SLP-76 as a Scaffold for Signaling Complexes
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
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
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.
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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