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 *

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...