BLNK: molecular scaffolding through 'cis'-mediated organization of signaling proteins

Abstract

Assembly of intracellular macromolecular complexes is thought to provide an important mechanism to coordinate the generation of second messengers upon receptor activation. We have previously identified a B cell linker protein, termed BLNK, which serves such a scaffolding function in B cells. We demonstrate here that phosphorylation of five tyrosine residues within human BLNK nucleates distinct signaling effectors following B cell antigen receptor activation. The phosphorylation of multiple tyrosine residues not only amplifies PLCgamma-mediated signaling but also supports 'cis'-mediated interaction between distinct signaling effectors within a large molecular complex. These data demonstrate the importance of coordinate phosphorylation of molecular scaffolds, and provide insights into how assembly of macromolecular complexes is required for normal receptor function.

Inhibition of constitutive and BCR-induced Syk activation downregulates Mcl-1 and induces apoptosis in chronic lymphocytic leukemia B cells

“…We also show that Y352-phosphorylated Syk is enzymatically active, as evidenced by experiments with the Syk inhibitor R406, which showed inhibition in the basal phosphorylation of the direct Syk targets BLNK and Cbl. 44,45 In addition, the Constitutive activation of Syk in B-CLL S Gobessi et al treatment of CLL cells with R406 decreased the basal activity of several downstream signaling molecules that play important roles in regulating CLL-cell survival, such as the Akt, ERK and GSK-3 kinases and the FoxO1/3a transcription factors. 25,26,37,46,47 The specificity of R406 in inhibiting Syk-mediated activation of Akt and ERK was further confirmed in experiments with a constitutively active TEL-Syk protein.…”

Cutting Edge: Feed-Forward Activation of Phospholipase Cγ2 via C2 Domain–Mediated Binding to SLP65

“…Negatively charged amino acids guide tyrosine kinases to a C-terminally located substrate site (11). The 4G10 anti-pTyr Ab weakly recognizes phospho-Y 119 (data not shown) so that it was missed in an earlier study (4).…”

“…Phospho-SLP65 nucleates the assembly of downstream effectors into larger complexes to control a plethora of signaling pathways (3). For mobilization of the Ca 2+ second messenger, SLP65 simultaneously recruits phospholipase Cg2 (PLCg2) and Bruton's tyrosine kinase (Btk) to distinct phosphotyrosine (pTyr) residues by virtue of the enzymes' Src homology (SH) 2 domains (4)(5)(6). This structural organization allows Btk to phosphorylate and activate PLCg2.…”

“…However, SLP65 phosphorylation lasts longer than intracellular Ca 2+ fluxes, suggesting that the activity of PLCg2 or its inducible association to SLP65 is subject to further regulation. Moreover, the earliest and most prominent pTyr site of avian SLP65, tyrosine 138 (Y 138 ) (8), is not involved in SH2 domain binding (4) but is conserved between SLP65 orthologs and embedded within the consensus sequence EYFDN (singleletter code for amino acids with F representing A, V, or I). A similar motif can be found in the T cell paralog SLP76 (3), albeit at a different position (i.e., Y 173 in murine SLP76) and without a negatively charged residue N terminal to the tyrosine.…”

Fc gamma receptor IIb modulates the molecular Grb2 interaction network in activated B cells

“…Engagement of the BCR leads to phosphorylation of ITAMs and subsequent activation of the spleen tyrosine kinase (Syk) [1]. A bona fide Syk substrate is the adapter protein Src homology 2 (SH2) domain-containing lymphocyte protein of 65 kDa (SLP-65) [2] that upon phosphorylation provides docking sites for the SH2 domains of Bruton's tyrosine kinase (Btk) and phospholipase C-γ2 (PLC-γ2) [3,4]. Formation of this trimolecular complex leads to mobilization of Ca 2+ ions and activation of the NF κ -B and MAPK pathways [5].…”