1 The Dok-3/Grb2 signal module attenuates Lyn-dependent activation of Syk in B cell antigen receptor microclusters Marion Lösing 1,5 , Ingo Goldbeck 1,6 , Birgit Manno 1 , Thomas Oellerich 1,7 , Tim Schnyder 2 , Hanibal Bohnenberger 1 , Björn Stork 1,8 , Henning Urlaub 3,4 , Facundo D. Batista 2 , Jürgen Wienands 1# , and Michael Engelke 1# 1 Georg August University of Göttingen, Institute of Cellular and Molecular Immunology, Humboldtallee 34, 37073 Göttingen, Germany 2 Lymphocyte Interaction Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, London, WC2A 3LY, UK. 3 Max Planck Institute of Biophysical Chemistry, Bioanalytical Mass Spectrometry, Am Fassberg 11, 37077 Göttingen, 4 Bioanalytics Department of Clincal Chemistry, University Medical Center Goettingen, Robert-Koch- Straße 40, 37075 Göttingen, Germany 5 Current address: Vivo Science GmbH, Fabrikstr. 3, 48599 Gronau, Germany 6 Current address: Richter-Helm BioLogics, Habichthorst 30, 22459 Hamburg 7 Current address: Johann Wolfgang Goethe University of Frankfurt, Department of Hematology and Oncology, Theodor-Stern-Kai 7, 60595 Frankfurt, Germany 8 Current address: University of Düsseldorf, Institute of Molecular Medicine, Universitätsstr. 1, 40225 Düsseldorf Running title: The Dok3/Grb2 module inhibits Syk avtivity in B cells # to whom the correspondence should be addressed: Jürgen Wienands, Georg August University of Göttingen, Institute of Cellular and Molecular Immunology, Humboldtallee 34, 37073 Göttingen, Germany. Phone +49 (0) 551 395812; fax: +49 (0) 551 395843; e-mail address: jwienan@gwdg.de Michael Engelke, Georg August University of Göttingen, Institute of Cellular and Molecular Immunology, Humboldtallee 34, 37073 Göttingen, Germany. Phone +49 (0) 551 3913858; fax: +49 (0) 551 395843; e-mail address: mengelk@gwdg.de Keywords: BCR, Lyn, Syk, Dok-3, Grb2, SHIP Background: The signal module comprising Dok-3 and Grb2 controls differential BCR signal intensity. Results: Dok-3/Grb2 transolocate 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 towards 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. SUMMARY Recruitment of the growth factor receptor-bound protein 2 (Grb2) by the plasma membrane-associated adapter protein downstream of kinase-3 (Dok-3) attenuates signals transduced by the B cell antigen receptor (BCR). Here we describe molecular details of Dok-3/Grb2 signal integration and function showing that the Lyn-dependent activation of the BCR transducer kinase Syk is attenuated by Dok-3/Grb2 in a site-specific manner. This process is associated with the SH3 domain-dependent translocation of Dok- 3/Grb2 complexes into BCR micro- signalosomes and augmented phosphorylation of the inhibitory Lyn target SHIP. Hence, our findings imply that Dok-3/Grb2 modulates the balance between activatory and inhibitory Lyn functions with the aim to adjust BCR signaling efficiency. B cell development and function is strictly dependent on signals that are transduced by the B cell antigen receptor (BCR) consisting of a transmembrane immunoglobulin component and an Ig/Ig heterodimer (1,2). Antigen binding to