Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

SLP-76

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_474

Synonyms

Historical Background

SLP-76 was first identified as a substrate of the protein tyrosine kinases that are activated by T-cell antigen receptors (TCRs) (Jackman et al. 1995). SLP-76, which functions as a signal transducer downstream of TCRs, is an adaptor protein that has three modular domains: an amino (N)-terminal acidic domain with three tyrosine (Y) phosphorylation motifs, a central proline-rich region, and a carboxy (C)-terminal SH2 domain (Fig. 1). Two other proteins, B-cell linker protein (BLNK/BASH/BCA/SLP-65) and cytokine-dependent hematopoietic cell linker (CLNK/MIST), constitute a family of SLP-76 adaptors, each expressed exclusively in hematopoietic cells. The three family members have only a moderate degree of sequence homology, but share a characteristic domain structure (Fig. 1). These SLP-76 family members are critical for nucleation of large signaling complexes in diverse hematopoietic cell types.
SLP-76, Fig. 1

Domain structures of SLP-76 adaptor family members. Domains found in the SLP-76 adaptor family members are schematically shown. LZ leucine-zipper motif domain, SAM sterile alpha motif domain, SH2 Src homology 2 domain, PR proline-rich region. The amino acid position numbers are indicated in SLP-76

SLP-76 as a Scaffold for Signaling Complexes

Investigation into the three functional domains of SLP-76 has revealed how SLP-76 operates in TCR signaling. SLP-76 regulates T-cell function in various ways through its binding partners (Fig. 2).
SLP-76, Fig. 2

Binding partners for functional domains of SLP-76. Binding partners for each domain structure of SLP-76 and the consequent functional outcomes are shown. ADAP adhesion- and degranulation-promoting adaptor protein, Gads Grb2-related adaptor proteins, HPK1 hematopoietic progenitor kinase 1, Itk interleukin 2-inducible T-cell kinase, LAT linker for activation of T cells, NCK non-catalytic region of tyrosine kinase, PAK1 p21-activated kinase 1, PI3K phosphatidylinositol 3-kinase, PLC phospholipase C, WASP Wiskott-Aldrich syndrome protein

The amino-terminal tyrosines of SLP-76 are essential for SLP-76 function. Three tyrosines at amino acids 112 (113 in humans), 128, and 145 become phosphorylated after TCR stimulation. Mutation of tyrosine 112 and/or 128 abrogates the inducible association of SLP-76 with the guanine nucleotide exchange factor (GEF) Vav1 (Wu et al. 1996), the adaptor non-catalytic region of tyrosine kinase (NCK) (Wunderlich et al. 1999), and the p85 subunit of phosphatidylinositol 3-kinase (PI3K) (Shim et al. 2004). Mutation of tyrosine 145 results in the loss of binding to Tec family tyrosine kinase interleukin 2-inducible T-cell kinase (Itk) (Su et al. 1999). Recently, a sterile-α motif (SAM) was identified at amino acids 12–78 just proximal to the three tyrosines, but it is not currently known what interaction(s) the SAM domain of SLP-76 mediates.

The central proline-rich region of SLP-76 contains a characterized SH3-domain-binding motif (RxxK) that mediates constitutive interaction with Grb2-related adaptor proteins (Gads) (Berry et al. 2002). In addition to the Gads-binding site, the P1 domain at amino acids 157–223 was defined as a region necessary for the basal association of SLP-76 with phospholipase C-γ1 (PLCγ1) (Yablonski et al. 2001). However, the critical aspect of the P1 domain probably lies more in its role as a molecular spacer, preventing the premature recruitment of PLCγ1 to the cell membrane by associating with the transmembrane adaptor linker for activation of T cells ( LAT), rather than as a mediator of an essential protein/protein interaction (Gonen et al. 2005; Jung et al. 2010). The Src family kinase Lck also binds the proline-rich domain of SLP-76 (Sanzenbacher et al. 1999). Recently, serine 376 within the proline-rich region was shown to be phosphorylated by the serine-threonine kinase hematopoietic progenitor kinase 1 (HPK1), one of the binding partners of the SLP-76 SH2 domain. Phosphorylation at this site results in decreased TCR signal transduction (Shui et al. 2007), but the mechanism has yet to be defined.

The carboxy-terminal SH2 domain of SLP-76 inducibly associates with adhesion- and degranulation-promoting adaptor protein ( ADAP) (Musci et al. 1997) and HPK1 (Sauer et al. 2001). The SLP-76 SH2 domain can also associate with the cytoplasmic tail of the T-cell surface receptor CD6 (Hassan et al. 2006).

Role of SLP-76 in TCR Signaling

TCR ligation results in activation of the Src family kinase Lck, which phosphorylates tyrosines in the immunoreceptor tyrosine–based activation motifs (ITAMs) present within the  CD3 chains of the TCR complex. Phosphorylated ITAMs recruit the Syk family kinaseζ-chain-associated protein kinase of 70 kDa ( ZAP-70), leading to phosphorylation of SLP-76 and LAT, which is a critical step in the assembly of the signaling complex by the two adaptors. Phosphorylation of LAT indirectly recruits SLP-76 via Gads binding, and the two adaptors nucleate a multimolecular complex of several signaling proteins and transport this complex to the cell membrane, where LAT is constitutively localized within microdomains known as lipid rafts or glycolipid-enriched membrane domains (GEMs). For PLCκ1 enzyme activation, recruitment to the GEM and tyrosine phosphorylation is induced by binding to the phosphotyrosine of LAT via its SH2 domain, and SLP-76 recruitment to LAT via Gads is also required for this process (Yablonski et al. 1998). Thus, LAT, SLP-76, and Gads appear to constitute an integrated signaling unit that couples TCR-mediated activation of cytoplasmic protein kinases to PLCγ1 enzyme activation (Fig. 3).
SLP-76, Fig. 3

Role of SLP-76 in T-cell function. SLP-76-mediated signaling pathways downstream of the T-cell receptor: cellular activation and inside-out signaling are shown. AP1 activator protein 1, DAG diacylglycerol, IP3 inositol-1, 4, 5-trisphosphate, MAPK mitogen-activated protein kinase, NFAT nuclear factor of activated T cells, NF-κB nuclear factor-κB, PKC protein kinase C, Rap1 Ras-proximity-1, RasGRP Ras guanyl-releasing protein, RIAM Rap1-GTP-interacting adaptor molecule, SKAP Src kinase–associated phosphoprotein, Sos son of sevenless, TCR T-cell receptor

The role of SLP-76 in TCR signaling was determined from overexpression studies in the Jurkat human leukemic T-cell line, demonstrating that SLP-76 is a positive regulator of TCR signaling. Complementary loss-of-function studies in SLP-76-deficient Jurkat cells known as J14 cells (Yablonski et al. 1998) revealed considerably reduced phosphorylation of PLCγ1, but not of ZAP-70, LAT, and Itk, showing that SLP-76 links proximal signaling molecules and downstream effectors. Thus, signaling events due to PLCγ1 activation, such as the generation of inositol-1, 4, 5-trisphosphate (IP3) and diacylglycerol (DAG) and resulting intracellular calcium flux, the activation of protein kinase C (PKC) family members, and indirect activation of Ras through Ras guanyl-releasing protein ( RasGRP), are all reduced. Upregulation of a T-cell activation marker CD69, transcriptional activity of nuclear factor of activated T cells ( NFAT), and activator protein 1 (AP1) is significantly reduced with SLP-76 deficiency (Yablonski et al. 1998).

The SLP-76-binding partners Vav1 and NCK, which inducibly associate with the amino-terminal tyrosines of SLP-76 after TCR stimulation, regulate cytoskeletal organization via Cdc42 recruitment through p21-activated kinase 1 (PAK1) and Wiskott-Aldrich syndrome protein (WASP). This model of TCR-mediated cytoskeletal rearrangement implies that SLP-76 regulates actin polymerization in response to TCR stimulation by regulating colocalization of Vav1 with NCK (Zeng et al. 2003) (Fig. 3).

SLP-76 has been implicated in TCR-induced integrin activation, so-called “inside-out signaling” because of its association with ADAP through the SH2 domain. Mutational analysis of ADAP at sites of interaction with SLP-76 diminishes the ability of T cells to adhere to integrin-coated surfaces. The molecular mechanism of SLP-76-mediated inside-out signaling to integrins is thus likely ADAP dependent. Recently, direct involvement was shown for SLP-76 in membrane targeting and activation of the small GTPase Ras-proximity-1 (Rap1), which promotes clustering and affinity modulation of integrins for their full activation, although the domain requirements have not been identified (Horn et al. 2009) (Fig. 3).

SLP-76 Signaling in Other Receptor Systems

The amino-terminal tyrosine motifs of SLP-76 are the earliest phosphorylation substrates of Src and Syk family kinases and are activated by ligation of tyrosine kinase–associated receptors on a variety of hematopoietic cell types. This tyrosine phosphorylation of SLP-76 induces associations with SH2 domain–containing proteins. The central proline-rich sequences mediate constitutive interactions with SH3 domain–containing proteins, and the carboxy-terminal SH2 domain of SLP-76 induces phosphotyrosine-dependent interactions with additional binding partners. Consequently, the SLP-76 adaptor nucleates large signaling complexes and plays critical roles in a variety of tyrosine kinase–associated receptor signaling pathways in various hematopoietic cell types (Fig. 4).
SLP-76, Fig. 4

Role of SLP-76 in signaling triggered by other surface receptors in hematopoietic cell types. SLP-76 signaling in other receptor systems that are associated with a variety of tyrosine kinases is shown

SLP-76 expressed in platelets is phosphorylated after stimulation of the ITAM-bearing collagen receptor, glycoprotein (GP) VI. As in T cells, SLP-76 lies downstream of the Src and Syk family kinases but upstream of PLCγ activation. SLP-76 is also inducibly phosphorylated upon ligation of the platelet integrin αIIβB3, which is required for normal platelet spreading on its ligand fibrinogen. Unlike TCR signaling, which depends critically on SLP-76 and LAT as well as the inducible interaction between these two adaptor proteins, collagen receptor and integrin signaling in platelets rely more on SLP-76 than on LAT for their function (Judd et al. 2002).

In addition to T cells and platelets, SLP-76 is expressed in multiple hematopoietic lineages including neutrophils, mast cells, macrophages, and natural killer (NK) cells (Jackman et al. 1995). The predominant ITAM-bearing receptors on these cells are Fc receptors (FcRs) including FcɛRI in mast cells and FcγRs in other cell types. The signaling molecules that are both upstream and downstream of SLP-76 are similar among these receptors and across cell types. However, important differences exist in how SLP-76 coordinates signal transduction across different cell and receptor types.

Cross-linking of FcɛRI, which constitutively interacts with the Src family kinase Lyn, with antigen-bound IgE induces activation and recruitment of Lyn and Syk. Consequently, tyrosine phosphorylation of SLP-76 and nucleation of a signaling complex similar to the one formed in T cells upon TCR activation occur. This complex includes LAT, Vav1, Btk, Gads, PLCγ1/2, and ADAP. SLP-76-deficient bone marrow–derived mast cells degranulate poorly and produce negligible amounts of the cytokine interleukin-6 and also show biochemical defects such as diminished calcium flux and phosphorylation of PLCγ2 (Pivniouk et al. 1999).

Upon FcγR stimulation in neutrophils, signals and effector functions are induced by activation of the Src family kinases Hck and Fgr, followed by Syk activation. These responses in neutrophils are synergistically augmented by cell adhesion. SLP-76-deficient neutrophils show reduced calcium flux and production of reactive oxygen species in response to FcγR stimulation and also fail to spread upon stimulation with integrins (Newbrough et al. 2003). These data suggest that SLP-76 is involved in both FcγR and integrin signaling in neutrophils.

In contrast to neutrophils, SLP-76-deficient macrophages respond normally to FcγR signals, leading to intact phagocytosis function and production of reactive oxygen species (Nichols et al. 2004). Similarly, SLP-76 appears dispensable for the FcγRIIIA-mediated killing function in NK cells (Peterson et al. 1999).

SLP-76-Deficient Mice and Defects in Multiple Lineages

The importance of SLP-76 as a regulator of signaling pathways for cellular function was highlighted by SLP-76-deficient mice. SLP-76-deficient mice present in Mendelian ratios in utero suffer perinatal mortality in more than 60% of the mice. SLP-76-deficient fetuses show varying degrees of subcutaneous hemorrhage, and surviving mice have blood in the peritoneum (Clements et al. 1998). Adult SLP-76-deficient mice have very small thymi and lack peripheral T cells and lymph nodes. By contrast, normal numbers of macrophages and NK cells are seen in these mice (Clements et al. 1998).

SLP-76-deficient thymocytes are unable to induce allelic exclusion and β-selection due to the failure to transduce pre-TCR signals, resulting in a complete block of thymocyte development at the double-negative 3 stage (Clements et al. 1998). A possible role for SLP-76 in pre-B-cell receptor signaling at the pre-B-cell stage has been shown. Despite hemorrhage in the peritoneum in SLP-76-deficient mice, normal platelet development and normal bleeding times are observed (Clements et al. 1998). Loss of SLP-76 in platelets results in decreased PLCγ2 activation, aggregation, and degranulation in response to collagen as well as decreased spreading on fibrinogen. SLP-76-deficient neutrophils show defective responses to integrin stimulation, with a loss of reactive oxygen species production and failure to spread, as well as a reduced response to FcγR stimulation (Newbrough et al. 2003). SLP-76-deficient mice have a normal number and distribution of mast cells, but SLP-76 deficiency results in loss of mast cell–dependent responses: reduced degranulation, cytokine production, and consequent loss of passive systemic anaphylaxis (Pivniouk et al. 1999).

The abnormal subcutaneous hemorrhagic phenotype in SLP-76-deficient mice is due to a failure of separation of the lymphatic and blood vascular networks during development (Abtahian et al. 2003). SLP-76-deficient mice have chimeric vessels composed of lymphatic and blood endothelial cells, with blood-filled lymphatics sometimes observed. The exact role of SLP-76 for vascular development has yet to be determined.

Summary

SLP-76 was first cloned in 1995 as a substrate of the TCR-activated protein tyrosine kinases and a binding protein for Grb2. Since then, initial characterization of the role of SLP-76 in TCR signaling has documented SLP-76 as an adaptor protein containing multiple protein interaction domains, creating a molecular scaffold on which key signaling complexes are built. The adaptor protein SLP-76 is expressed in multiple lineages of hematopoietic cells including T cells, platelets, and neutrophils. The details of how SLP-76 functions were elucidated both in T cells and other hematopoietic lineages using cell lines and SLP-76-deficient mice. Besides having critical roles for signaling downstream of both immunoreceptors and integrins, SLP-76 may function as a regulator of additional cell surface receptor signals such as those in the vasculature. Insights from studies of how SLP-76 regulates immune cell development and coordinates signal transduction across different cell and receptor types will provide important information about how the immune system uses various strategies for optimal host defense. Based on these observations, potential therapeutic targets can be explored.

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Department of Life Science, College of Natural SciencesEwha Womans UniversitySeoulKorea