Spatial and temporal modulation of intracellular Ca2+ fluxes controls the cellular response of B lymphocytes to antigen stimulation. Herein, we identify the hematopoietic adaptor protein Dok-3 (downstream of kinase-3) as a key component of negative feedback regulation in Ca2+ signaling from the B-cell antigen receptor. Dok-3 localizes at the inner leaflet of the plasma membrane and is a major substrate for activated Src family kinase Lyn. Phosphorylated Dok-3 inhibits antigen receptor-induced Ca2+ elevation by recruiting cytosolic Grb2, which acts at this location as a negative regulator of Bruton's tyrosine kinase. This leads to diminished activation of phospholipase C-gamma2 and reduced production of soluble inositol trisphosphate. Hence, the Dok-3/Grb2 module is a membrane-associated signaling organizer, which orchestrates the interaction efficiency of Ca2+-mobilizing enzymes.

Background:The signal module comprising Dok-3 and Grb2 controls differential BCR signal intensity. Results: Dok-3/Grb2 translocate to BCR microsignalosomes and inhibit Lyn-dependent activation of the BCR transducer kinase Syk. Conclusion: Dok-3/Grb2 change the balance of activatory and inhibitory Lyn functions toward BCR signal inhibition. Significance: Learning how adapter proteins translocate to and change signal processes in BCR microsignalosomes is important to understand the regulation of antigen-induced B cell activation.

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 *

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