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

Abstract

The traditional view of how intracellular effector proteins are recruited to the B cell antigen receptor (BCR) complex at the plasma membrane is based on the occurrence of direct protein-protein interactions, as exemplified by the recruitment of the tyrosine kinase Syk (spleen tyrosine kinase) to phosphorylated motifs in BCR signaling subunits. By contrast, the subcellular targeting of the cytosolic adaptor protein SLP-65 (Src homology 2 domain-containing leukocyte adaptor protein of 65 kD), which serves as a proximal Syk substrate, is unclear. We showed that SLP-65 activation required its association at vesicular compartments in resting B cells. A module of ∼50 amino acid residues located at the amino terminus of SLP-65 anchored SLP-65 to the vesicles. Nuclear magnetic resonance spectroscopy showed that the SLP-65 amino terminus was structurally disordered in solution, but could bind in a structured manner to noncharged lipid components of cellular membranes. Our finding that preformed vesicular signaling scaffolds are required for B cell activation indicates that vesicles may deliver preassembled signaling cargo to sites of BCR activation.

Tripartite phase separation of two signal effectors with vesicles priming B cell responsiveness

“…Tripartite phase separation at physiological concentrations. In accordance with previously published data 13,15 , citrine-tagged wild-type SLP65 but not mutant versions of SLP65 lacking either the N-terminal lipid binding domain or the CIN85-interacting PRMs formed submicrometer granules in resting B cells (Fig. 1a).…”

“…1). Hence, the formation of granules through coordinated interactions of SLP65 with both vesicles and CIN85 appeared requisite for proper SLP65 signaling as suggested previously 13,15 . Next, we investigated by confocal fluorescence microscopy, whether granule formation can be recapitulated in vitro using recombinantly expressed SLP65 and CIN85 and synthetic lipid vesicles in different combinations.…”

“…However, the vesicular compartmentalization of SLP65 appeared to be critical for the stimulation-independent (i.e. constitutive) association of SLP65 with CIN85 13,15 . The association of SLP65 and CIN85 is caused by atypical SH3 domain docking motifs in SLP65 that associate in a promiscuous manner with the three N-terminal SH3 domains of CIN85 15 .…”

“…More recently, we found that the signaling function of SLP65 relies on its interaction with intracellular vesicles to which it is attached by virtue of an N-terminal lipid binding moiety 13,14 . A majority of these vesicles stained positive for vesicle-associated membrane protein (VAMP) 7 and quinacrine, but there was no unique marker or combination of markers characterizing a particular cellular vesicle pool that specifically attracts SLP65 molecules.…”

Elucidation of tonic and activated B-cell receptor signaling in Burkitt’s lymphoma provides insights into regulation of cell survival

“…We have shown here that cytoskeleton regulators represent one of the largest groups of phosphoacceptor proteins and have identified putative candidates that might control these processes, including ACTN4. Moreover, a recent study indicates that vesicles deliver preassembled SH2-domain-containing leukocyte protein of 65 kD (SLP65) signaling cargos to sites of BCR activation (39). Thus, the down-regulation of ARFGEF2 expression might interfere with vesicle trafficking and thereby disturb tonic BCR signaling.…”