Important Cytosolic Adaptors In Inside Out Signalling Biology Essay

The cytosolic adaptor proteins SLP-76 and Gads, initially isolated from T-cell lysates as Grb2-associated molecules, are predominantly expressed in hematopoietic cells, particularly T-cells, in addition to monocytes/macrophages, NK cells, mast cells and platelets. Following TCR engagement, SLP-76 becomes tyrosine phosphorylated by ZAP-70 (Bubeck Wardenburg et al., 1996) and thus generates binding sites for the SH2 domains of several other cytoplasmic adaptors including the Rac/Rho family of GTPases, Vav, Nck, and the tec-family protein tyrosine kinase Itk (Ménasché et al., 2007). Upon T-cell activation, SLP-76 interacts via its SH2-domain with the cytosolic adapter SLAP-130 (SLP-76–associated phosphoprotein of 130 kDa; also known as FYB, for Fyn-binding protein; or, as recently suggested, ADAP, for adhesion- and degranulation-promoting adapter protein) (Ref). SLAP-130 interacts constitutively with SKAP55 (Src kinase–associated phosphoprotein of 55 kDa) (Ref). The interaction between SLAP-130 and SKAP55 seems to be of high stoichiometry and to involve a proline-rich region of SLAP-130 and the C-terminal domains of SKAP55 (Ref). The role of SLP-76 as a positive regulator of lymphocyte function was confirmed by its ability to augment TCR-induced IL-2 promoter activity when overexpressed in T-cells (Ref). An absolute requirement for SLP-76 in TCR-mediated stimulation has been demonstrated in SLP-76-deficient T-cells (Ref). SLP-76-deficient T-cells fail to induce PLCγ1 phosphorylation, Ca2+ influx, ERK activation or the upregulation of IL-2 promoter activity in response to TCR engagement. SLP-76-/- mice lack mature T-cells, revealing its critical role in thymocyte development (Ref) presumably due to a defect in pre-TCR signalling. Additionally, the proline-rich domain of SLP-76 mediates its constitutive association with Gads (Ref), which inducibly binds to phosphorylated LAT following TCR stimulation (Ref). Since LAT is required for SLP-76 phosphorylation (Ref), it is possible that Gads recruits SLP-76 to LAT where it can then be phosphorylated by active ZAP-70. Moreover, SLP-76 may be required for the stable association of LAT with its other binding partners. SLP-76 binds to the SH3 domain of Lck (Ref), suggesting that SLP-76 may mediate an interaction between this protein tyrosine kinase and potential substrates (Figure 4).

An important role for Gads in TCR-mediated signalling leading to LFA-1 activation has been suggested in studies where Gads and SLP-76 act in synergy to augment TCR-stimulated NFAT and IL2 promoter activity when coexpressed in Jurkat T-cells. The amino acid sequence of Gads shares the highest degree of homology with Grb2 and encodes an SH2 domain flanked by two SH3 domains (SH3–SH2–SH3). The SH2 domain of Gads also mediates a TCR-inducible association with phosphorylated LAT, providing a means to couple SLP-76 and LAT in response to TCR stimulation. The C-terminal SH3 domain of Gads constitutively binds a proline-rich sequence of SLP-76, which maps to the same binding site for the SH3 domain of Grb2 (Ref). Furthermore, overexpression of a Gads mutant that lacks a functional SH2 domain results in a dominant inhibition of TCR-induced NFAT activity, suggesting that recruitment of SLP-76 to phosphorylated LAT is a critical component of its function in T-cells (Ref).

ADAP is another important cytosolic adaptor protein shown to become tyrosine phosphorylayed in human T-cells after triggering of β1 integrins (Ref). It colocalizes with LFA-1 in the IS (Wang et al, 2004). ADAP interacts with SLP-76, and this interaction seems to be crucial for T-cell migration and functions. In fact, T-cells from mice deficient in ADAP show pronounced defects in proliferation, IL-2 production and adhesion, although many signalling pathways that are required for T-cell activation remain intact, including F-actin polymerization at the immunological synapse (Ref 105, 106). LFA-1 clustering was found to be lost and T-cell activation was severely impaired in T-cells from ADAP-/- mice (Ref), suggesting a specific role of ADAP in TCR-mediated integrin activation, rather than in affecting global TCR signalling. Interestingly, a mutant form of ADAP, which is unable to bind SLP-76, was fully functional, which indicates that the recruitment of ADAP to the LAT-SLP-76 signalling complex might not be required for adhesion. In addition, ADAP can bind to the actin regulators ENA and VASP, which might result in actin cytoskeletal rearrangement leading to integrin activation. ADAP also interacts with an adaptor protein SRC-kinase-associated protein of 55 kDa (SKAP55, also known as SKAP1) to regulate TCR-mediated LFA-1 activation (Wang et al, 2003). The association between ADAP and SKAP55 is constitutive and occurs primarily between the SH3 domain of SKAP55 and a proline-rich motif found in ADAP, and a recent study indicates that this interaction is required for the stabilization of these proteins. The mechanism by which ADAP/SKAP55 controls integrin activation has remained elusive for many years, but a recent links ADAP/SKAP55 complex to the membrane localization of Rap1 following TCR engagement (Menashe et al, 2007). Following T-cell activation ADAP/SKAP55 complex binds RIAM (a Rap1 effector) and this new complex provides a scaffold for the recruitment of active Rap1, thereby facilitating integrin activation. In another study, Jurkat T-cells in which SKAP55 has been depleted with RNAi are also impaired in TCR-mediated adhesion, although normal calcium flux and activation of several important TCR-mediated pathways is maintained. The possibility that the SLP-76/ADAP/SKAP55 complex is involved in regulation of T-cell adhesiveness might also provide new clues to the propagation of TCR-mediated signals that lead to the high avidity state of the integrin LFA-1. Among several other cytoplasmic adaptors, role of talin, α-actinin and kindlin-3 in chemokine mediated integrin activation in human leukocytes has also been established (Ref).

LFA-1 linked signalling complex in outside-in signalling

Following adhesive interactions between LFA-1 and extracellular ligand(s), T-cells acquire and maintain a polarized morphology and functional asymmetry, forming a leading lamellipodium and a rear compartment or uropod. This dramatically changing migratory morphological asymmetry in T-cells is achieved by selective, highly ordered and coordinated activities of numerous intracellular molecules, and several distinct and integrated biochemical pathways with concomitant cytoskeletal-based rearrangements (Westermann et al., 2001; Cose et al., 2006; Cose, 2007; Woodland and Kohlmeier, 2009; Geginat et al., 1999; Kelleher et al., 1995; Rodriguez-Fernadez et al., 1999). Unfortunately, the integration of the LFA-1 signal with cytoskeletal control still remains a poorly understood area of research, specifically how adaptor and linker proteins are implicated in LFA-1 outside-in signalling. Our research group and others have established an important role of LFA-1 in redistributing T-cell adaptor and linker proteins to propagate signal transduction in the organization and dynamics of the microtubule and actin cytoskeletal systems (Volkov et al., 2001; Ref). The interaction between LFA-1 with its ligand ICAM-1 triggers a complex but defined set of intracellular changes, including translocation of PKC isoforms β and δ to the microtubule cytoskeleton in migrating T-cells (Volkov et al., 2001). Moreover, LFA-1 outside-in signals have been associated with the formation of new phenomenon called the "actin cloud". LFA-1 ligation also induces phospholipid biosynthesis that regulates the formation and strengthening of adhesion sites important for maintaining cell polarity during migration (Geiger et al., 2001). In this regard, adaptor proteins are not only important for recruiting required surface receptors, but also in mediating LFA-1 outside-in signalling leading to cytoskeletal remodelling necessary for an efficient migration of T-cells. Both the combination of adaptor proteins and the sequence of their binding to the LFA-1 cytoplasmic tail are important in propagating LFA-1-mediated downstream signalling.

Mechanism of LFA-1-mediated outside-in signalling:

There are various regulatory mechanisms that control the spatial and temporal binding of a large number of adaptors to the limited binding sites on the LFA-1 tail. Phosphorylation of the LFA-1 cytoplasmic tail is one of the possible regulatory mechanisms, and both α and β chains of LFA-1 contain several potential phosphorylation sites that are phosphorylated after cell stimulation by pharmacological activators (e.g. phorbol esters), ICAMs or TCR ligation (Fargerholm et al., 2004). LFA-1 has also been shown to activate protein kinase C isoforms in T-cells, and the activation of this family of kinases is associated with LFA-1 phosphorylation at β2 chain S745 and T758-T760 (Fagerholm, 2002; Perez et al., 2003). S745 phosphorylation is important for LFA-1-dependent c-Jun activation and subsequent AP-1 activity (Figure 5). Further, this phosphorylation leads to physiological release of the transcriptional coactivator JAB-1 (also called COP9 signalosome subunit 5, CSN5) from LFA-1, which enables JAB-1 to participate in downstream signalling events (Perez et al., 2003; Kinoshita et al., 2012). As a signalosome component, JAB-1 is involved in inflammatory cascades, cell cycle regulation, apoptosis, carcinogenesis and immunity (Bianchi et al., 2000; Claret et al., 1996; Kleemann et al., 2000; Shackleford and Claret, 2010; Wei et al., 2008). The phosphorylation of threonine triplet T758-T760 in the β2 cytoplasmic tail of LFA-1 is required for its interactions with the actin cytoskeleton and for modulation of cell spreading and adhesion (Peter and O’Toole, 1995). In addition, LFA-1 T758 phosphorylation is required for its binding to 14-3-3, while preventing the binding of filamin. Although, this phosphorylation site does not directly regulate talin binding, 14-3-3 can outcompete talin for binding to phosphorylated β2 tail (Takala et al., 2008).

Important signal transduction processes that follow LFA-1 engagement include activation of various kinases and GTPases including PLCγ1 (Kanner et al., 1993), Src family tyrosine kinase Lck, Syk family tyrosine kinase ZAP-70 (Soede et al., 1993; Ref.), Pyk-2, FAK, ROCK, and RAP1 (Rodriguez-Fernandez et al., 1999) (Figure 5). Deficiency of ZAP70 through pharmacological inhibition or knockdown in T-cells prevents the conversion to high-affinity LFA-1, decreases the speed of migration on ICAM-1, and reduces firm adhesion under shear-flow conditions (Evans et al., 2011). These data suggest that active ZAP-70 is important for ligand-binding affinity of LFA-1 and LFA-1 induced T-cell migration. Blocking ROCK in T-cells also results in a failure of the trailing edge to detach (Smith et al., 2003). Although ROCK has many potential downstream effectors (Riento and Ridley, 2003), MLC is a key target in migrating T-cells. Pharmacological inhibition of ROCK blocks Ser19 phosphorylation of MLC (Smith et al., 2003), resulting in the enhanced adhesion of T-cells possibly by promoting integrin clustering (Rodriguez-Fernandez et al., 2001). There is convincing evidence for a role of RAP1-activated RAPL in the second phase of LFA-1 adhesion strengthening (Ebisuno et al. 2010). RAPL has been identified as a major effector of RAP1 responsible for delivering both RAP1 and LFA-1 to the membrane of migrating T-cells and to the immunological synapse. The RAPL binding site on the LFA-1 α-subunit tail features two lysine residues (K1097 and K1099) that are close to the membrane, as well as the conserved GFFKR motif (Katagiri et al., 2003). This suggests an important role of RAPL in the subsequent allosteric changes that separate the α- and β-subunit cytoplasmic tails leading to the high-affinity conformation. LFA-1 may therefore be stabilized by RAPL binding to the opposing LFA-1 α-subunit. These kinases and GTPases can therefore be considered as early, if not the first, mediators of outside-in signalling.

Additionally, adaptor proteins SLP-76, ADAP and Vav1 also play important roles in the LFA-1-mediated outside-in signalling (Suzuki et al., 2007). SLP-76 nucleates the formation of a ternary complex comprising SLP-76, Vav, and Nck (Ref). Binding of Vav and Nck to phosphorylated SLP-76 brings Vav in proximity with the GTPases Rac1 and CDC42, which in turn activates an evolutionary conserved family of serine/threonine protein kinases, PAK or p21-activated kinase (Hofman et al., 2004; Ref50). Another recent study proposes SLP-76 and Nck independent activation of PAK1 (Ref53). PAK kinases are strongly implicated in the regulation of cytoskeletal architecture (Hofman et al., 2004), focal adhesion contacts, cell motility and lymphocyte chemotaxis (Weiss-Haljiti et al., 2004; Volinsky et al., 2006). Other than cytoskeletal proteins tubulins and actin, PAK1 has been shown to interact with a number of migration-related proteins including Profilin, Cofilin, Importin, 14-3-3, Paxilin, Dynactin, Dynanin, Grb2 and PAK-interacting exchange factor PIX (Hofmann et al., 2004; Mayhew et al., 2006). Activation of PAK1 activates LIM kinase, which phosphorylates an inhibitory residue on Cofilin preventing Cofilin-mediated disassembly of actin filaments.

FERM-containing cytoplasmic adaptors in LFA-1 outside-in signalling:

The FERM-containing cytoplasmic adaptor proteins talin and kindlin3 are of fundamental importance in LFA-1-mediated signalling. Whereas the F3 sub-domain of talin binds directly to the proximal NXX(Y/F) motif in the integrin β tails, kindlin binds to the membrane-distal NXX(Y/F) motif in the integrin β tails. Although it is not known whether they bind sequentially or simultaneously, talin binding to β-subunit cytoplasmic tails of LFA-1 causes the separation of the integrin α and β tails, which triggers further structural changes in the integrin that involves unpacking of the transmembrane domains and unfolding of the ectodomain. Talin binding to LFA-1 is a highly regulated process (Ref) that increases LFA-1 ligand binding affinity and activates integrin functions. Kindlin-1 and -2 (Harburger et al., 2009) and integrin-linked kinase (ILK) (Honda et al., 2009) have been shown to control talin binding to β integrin cytoplasmic tails. The detailed mechanism(s) by which kindlin3 regulates the ligand binding affinity of integrins and whether both talin and kindlin serve the same or different functions in regulating conformational rearrangement of the LFA-1 ectodomain are still not clear. Studies have shown that the expression of talin is essential for the integrity of the LFA1 high-affinity focal zone in T-cells migrating on ICAM-1 (Ref. 63), and Talin1-/- mice are unable to make LFA-1-mediated contacts between T-cells and APCs (Ref 64). LFA-1 binding to ICAM-1 induces integrin αLβ2 micro-clustering and promotes physical association of kindlin3 PH domain with a scaffold protein RACK1, which stabilizes the activated forms of PKCs and enables the translocation of PKCs to different sites in the cell. Thus, it is possible that RACK1 recruits activated PKCs to the LFA-1/ICAM-1 induced ternary complex containing integrin β2 tail, RACK1 and kindlin3 that may ultimately influence both ligand binding and signalling activities of LFA-1. Surprisingly, kindlin-3 (absent in immune cells from LAD-III patients) is not required for T-cell motility over chemokines, nor for the collapse of microvilli by chemokine signals (Ref).

Paxicillin regulation of LFA-1-mediated outside-in signalling:

Talin regulation of LFA-1-mediated outside-in signalling (a separate paragraph required?)

CG-NAP regulation of LFA-1-mediated outside-in signalling (a separate paragraph required?)

STAT3 regulation of LFA-1-mediated outside-in signalling (a separate paragraph required?)

LFA-1-mediated outside-in signaling generates the "actin cloud":

LFA-1 stimulation in T-cells promotes the generation of a unique phenomenon of called "actin cloud", which is independent of TCR activation and proximal signalling. The actin cloud is localized at the centre of T-cell-APC interface, where it accumulates LFA-1 and tyrosine-phosphorylated proteins (Suzuki et al., 2007). The LFA-1-induced actin cloud formation involves ADAP phosphorylation through LFA-1/ADAP assembly, Src kinases, SLP-76, and MAPKs, including extracellular signal-regulated kinase 1 (Erk1)/Erk2 via the activation of LFA-1-associated cytohesin-1 and Jun, which is mediated by JAB-1 and c-Jun N-terminal kinase (JNK). Formation of actin cloud lowers the threshold for subsequent T-cell activation, which is impaired following LFA-1 stimulation of ADAP-deficient T-cells. Further, phosphorylated ADAP and clustured LFA-1 were also found in complex and ADAP co-immunoprecipitated with LFA-1 (Suzuki et al., 2007). Since ADAP has been shown to associate with and be phosphorylated by Fyn, LFA-1-mediated phosphorylation of ADAP may occur via Fyn, which provides the binding site for the SH2 domain of SLP-76 as the mechanism of TCR- and ZAP-70-independent SLP-76 phosphorylation. Thus, the actin cloud induced by LFA-1 engagement may serve as a possible platform for LFA-1-mediated costimulatory function for T-cell activation.

LFA-1 transduces T-cell migration signals through cytohesin-1

The adaptor protein cytohesin-1, which has a PH domain at the C-terminus and a guanidine exchange factor for a small GTPase, ADP ribosylation factor, has been reported to associate with LFA-1 and mediate outside-in signalling. It selectively binds to the integrin β2-chain through its Sec-7 and C-terminal PH domains (Ref). Additionally, cytohesin-1 colocalizes with active LFA-1 and regulates the activity of β2 integrins, especially LFA-1 in immune cells such as Jurkat cells or DCs (Ref). It also regulates LFA-1-dependent functional responses, including leukocyte adhesion to ICAM-1 or endothelial cells, rolling along, and transmigration through endothelial cell layers (Kolanus et al., 1996; Geiger, 2000; Weber et al., 2001; Quast et al., 2009). Overexpression of cytohesin-1 or its Sec-7 domain enhances adhesion of Jurkat T-cells to ICAM-1 (Kolanus et al., 1996), as well as chemokine-stimulated cell capture on and migration across endothelium (Weber et al., 2001). Since the PH domain of cytohesin-1 is also important for its association with the cell membrane and over-expression of the recombinant PH domain blocks LFA-1 function (Nagel et al., 1998), it can be suggested that cytohesin-1 must associate with the membrane to modulate LFA-1 activity. A cytohesin-1 binding protein CYTIP removes cytohesin-1 from the membrane and thereby down-regulates adhesion of T-cells to ICAM-1 (Boehm et al., 2003). The Sec-7 domain of cytohesin-1 also acts as a GEF for the ARF family of vesicle trafficking GTPases and, through ARF-6, causes spreading of Jurkat T-cells (Weber et al., 2001). Additionally, cytohesin-1 associates with actin following phosphorylation by PKCδ, and thus could provide a link between LFA-1 and the cytoskeleton (Dierks et al., 2001).

Crk adaptor proteins in LFA-1 outside-in signalling

The CT10 regulator of kinase (Crk) family of adaptors that includes CrkI, CrkII, CrkIII and Crk-Like (CrkL) proteins are of interest because they play major roles in signal transduction pathways mediated via both LFA-1 and α4 integrins, leading to lymphocyte adhesion and migration. They bind to a variety of key signalling molecules including the tyrosine phosphorylated paxillin, p85-PI3K, ZAP-70, Gab1 and JNK1, and thus coordinate multiple signalling pathways in T-cells. Crk family of proteins interact with a number of kinases in activated T-cells. They activate a serine-threonine kinase hematopoietic progenitor kinase 1 (HPK1), which functions as an upstream regulator of the JNK and plays a critical role in the signalling pathway leading to IL-2 induction (Ref). Crk and CrkL can also transiently associate with the ZAP-70 (Ref) and the Syk (Ref) in a lymphocyte activation-dependent manner. The observation that Crk can simultaneously interact with membrane-associated ZAP-70 and cytosolic WASP (Ref) has led to the assumption that Crk regulates WASP recruitment to the plasma membrane, thereby promotes actin polymerization and cytoskeletal rearrangement (Ref), which may play an important role in the formation and organization of the immunological synapse. In T-cells, the interaction of LFA-1 with ICAM-1 results in tyrosine phosphorylation and association of T-cell p130 Crk-associate substrate (p130Cas) homologus protein p105Cas with Crk or CrkL (Ref), which play pivotal roles in tyrosine kinase-based signalling pathways mediating cell adhesion and induction of cell migration (Ref). An additional docking protein that plays an important role in LFA-1-mediated signalling pathways in T-cells is the DOCK2, which associates with CrkL and regulates the activity of GEF for rac1, thereby promotes T-cell attachment (Ref).

Adapters in as a potential drugable targets in human diseases

The emergence of adaptor proteins as important regulators of LFA-1-mediated signalling opens novel avenues for potential targeted therapies that are designed to control T-cell migration. The integrin LFA-1 have been recognized to play vital roles in both health as an immune response regulator and diverse range of diseases including infection, cancer, thrombosis and autoimmune disorders. Initial success and expectations that antagonists directly targeting LFA-1/ICAM-1 interactions would be blockbuster drugs, led the development of efalizumab (Raptiva, Genetech), a humanized mAb specific for the αL subunit of LFA-1 approved in 2003 for the treatment of moderate to severe psoriasis. However, in contrast to its success in long-term trials, patients had worse outcomes owing to an increased chance of the development of PML, a potentially fatal side effect thought to be related to immune suppressive properties and led to its withdrawal in 2009 from the market. With the continued advances in the understanding of LFA-1-mediated signalling pathways, the concept of blocking downstream signalling components is becoming attractive and might prove to be more fruitful.

Although several therapeutic strategies are aimed at blocking LFA-1/ICAM-1 ligation, there is a recent appreciation that an alternative strategy may be to target intracellular signalling mechanisms to inhibit LFA-1 activation, thereby suppressing its binding to ICAM-1 or to prevent the downstream signalling cascades. The emergence of adaptors as important regulators of LFA-1 signal transduction suggests the possibility of their targeting as novel strategies for controlling integrin-mediated lymphocyte migration. Several research groups are engaged in studies that target rational drug design to block the ability of some adaptor proteins to form interactions with cellular signaling proteins. In spite of some success, clinical studies targeting individual adaptors in inflammatory diseases have been inconclusive and a combinatorial approach may be warranted. Studies examining the signalling mechanisms underlying the action of these adaptors on LFA-1 mediated T-cell migration have revealed that common pathways seem to be used. Moreover, some of the adaptors may perform cell-type or tissue specific functions. Given the important role of adaptor proteins in propagating cellular signals, it is quite likely that we will see much progress in this area of research in the near future and we will obtain a greater appreciation and understanding for the role of adaptor proteins in signal transduction and as potential targets for therapy. Recent advances in the understanding of LFA-1-mediated signalling have created substantial opportunities of targeting several other intracellular signalling processes as an alternative strategy to regulate T-cell trafficking. A better understanding of the functional versatility of adaptor proteins, their heterogeneity, cellular localization, and the mechanism by which they regulate T-cell migration will help appreciate how adaptors deliver functions to lymphocyte trafficking, ultimately offering opportunities for selective pharmacological manipulations.

While it could be hoped that adaptor therapy would be sufficient alone to regulate T-cell migration or to cure chronic inflammatory diseases, it is extremely unlikely. Although adaptors play important roles in various signalling processes, identifying the specific adaptors involved and their precise role in LFA-1-mediated T-cell motility is difficult because of multifactorial nature of the integrin LFA-1 and its involvement in multiple pathways. Existing studies suggest that some adaptors can play crucial roles in many physiological systems and any inhibition of these may result in serious adverse effects. In addition, recent studies have also identified human diseases that are caused by the loss of expression or function of particular adapter proteins. Alterations in the delicate balance of signals that are mediated by adapters could have severe consequences for immune homeostasis in vivo, such as the development of autoimmunity, immunodeficiency or cancer. Therefore, more developments is needed before adaptor targeted treatments can move from the laboratory bench to the bedside. The diversity of adaptor proteins and their complex role in LFA-1-mediated signalling suggests great potential for this super-family as drugable targets. We believe that, in situation where adaptors functions are known, they could be used in combination with other immunotherapy to overcome known side effects.