SLP-65 Signal Transduction Requires Src Homology 2 domain-mediated Membrane Anchoring and a Kinase-independent Adaptor Function of Syk

Abstract

The SH2 domain-containing leukocyte adaptor proteins, SLP-65 (1) (BLNK (2), BASH (3)), and SLP-76 (4), are instrumental to integrate and collect signals from antigen receptors on B and T lymphocytes, respectively. In their N-terminal half, the two effector proteins encompass a variety of similar consensus motifs for either inducible tyrosine phosphorylation by Syk/ ZAP-70 family kinases or constitutive association to proteins with Src homology (SH) 3 3 domains. Both SLP adaptors possess a C-terminal SH2 domain with a very high degree of sequence similarity and reported binding specificity for phosphorylated tyrosine residues in the consensus motif YXDV (in single letter code for amino acids, with X being any amino acid). The similar overall structure of the two SLP family members matches their closely related signaling roles. Most prominently, their scaffolding functions are mandatory for the correct subcellular localization and activation of phospholipase C-␥ (PLC-␥) isoforms to induce mobilization of the second messenger Ca 2ϩ . Surprisingly and despite the common protein architecture, the molecular mechanisms through which SLP-65 and SLP-76 assemble and target the Ca 2ϩ initiation complexes in B and T cells, respectively, appear to be different (5).Downstream of the B cell antigen receptor (BCR), phosphorylated SLP-65 provides docking sites for the SH2 domains of Bruton's tyrosine kinase (Btk) and PLC-␥2 (2, 6 -9). Simultaneous recruitment of both enzymes must occur in cis, i.e. to a given SLP-65 molecule because only tri-molecular complex formation enables Btk to phosphorylate and thereby activate its target PLC-␥2 (9). To provide PLC-␥2 with access to its lipid substrate phosphatidylinositol 4,5-bisphosphate, the assembled Ca 2ϩ initiation complex needs to be tethered at the plasma membrane. As shown more recently, membrane anchoring requires an N-terminal leucine zipper motif in SLP-65, but the exact mechanism remains elusive (10). However, the N terminus of SLP-76 does not contain such a leucine zipper. In fact, nucleation of the Ca 2ϩ initiation complex upon engagement of the T cell antigen receptor (TCR) involves not only the intracellular adaptor SLP-76 but also (the transmembraneous linker of activated T cells) LAT (5,11,12). Tyrosine-phosphorylated LAT binds PLC-␥1 directly and associates simultaneously with a SLP-76/Itk complex through the Grb2 family member Gads. Itk is the T cell paralogue of Btk. LAT-and Gads-related molecules in B cells are NTAL (13) (alternatively named Lab (14)) and Grb2 itself. We recently showed that the NTAL/Grb2 module is indeed critically involved in BCR-triggered Ca 2ϩ elevation but through a SLP-65-independent mechanism (15, 16).One mode of stimulation-dependent membrane recruitment of SLP-65 involves its SH2 domain, which directly binds the *

Signaling of the ITK (Interleukin 2-inducible T Cell Kinase)-SYK (Spleen Tyrosine Kinase) Fusion Kinase Is Dependent on Adapter SLP-76 and on the Adapter Function of the Kinases SYK and ZAP70

“…Likewise, in normal cells, the importance of the adapter function of ZAP-70 has been shown for the development of immature CD4 ϩ CD8 ϩ thymocytes having limited expression of LCK (37). In SYK, the importance of an adapter function for calcium mobilization was demonstrated in the chicken cell line DT-40 cells as well as in mouse B lymphocytes (29,33). In a recent report, suppression of Treg cells was also demonstrated to be independent of ZAP-70 kinase activity (38).…”

Macromolecular assembly of the adaptor SLP-65 at intracellular vesicles in resting B cells

“…S1) (11,12). To investigate the underlying mechanism, we visualized the subcellular distribution of citrine-tagged versions of wild-type and N-terminally truncated SLP-65 (DN-SLP-65) with laser scanning confocal microscopic analysis of resting and BCR-activated transductants of the chicken B cell line DT40 (Fig.…”

“…Mutational analyses showed that a short N-terminal SLP-65 segment of about 50 amino acid residues is indispensable for SLP-65 signaling (11). Furthermore, deletion of the N terminus of SLP-65 interfered neither with the functionality of the SLP-65 SH2 domain nor with the ability of SLP-65 to form a complex with CIN85 (12,13). These findings demonstrated that, in addition to the SH2 domain and the CIN85-binding sites, the N-terminal region of SLP-65 constitutes a third and separate targeting device that controls a nonredundant route of SLP-65 activation.…”

SLP-65 Phosphorylation Dynamics Reveals a Functional Basis for Signal Integration by Receptor-proximal Adaptor Proteins

“…This enables SLP-65 to nucleate the formation of a multiprotein complex by recruiting several SH2 domain-containing effector proteins such as phospholipase (PLC)-␥2 and Bruton's tyrosine kinase (21). SLP-65 not only assembles this signalosome but is also critical for its stimulation-induced translocation from the cytosol to the plasma membrane (22,23). Assembly and membrane targeting of this complex are both requisites for PLC-␥2 to hydrolyze membrane phospholipids resulting in the generation of diacylglycerol and inositol triphosphate, which in turn induces the release and entry of Ca 2ϩ ions from intra-and extracellular sources, respectively (24 -26).…”