrin repeat kinase (SHARK)) by SH2 SYK SYK domain‑mediated recruitment to phosphorylated tyrosine residues. The kinase responsible for phosphorylation of the ROS ? Draper ITAM is the D. melanogaster SRC family kinase SRC42A. b | The mechanism of pro‑inflammatory responses CARD9 triggered by recognition of fungal pathogens by C‑type lectins is illustrated. CLEC7A binds SYK directly through an intracellular hemITAM, whereas CLEC6A and CLEC4E BCL-10 Phagocytosis engage SYK through FcRγ. Pathogens trigger SYK‑mediated NLRP3 phagocytosis, cytokine and chemokine production, and ROS Inflammasome generation. SYK‑mediated nuclear factor‑κB (NF‑κB) MALT1 ASC signalling is controlled by the CARD9–BCL‑10–MALT1 complex. In addition, SYK is required for interleukin‑1β (IL‑1β) processing by the NLR family, pyrin domain‑containing 3 (NLRP3) inflammasome, which is activated by Pro-caspase 1 SYK‑dependent reactive oxygen species (ROS) production, NF-κB K+ efflux and presumably additional unknown danger signals. ASC, adaptor protein apoptosis‑associated speck‑like protein containing a CARD; BCL‑10, B cell lymphoma 10; Chemokines and cytokines Pro-IL-1β IL-1β CARD9, caspase‑recruitment domain 9; DC, dendritic cell; MALT1, mucosa‑associated lymphoid tissue lymphoma translocation protein 1; TDM, trehalose 6,6ʹ‑dimycolate. Nature Reviews | Immunology

unidentified components are probably also required for kinase SH2 domain ankyrin repeat kinase (SHARK) the activation of the NlRP3 inflammasome following (FIG. 4a), which is essential for Draper function in glial yeast infection (FIG. 4b). cells in vivo4. Taken together, these pathways indicate that the ITAM–SYK‑mediated damage recognition Recognition of bacteria and viruses. There is increasing pathway is an ancient mechanism that it shared between evidence that SYK‑coupled PRRs also have important insects and mammals and that this pathway overlaps roles in innate recognition of bacteria, with CleC7A, with innate recognition of pathogens. CleC6A and CleC4e all implicated in sensing of myco‑ bacterial PAMPs46 (FIG. 4a). Although the mycobacterial Other immune functions of SYK structures that are recognized by CleC7A and CleC6A SYK has been implicated in several other signalling path‑ remain to be identified, CleC4e has been reported to ways in immune cells. The DAP12‑associated trigger‑ be the long‑sought receptor for the mycobacterial cord ing receptor expressed on myeloid cells (TReM) family factor trehalose 6,6ʹ‑dimycolate (TDM), the most thor‑ molecules participate in several inflammation‑related oughly studied immune stimulant of Mycobacterium biological processes, including modulation of Toll‑like tuberculosis107,111. Myeloid cell activation by TDM, or receptor function, through activation of SYK119. CleC4C its less toxic analogue trehalose 6,6ʹ‑dibehenate, uses (also known as BDCA2), an FcRγ‑associated receptor ITAM–SYK signalling through FcRγ, SYK and the expressed on DCs, also signals through SYK but, in con‑ CARD9–BCl‑10–MAlT1 complex106,107. trast to C‑type lectins involved in pathogen recognition, In addition to CleC7A, CleC6A and CleC4e, there it does not activate NF‑κB46. Other well known immune are more myeloid C‑type lectin receptors with as yet uni‑ cell receptors do not contain ITAMs but also seem to use dentified functions46, and other innate immune recep‑ ITAM–SYK‑mediated accessory signals. Interferon‑γ tors have also been proposed to signal through SYK112. (IFNγ)‑mediated enhancement of IFNα‑induced signal Furthermore, genetic deletion of SYK has been shown to transducer and activator of transcription 1 (STAT1) acti‑ attenuate antibacterial host defence in mice113. Dengue vation requires DAP12, FcRγ and SYK, possibly through virus is detected by the C‑type lectin CleC5A (also interaction of DAP12‑ and FcRγ‑containing receptors known as MDl1), which induces pro‑inflammatory with the IFNα receptor120. Basophils lacking either FcRγ cytokine production through DAP12 (REF.. 114) (FIG. 4a). or SYK show decreased Il‑3‑induced Il‑4 secretion (but Therefore, SYK is presumably also involved in signalling not proliferation), supposedly through direct association PRR‑mediated recognition of certain viruses. between FcRγ and the Il‑3 receptor common β‑chain121. SYK and DAP12 have been implicated in signalling from Signalling tissue damage the colony‑stimulating factor 1 receptor (also known as The innate immune system is also activated by non‑ FMS) in an ITAM‑dependent manner also requiring infectious stimuli, such as cell death caused by sterile PYK2 (REFS 78,122). MHC class II molecules on B cells injury or developmental processes, or sterile inflamma‑ associate with, and trigger the phosphorylation of, the tion during autoimmune diseases. ITAM‑bearing Igα and Igβ adaptors123. There is accumulating evidence that SYK‑coupled NK cells express SYK and ZAP70, both of which are C‑type lectins can detect host‑derived molecules from involved in FcγR‑mediated ADCC responses34. NK cells damaged cells. CleC4e, an FcRγ‑associated fungal and also express the ITAM‑bearing transmembrane adaptor mycobacterial PRR (see above), can sense necrotic cells DAP12, which is coupled to several activating NK cell after prolonged culture in vitro115, and it is reasonable receptors. However, although SYK and/or ZAP70 are to assume that this response also requires the ITAM‑ required for calcium signalling and cytokine release follow‑ mediated activation of SYK (FIG. 4a). The CleC4e ligand ing activation of DAP12‑associated receptors124,125, NK cells expressed by necrotic cells has been identified as splice‑ lacking both SYK and ZAP70 can kill target cells through a osome‑associated protein 130 (SAP130)115. A second SYK‑ DAP10‑ and PI3K‑mediated parallel pathway125,126. coupled receptor for recognition of dead cells is CleC9A Crystals of monosodium urate activate DCs by a (also known as DNGR1). like CleC7A, CleC9A has a receptor independent pathway127, which involves aggre‑ cytoplasmic hemITAM with a single Tyr‑X‑X‑leu motif gation of cholesterol and activation of a SYK‑dependent (FIG. 4a). CleC9A is highly expressed by subsets of DCs signalling pathway. The mechanism of this activation is and monocytes and recognizes as yet unidentified ligands incompletely understood, but might involve the recruit‑ that are exposed during cell necrosis116. CleC9A promotes ment of ITAM‑associated receptors. Importantly, this DC‑mediated cross‑presentation of dead cell‑derived mechanism may contribute to the development of antigens through a hemITAM–SYK interaction117. crystal‑induced diseases such as gout. The ITAM or hemITAM–SYK‑mediated damage SYK had also been proposed to participate in G recognition pathway is probably an ancient mecha‑ protein‑coupled receptor signalling128. However, genetic nism as it is also found in invertebrates. The clear‑ studies of this pathway in neutrophils and mast cells128 ance of neuronal cell corpses by phagocytic glial cells argue against that possibility. Monosodium urate in the brains of D. melanogaster requires Draper, an A monosodium salt of uric acid ITAM‑containing phagocytic receptor118 (FIG. 4a). Non-immune functions of SYK that has a tendency to precipitate in needle-shaped Draper ligation by phagocytic targets triggers ITAM Although ITAM–SYK signalling has long been regarded crystals in the joints, leading to phosphory lation by the SRC‑related kinase SRC42A, to be specific for the immune system, recent studies the inflammatory disease gout. leading to subsequent association with the SYK‑related indicate several non‑immune functions of SYK.

Bone metabolism. Osteoclasts are macrophage‑related progenitor cells (which share their origin with true blood bone‑resorbing cells that develop from early myeloid cells)136 or that the vascular phenotype is due to a gain precursors under the control of the TNF‑related protein of function effect, whereby tissue accumulation of Syk–/– receptor activator of NF-κB (RANK) ligand expressed by myeloid cells leads to excessive release of pro‑angiogenic osteoblasts. Differentiated pre‑osteoclasts fuse to form factors that trigger hyperproliferation of the lymphatic large multinucleated cells, which then bind to and resorb vasculature137. However, the mechanism attributable to Receptor activator of NF-κB the bone surface. Importantly, αVβ3 integrin participates the circulating endothelial progenitor cells contradicts the (RANK). A cytokine receptor in diverse functions of osteoclasts, including sealing the lack of SYK expression in the endothelial cell lineage137, that is closely related to tumour necrosis factor resorption area from the surrounding environment. loss whereas the gain of function mechanism does not seem receptors and that is mainly of function mutations of DAP12 cause bone abnormalities to explain the correction of the vascular phenotype by the expressed by osteoclast in humans (Nasu-Hakola disease)129, which indicates a pos‑ presence of only 5–10% wild‑type cells in the haematopoi‑ lineage cells. Ligation of sible role of ITAM‑based signalling in bone metabolism. etic compartment in mixed bone marrow chimaeras3. RANK on early macrophage Indeed, DAP12‑deficient mice show moderate osteopet- A series of recent studies propose an exciting third precursors by RANK ligand rosis130,131 (FIG. 5c) expressed on osteoblasts , whereas mice lacking both DAP12 and FcRγ explanation . Blood and lymph vessels fail to sepa‑ 5,132 138 triggers re-programming of show severe osteopetrosis . Although FcRγ is involved rate in platelet‑deficient fetuses , as well as in mice lack‑ the cell leading to osteoclast in osteoblast–osteoclast interactions, DAP12 is responsible ing either the platelet receptor CleC2 (REF. 139) or the development. for relaying an osteoblast‑independent signal5,132 (FIG. 5a). CleC2 ligand podoplanin, which is expressed on lymphatic 139–141 Osteoblasts Importantly, SYK, which is activated downstream of (but not blood) endothelial cells . A similar vascular Bone-lining fibroblast-related DAP12 and FcRγ in an ITAM‑dependent manner, is also phenotype is also induced by platelet‑specific deletion of cells of non-haematopoietic required for osteoclast development and function5,85,133 SlP76 (REF. 139) (the deficiency of which also leads to a origin that are specialized for (FIG. 5a). Although the precise mechanism of DAP12 and blood–lymphatic vascular separation defect3) or of SYK the generation of bone matrix FcRγ activation is poorly understood, it probably involves itself (e. Schweighoffer and V. T., unpublished observa‑ and the regulation of bone metabolism. Through the FcRγ‑associated receptors osteoclast‑associated recep‑ tions; and C. Bertozzi and M. Kahn, personal communica‑ expression of RANK ligand, tor (OSCAR) and paired immunoglobulin‑like receptor A tion). lymphatic endothelial cells also induce aggregation they also promote the (PIRA), and the DAP12‑associated molecule TReM2 of platelets in a SlP76‑dependent manner139. Together development and activation (REF. 132) (FIG. 5a). This ITAM‑mediated pathway has with the ability of podoplanin to activate platelets through of osteoclasts. been proposed to be a co‑stimulatory pathway for RANK CleC2 (REF. 142) and the finding that CleC2 signals 132 Nasu-Hakola disease ligand signalling , as well as a mediator of αVβ3 integrin through SYK and PlCγ2 (REF. 42) (FIG. 5b), these results sug‑ (Also known as polycystic signal transduction5,85 (FIG. 5a). gest that podoplanin on lymphatic endothelial cells trig‑ lipomembranous gers platelet activation and aggregation through CleC2, osteodysplasia with sclerosing Platelet functions. Several platelet functions rely on SYK SYK, SlP76 and PlCγ2 (FIG. 5b,c), thereby closing off any leukoencephalopathy (PLOSL)). An inherited human disease signalling. GPVI, a major collagen receptor of platelets, is blood–lymphatic shunts by a yet unidentified mechanism characterized by presenile an FcRγ‑associated receptor that is closely related to FcαRs, that possibly involves platelet aggregation. As a conse‑ dementia and bone cysts. It is which may explain why GPVI, similar to other FcR family quence, newly formed lymphatic sacks are separated from caused by loss of function members, signals through SYK2 (FIG. 5b). The C‑type lectin the blood vasculature, and a long‑term incompatibility mutations in the ITAM-containing adaptor CleC2 has also been shown to signal through SYK in between the two vascular systems is established. 42 protein DAP12 or the platelets . Similar to other hemITAM‑containing recep‑ DAP12-associated receptor tors, CleC2 has a single cytoplasmic Tyr‑X‑X‑leu motif Other non-haematopoietic functions. SYK is also TREM2. The clinical which engages both SH2 domains of SYK by dimerization expressed by cells of non‑haematopoietic origin including manifestations probably result of the receptor45 (FIG. 5b). SYK also mediates outside‑in normal mammary glands and mammary gland epithelial from the loss of DAP12 or 73 143 TREM2 in osteoclasts and signalling by αIIbβ3 integrin on platelets . The mecha‑ cell lines , as well as various other tissues (reviewed in microglial cells. nism of SYK activation by αIIbβ3 integrin was shown to REFS 144,145). Although studies have proposed that SYK be ITAM dependent84 (see above) and to require, at least has a role in processes including signalling by integrins Osteopetrosis in human cells, the ITAM‑containing FcγRIIA molecule88 and TNF receptors, transcriptional regulation and A rare inherited disease (FIG. 5b) (REF. 134) REF.144) characterized by pathological . As CleC2 and the SYK substrate SlP76 mitotic progression in these cells (reviewed in , accumulation of mineralized (REF. 135) are required for arterial thrombus formation, it this area is still poorly understood with little mechanistic bone matrix in the bones. is likely that SYK also participates in in vivo functions of information available. Despite the increased bone platelets during haemostasis. A SYK‑related kinase has also been proposed to par‑ mass, osteopetrotic bones are ticipate in gonadal development in the parasitic helminth fragile owing to abnormal bone 146 composition and structure. It is Vascular development. One of the most striking pheno‑ Schistosoma mansoni , suggesting that the role of SYK‑ caused by a lack or dysfunction types of SYK‑deficient fetuses is the appearance of blood‑ related kinases in non‑haematopoietic tissues may also of osteoclasts and therefore filled subcutaneous structures originally described as have an ancient evolutionary origin. can be cured by bone marrow petechial haemorrhages23,24 but later identified as blood‑ transplantation. filled lymphatic vessels3, and this indicated that SYK is SYK in disease pathogenesis and therapy Haemostasis required for the separation of lymphatic vessels from the Allergy and autoimmunity. The diverse roles of SYK in the The combination of events that general circulation. Surprisingly, this vascular defect could immune system suggest that it might participate in aller‑ result in cessation of bleeding. be transferred to lethally irradiated wild‑type recipients gic and/or autoimmune diseases. Indeed, SYK‑deficient Haemostatic processes include by bone marrow transplantation33, indicating a role for bone marrow chimaeras are protected from reverse passive constriction of blood vessels, Arthus reaction70 platelet aggregation and SYK expression by the haematopoietic compartment. Two and autoantibody‑mediated experimen‑ 147 coagulation (clotting) of the subsequent studies proposed different explanations, sug‑ tal arthritis . A supposedly SYK‑selective inhibitor, R112 blood. gesting either that the defect lies in circulating endothelial (Rigel), alleviates the symptoms of allergic rhinitis in

a b Collagen Rhodocytin Fibrinogen Podoplanin Osteoblast αIIbβ3 integrin RANK ligand αVβ3 integrin GPVI CLEC2 FcγRIIA ? β-chain α-chain OSCAR? TREM2? FcRγ PIRA? Other? Other? FcRγ DAP12 Platelet

RANK Osteoclast P P P P P P

P P P P

? SYK SYK SYK

SYK SYK SLP76 PLCγ2

Activation and PLCγ2 thrombus formation

c Ca2+

Blood vessel

Calcineurin ? ?

NFATc1

Fusion and Platelets Differentiation bone resorption

Podoplanin Lymph vessel

Figure 5 | non-immune functions of SyK. a | The role of DAP12 and Fc receptor γ‑chain (FcRγ) in osteoclasts is illustrated. In addition to a receptor activator of NF‑κB (RANK)–RANK ligand interaction, osteoblasts express yet unidentified ligands that Podoplanin engage a putative FcRγ‑associated receptor (most likely osteoclast‑associated receptor (OSCAR)Na ortur pairede Revie immunoglobulinws | Immunolog‑y A 43 kDa sialoglycoprotein like receptor A (PIRA)) on the osteoclast surface. Osteoclasts also express putative DAP12‑associated receptors (most likely originally identified in triggering receptor expressed on myeloid cells 2 (TREM2)), which bind to yet unidentified ligands that are not expressed by podocytes of the glomerular osteoblasts. Both FcRγ and DAP12 promote osteoclast development and function through immunoreceptor tyrosine‑based membrane but later shown to activation motif (ITAM)‑mediated spleen tyrosine kinase (SYK) activation and the phospholipase Cγ2 (PLCγ2)–calcineurin– be expressed by several other nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) pathway. b | SYK‑mediated signalling in platelets is illustrated. cell types including lymphatic Collagen activates the FcRγ‑associated receptor glycoprotein VI (GPVI) on platelets and triggers SYK activation in an endothelial cells and tumour ITAM‑mediated manner. The snake venom rhodocytin, endogenous podoplanin and possibly other platelet agonists activate cells. It is a natural ligand of the receptor CLEC2 (also known as CLEC1B), which recruits SYK to phosphorylated tyrosine in the CLEC2 hemITAMs with a CLEC2 and induces CLEC2- mediated platelet aggregation. 2/1 stoichiometry. Fibrinogen engages αIIbβ3 integrin, which associates with the ITAM‑containing receptor FcγRIIA. This complex probably activates SYK through both conventional ITAM‑mediated SYK activation pathways and by binding of the Reverse passive Arthus integrin β‑chain to the amino‑terminal SH2 domain of SYK in a phosphotyrosine‑independent manner. All three cases of SYK reaction activation lead to platelet activation through SH2 domain‑containing leukocyte protein 76 (SLP76) and PLCγ2. c | The role of An antibody-mediated local SYK in separation of blood and lymphatic vessels is illustrated. Lymphatic vessels express podoplanin which triggers platelet hypersensitivity reaction that is activation and aggregation. This and possibly other unknown mechanisms lead to closing off any blood–lymphatic shunts. triggered by systemic injection The mechanism of podoplanin‑mediated platelet activation involves CLEC2, which activates SYK through hemITAM of antigen followed by local phosphorylation, as well as SLP76 and PLCγ2, which function downstream of SYK. (subcutaneous) injection of antibody against the injected antigen. The formation of human patients148. An R112‑related inhibitor, R406 (Rigel), arthritis151. More recently, fostamatinib has shown benefi‑ immune complexes results in as well as its orally bioavailable prodrug, fostamatinib cial effects in autoantibody‑induced thrombocytopenia local oedema and inflammation, which is mediated by Fc (R788; Rigel/Astra Zeneca), delayed the onset and reduced in mice, and autoimmune thrombocytopenic purpura in 149,150 152 receptors and activation of the the severity of autoimmune arthritis in animal models humans . Various aspects of the mechanism of action of complement cascade. and provided clinical benefit in patients with rheumatoid SYK inhibitors in clinical use are discussed in BOX 2.

Box 2 | What is the mechanism of action of SYK inhibitors in clinical use? Transformation of mature B cells mostly leads to vari‑ ous forms of B cell lymphoma or B cell lineage chronic Spleen tyrosine kinase (SYK) inhibitors have shown positive results in the treatment of lymphocytic leukaemia (Cll). ITAM‑mediated tonic 148 151,152 166 allergy , autoimmune diseases and B cell lineage malignancies , but the BCR signalling is required for the survival of resting mechanism of their action is incompletely understood. This is in part due to the diverse mature B cells and certain B cell lymphomas39,158,159. roles of SYK in immunological functions. For example, B cell‑mediated antigen presentation and autoantibody formation, Fc receptor‑mediated myeloid cell functions This signalling probably functions through SYK, which and β2 integrin‑mediated leukocyte activation have all been implicated in the could explain the expression of constitutively active SYK 160–162 pathogenesis of rheumatoid arthritis and they all have been shown to require SYK in B cell lymphomas and B cell lineage Cll cells . (see REF. 147 for further details). The rapid effects of R112 (Rigel) treatment in allergic SYK is also required for processing stromal cell‑derived rhinitis148 and the effectiveness of SYK inhibitors in autoantibody‑induced passive pro‑survival signals, such as adhesion and homing, by models of arthritis149 and autoimmune thrombocytopenia152 also suggest that SYK Cll cells independently of BCR signalling163. These inhibitors target disease components other than B cell receptor (BCR)‑mediated B cell results suggest that SYK contributes to the survival and functions. Likewise, although initial studies suggested that the only role of SYK in maintenance of B cell malignancies (FIG. 6d). Accordingly, B cell lymphomas is to relay immunoreceptor tyrosine‑based activation motif abrogation of SYK activity inhibited the growth of vari‑ (ITAM)‑mediated tonic BCR signalling, a recent report suggested that SYK also ous B cell lymphomas160,164,165, and fostamatinib therapy contributes to the survival of B cell lymphomas through processing BCR‑independent adhesive and homing signals163. prolonged the survival of patients with diverse types of 166 A further complicating issue is that R406, the active metabolite of the prodrug B cell lymphoma or B cell lineage Cll . fostamatinib (R788; Rigel/Astra Zeneca), is an ATP‑competitive kinase inhibitor and, SYK might also participate in haematological malig‑ similar to most such inhibitors, has rather limited specificity towards SYK. Indeed, R406 nancies of non‑B cell origin. T cell lymphomas often (Rigel) has been shown to inhibit several other kinases and non‑kinase targets at overexpress enzymatically active SYK167, may contain concentrations comparable to those inhibiting SYK149. Of those targets, the tyrosine chromosomal translocations leading to the expression of kinases FMS‑related tyrosine kinase 3 (FLT3), KIT, LCK, Janus kinase 1 (JAK1) and JAK3, an Il‑2‑inducible T cell kinase (ITK)–SYK fusion pro‑ as well as the adenosine A3 receptor have been implicated in autoimmune diseases, tein168 and are sensitive to silencing of SYK expression169. A providing additional or alternative explanations for the clinical effect of fostamatinib in chromosomal translocation generating a Tel–SYK fusion human patients (see REF. 147 for further details). protein causes myelodysplastic syndrome, a pre‑leukaemic Taken together, it is reasonable to assume that the clinical effects of fostamatinib are probably mediated by inhibition of several SYK‑dependent and SYK‑independent state that predisposes to the development of acute myeloid 170 immune signalling pathways. leukaemia , and SYK seems to be an important compo‑ nent of disease pathogenesis in acute myeloid leukaemia itself 171. These results may further extend the possible use Haematological malignancies. SYK has been linked of SYK inhibitors in human therapy. to the development or maintenance of haematological malignancies including B cell lineage leukaemias and SYK signalling by viral oncogenes. SYK is also required lymphomas (FIG. 6). for the oncogenic activity of several viruses that encode The first study linking SYK to a haematological malig‑ ITAM‑containing proteins172. The ITAM of the viral pro‑ nancy described marked reduction of SYK expression in tein latent membrane protein 2A (lMP2A) and host cell highly aggressive childhood pro‑B cell acute lympho‑ SYK are both required for epstein–Barr virus‑induced blastic leukaemia (All) but not in more differentiated oncogenic transformation173,174. Similarly, the ITAM‑ pre‑B cell All153. As SYK is required for the transition bearing envelope protein of mouse mammary tumour of pro‑B to pre‑B cells23, it is tempting to speculate that virus contributes to virus‑induced transformation in an the loss of SYK contributes to transformation of B cell ITAM‑ and SYK‑dependent manner175,176. Furthermore, precursors by preventing further differentiation (FIG. 6a), the ITAM‑containing K1 protein of Kaposi’s sarcoma‑ as has been proposed for SlP65, the lack of which con‑ associated herpesvirus initiates ITAM‑ and, presumably, tributes to pre‑B cell leukaemia in both humans and SYK‑dependent signalling pathways to promote lytic mice154,155. However, the lack of an independent con‑ viral replication in infected B cells177,178. The possible firmation of SYK deficiency in pro‑B cell All and the role of these and other viruses in breast cancer179 raise uncertainties related to extrapolating prior findings on the possibility of a tumour‑promoting role for SYK in SlP65 to SYK point to the need for further studies on the certain tumours. role of SYK in pro‑B cell leukaemia. SYK has different and better characterized roles Other non-haematopoietic tumours. In contrast to a pos‑ in lymphomas derived from B lineage cells beyond sible tumour‑promoting role for SYK in virus‑induced the pro‑B cell stage. Probably related to the role of tumours, it has been proposed to suppress the growth SYK in promoting proliferation and blocking apopto‑ of other (supposedly non‑virus‑induced) tumours of sis in pre‑B cells, exogenous expression of SYK could non‑haematopoietic origin. SYK expression inversely transform normal pre‑B cells, and endogenous SYK correlated with invasiveness in breast cancer cell lines143 Rhodocytin was required for the transformation of pre‑B cells by and exogenous expression of SYK inhibited tumour A snake venom toxin that exogenous MYC156 (FIG. 6b). Furthermore, SYK inhi‑ growth, whereas expression of a dominant‑negative SYK triggers platelet activation by bition promoted apoptosis of primary human B cell was found to promote tumorigenesis143. SYK expression binding to CLEC2 on platelets. precursor All (most likely pre‑B cell All) cells both also inversely correlated with the severity of breast can‑ It is a disulphide-linked heterodimer with features in vitro and in vivo, suggesting a potential therapeutic cer and other epithelial cell‑derived tumours in human 143,144,180–182 common to certain C-type application of SYK inhibitors in certain patients with patients , with particularly poor prognosis when lectins. B cell All157. low nuclear levels of SYK were present183,184.

a Pro-B cell b Pre-B cell c Immature B cell d Resting mature B cell Pre-BCR forming Pre-BCR BCR BCR

Igα or Igβ Igα or Igβ Igα or Igβ

ITAM P P P P P P P P

SYK SYK SYK SYK

Differentiation Proliferation and survival Maturation Survival and transition SYK deficiency Dysregulated Pre-BCR-mediated BCR-mediated tonic ↓ or constitutive SYK activation SYK activation Blocked differentiation SYK activation ↓ ↓ ↓ Proliferation Survival Other transformation Proliferation and survival event(s) and survival SYK-dependent Other non-BCR survival signals transformation event(s) Other transformation ? event(s)

B cell lymphoma Pro-B cell ALL Pre-B cell ALL B lineage CLL Figure 6 | The role of spleen tyrosine kinase (SyK) in B cell development and malignancies of the B cell lineage. a | SYK is required for normal transition from pro‑B cells to pre‑B cells. When other transforming factors are present, loss of SYK in pro‑B cells may contribute to the development of pro‑B cell acute lymphoblastic leukaemia (ALL), possibly by blocking further differentiation. b | After successful pre‑B cell receptor (BCR) heavy chain rearrangement, SYK signals proliferation and survival of pre‑B cells. However, dysregulated or constitutive activation of SYK can transform pre‑B cells to pre‑B cell ALL. In addition, pre‑BCR‑induced SYK activation is required for the transformation of pre‑B cells to pre‑B cell ALL by other transforming factors (such as MYC). c | Although SYK is required for maturation of immature B cells and their transition through the spleen, these processes have not yet been linked to any malignant transformation events. d | SYK is Nature Reviews | Immunology also required for the survival of normal mature B cells, as well as of mature B cell lymphomas and B cell lineage chronic lymphocytic leukaemias (CLLs), presumably owing to its role in ‘tonic’ BCR signalling and other SYK‑dependent, but BCR‑independent, signals. ITAM, immunoreceptor tyrosine‑based activation motif.

The decreased level of SYK expression in cancer cells In addition to the diverse roles of SYK in basic bio‑ probably results from hyper‑methylation of the SYK pro‑ logical processes, it is also involved in the pathogenesis of moter, rather than mutations or genetic deletion of SYK several human diseases, including allergy, autoimmunity itself 144,180,182, whereas the loss of nuclear localization of and various haematological malignancies. The promising SYK is probably due to alternative splicing, leading to the results of SYK inhibitors in recently completed human expression of the SYK‑B isoform, which lacks a nuclear clinical trials suggest that SYK may become an important localization signal185. However, it is presently unclear therapeutic target in those diseases in the future. how loss of SYK expression results in poor prognosis The new observations of diverse biological functions in epithelial cell‑derived tumours, although it may be for SYK also raise a number of questions. How do the related to any of the previously mentioned possible physi‑ initial steps of ITAM signalling occur? How do integrins ological functions of SYK in these lineages (reviewed in and selectins link to an ITAM‑based signalling pathway? REF.144). How are DAP12 and FcRγ coupled to osteoclast develop‑ ment? How does SYK in platelets contribute to vascular Conclusions and perspective development? what downstream pathways mediate the The SYK tyrosine kinase was originally thought to only diverse functions of SYK? what are the roles of SYK in contribute to signalling responses of immunoreceptors epithelial cells and are ITAMs involved in these func‑ of the adaptive immune response. However, more recent tions? Is SYK a suitable target in thrombocytic diseases studies have identified a large number of new functions of or pathological bone loss? would targeting SYK be effec‑ SYK both in the immune system and beyond. Moreover, tive in treating metabolic disorders of inflammatory ori‑ the identification of a similar pathway in D. melanogaster gin? will long‑term treatment with SYK inhibitors result indicates that this signalling pathway emerged sub‑ in unexpected side effects, such as those related to the stantially earlier than the adaptive immune response. role of SYK in innate immunity or its tumour suppressor Collectively, these studies indicate that SYK has a much function in epithelial cells? Answering these and other more diverse role in vertebrate and invertebrate biology questions will keep scientists interested in the biological than previously anticipated. functions of SYK busy for many years to come.

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T., Chen, C. L. & Qazi, S. dependent signaling by Kaposi’s sarcoma-associated cgi?id=MGI:99,515 Targeting SYK kinase-dependent anti-apoptotic herpesvirus K1 protein: effects on lytic viral All lInKS Are AcTIve In The onlIne Pdf resistance pathway in B-lineage acute lymphoblastic replication. J. Virol. 75, 5891–5898 (2001).