Human SLP-65 isoforms contribute differently to activation and apoptosis of B lymphocytes

Abstract

The SH2 domain-containing leukocyte adaptor protein of 65 kDa (SLP-65) is the key effector for signaling downstream of the B-cell antigen receptor (BCR). SLP-65 controls not only B lymphopoiesis and humoral immunity but also possesses a yet poorly defined tumor suppressor activity that is lost in many cases of acute lymphoblastic leukemia. We found that the 2 isoforms of human SLP-65 are differentially involved in positive and negative B-cell signaling. Reconstitution experiments revealed that an atypical SH3 domain-binding motif, which is present in the long but not in the short SLP-65 isoform, mediates association to Grb2 and suppresses activation of mitogenactivated protein kinases p38 and JNK as well as up-regulation of c-Fos expression. In turn, the short isoform activates not only AP1-driven but also NF-B-driven gene transcription more potently than the long isoform. Conversely, the long rather than the short SLP-65 isoform promotes BCRinduced B-cell apoptosis. Our data further delineate the structural requirements of positive and negative SLP-65 signal transduction in normal and neoplastic cells. IntroductionProper development and immune effector functions of B cells rely on the intracellular adaptor protein SLP-65 1 (also named BLNK 2 or BASH 3 ). SLP-65 is used by the B-cell antigen receptor (BCR) on mature B lymphocytes as well as by the pre-BCR on precursor cells for coupling to a variety of intracellular signaling pathways. 4,5 Consequently, SLP-65 deficiency in engineered mouse mutants [6][7][8][9] or human patients 10 interferes with early B lymphopoiesis and causes severe defects in humoral immune responses. Moreover, the block of B-cell differentiation can lead to pre-B-cell acute lymphoblastic leukemia (pre-B ALL) due to continued pre-BCR signaling and perpetual V(D)J gene exon rearrangements. [11][12][13] To achieve the diverse signaling functions in a coordinated manner, SLP-65 accommodates modular protein interaction domains that allow the formation of multiple molecular signaling complexes in a stimulation-independent and stimulation-dependent manner. Proteins with Src homology (SH) 3 domains, such as Grb2, 1,2,14 can be constitutively recruited to the N-terminal half of SLP-65 by proline-rich binding motifs with the typical consensus sequence PxxP, where x can be any amino acid. Inducible protein associations of SLP-65 are mediated by phosphotyrosine/SH2-domain interactions in 2 ways. First, following BCR activation Syk-mediated phosphorylation of SLP-65 1,2 creates specific docking sites for SH2 domain-containing proteins such as the adaptor proteins Grb2, 1,2,14 Nck 1,2,14 and CrkL, 15 the guanine nucleotide exchange factor Vav, 1,2,14 the E3 ubiquitin ligase c-Cbl, 16 and, importantly, components of the Ca 2ϩ initiation complex, that is, Bruton tyrosine kinase (Btk) [17][18][19] and phospholipase C (PLC)-␥2. 2,[19][20][21] Second, the C-terminally located SH2 domain of SLP-65 binds the phosphorylated hematopoietic progenitor kinase HPK-1 22,23 and a non-ITAM phosphotyrosine residue in t...

SCIMP, a Transmembrane Adaptor Protein Involved in Major Histocompatibility Complex Class II Signaling

“…5D). Interestingly, even though SLP65 is expressed in two isoforms, SCIMP interacted only with the longer form, which contains a binding site for the SH3 domain of Grb2 (18). This implies that binding of SLP65 to SCIMP may require the formation of a trimolecular complex involving Grb2.…”

Repression of B-Cell Linker (BLNK) and B-Cell Adaptor for Phosphoinositide 3-Kinase (BCAP) Is Important for Lymphocyte Transformation by Rel Proteins

“…Reintroduction of BLNK restored BLNK À/À pre-B-cell differentiation and inhibited their capacity (54). The long isoform of BLNK was also implicated in promoting BCR-induced apoptosis (55). On the other hand, BCAP participates in the PI3K/Akt pathway that promotes B-cell proliferation and survival and also leads to activation of JNK upon BCR engagement (27,49).…”

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

“…Luciferase and ␤-galactosidase activities were assayed and standardized as described by Brummer et al (39) for resting DT40 B cells and cells stimulated through their BCR for 6 h with M4 monoclonal antibody at a concentration of 2 g/ml. Where indicated, cells were pretreated one hour prior to BCR stimulation with SB202190 or SP600125 (Biomol) to inhibit p38 or JNK, respectively (29).…”

“…Indeed, recent studies showed that SLP-65 is capable of regulating MAP kinase activity in an inositol triphosphate/diacylglycerol-independent manner (25,29). These data suggest that phosphotyrosine-dependent and phosphotyrosine-independent processes cooperate to modulate SLP-65 signal output for early and late activation events.…”