Volume 10, Issue 5, Pages 1057-1069 (November 2002) Amplification of B Cell Antigen Receptor Signaling by a Syk/ITAM Positive Feedback Loop  Véronique Rolli, Maike Gallwitz, Thomas Wossning, Alexandra Flemming, Wolfgang W.A Schamel, Christa Zürn, Michael Reth  Molecular Cell  Volume 10, Issue 5, Pages 1057-1069 (November 2002) DOI: 10.1016/S1097-2765(02)00739-6

Figure 1 Inducible Expression of Multiple Genes in Transiently Transfected S2 Cells (A) Western blot analysis of coexpressed proteins in the total cellular lysate of transiently transfected S2 cells induced for the indicated times (h) with copper sulfate. (B) Tyrosine phosphorylated proteins in transiently transfected S2 cells without (-; lane 1) or with (+; lane 2) coexpression of ΔSHP-1. Protein production was induced for 24 hr with copper sulfate. The total cellular lysates of transfected S2 cells were analyzed for tyrosine phosphorylation (upper panel) and protein expression (second to fourth panel). The S2 cells were cotransfected with 0.2 μg of the following plasmids: pDSLP-65 + pDhSyk + pDmLyn + pDscδm + pDflmb-1 + pDHAB29 + pDCbl + pDGFP ± pDΔSHP-1. (C) The flag-tagged protein flIg-α is transported on the surface of S2 cells only as part of the heterodimer or the complete BCR. Flow cytometry analysis of flIg-α (ordinate) and EGFP (abscissa) expression of S2 cells 24 hr postinduction. The S2 cells express Syk, SLP-65, and EGFP either alone (a) or in combination with flIg-α (b), flIg-α, and HAIg-β (c), or the complete scBCR (d). S2 cells were stained with the monoclonal anti-flag antibody M2 and Cy5-conjugated goat-anti-mouse IgG as secondary antibody. The numbers indicated the percentage of cells in the respective quadrant. (D) Comparison of Syk (upper panel) and SLP-65 (lower panel) expression in transiently transfected S2 cells (lanes 1–3) with that in the established B cell lines J558L (lanes 4 and 5), WEHI231 (lanes 6 and 7), and K46 (lanes 8 and 9). The S2 cells express Syk and SLP-65 and EGFP either alone (lane 1) or in combination with the flIg-α/HAIg-β heterodimer (lane 2) and the complete scBCR (lane 3). The protein content of each lysate was determined by Bradford and equal amounts of total proteins were loaded except in lanes 5, 7, and 9 where a 1:10 dilution of the indicated lysates was used. Molecular Cell 2002 10, 1057-1069DOI: (10.1016/S1097-2765(02)00739-6)

Figure 2 Activation of Syk Requires Coexpression of the scBCR Western blot analysis of proteins in the lysates of S2 cells expressing for 24 hr either SLP-65 and Syk alone (lane 1), or SLP-65, Syk, and the scBCR (lane 2), or SLP-65 and the scBCR (lane 3). Proteins were analyzed for tyrosine phosphorylation (first panel) and expression (second to sixth panel). Molecular Cell 2002 10, 1057-1069DOI: (10.1016/S1097-2765(02)00739-6)

Figure 3 A Single ITAM-Containing Sequence Is Sufficient for the Activation of Syk (A) Western blot analysis of tyrosine phosphorylation (upper panel) and SLP-65 expression (lower panel) in the total lysates of S2 cells coexpressing for 24 hr SLP-65 and Syk alone (lane 1) or together with only Ig-α (lane 2), only Ig-β (lane 3) and different combinations of the Ig-α/Ig-β heterodimer with full-length or tailless (tl) subunits as indicated on the top (lanes 4–7). (B) Western blot analysis of tyrosine phosphorylation (first panel), Ig-α (second panel), and SLP-65 (third panel) expression in the total lysates of 24 hr induced S2 cells coexpressing SLP-65 + Syk alone (lane 1) or together with a scBCR containing scδm and either a wt Ig-α/Ig-β heterodimer (lane 2) or mutated forms of the heterodimer with the indicate Y/F mutations of the ITAM two tyrosines (lanes 3–5). (C) S2 cells coexpressing SLP-65 + Syk alone (lane 1) or together with a scBCR containing an Ig-α/Ig-βtl heterodimer with a tailless Ig-β and either wt or mutated Ig-α carrying either the F1, F2 or F1, 2 mutation of the ITAM tyrosines as indicated on the top (lanes 2–5). (D) Same analysis as in (C) except that here Lyn instead of Syk was coexpressed in the S2 cells. Molecular Cell 2002 10, 1057-1069DOI: (10.1016/S1097-2765(02)00739-6)

Figure 4 The Scr-Family Kinase Lyn Does Not Increase the Syk Activity in S2 Cells Coexpressing a scBCR with a Wild-Type ITAM Sequence (A) Western blot analysis for tyrosine phosphorylation (first panel), Ig-α (second panel), and SLP-65 (third panel) expression in the total lysates of S2 cells coexpressing for 24 hr SLP-65, scδm, and Ig-βtl together with the proteins indicated above. In this experiment the scBCR contained an Ig-α with either a wt (lanes 2, 4, and 6) or an F2 mutated ITAM (lanes 3, 5, and 7). (B) Interaction of endogenous Drosophila kinases with the scBCR. Western blot analysis for tyrosine phosphorylation (upper panel) and Ig-α (lower panel) expression in the total lysates of S2 cells. Untransfected (lane 1) or transfected S2 cells coexpressing for 24 hr a scBCR with Ig-βtl and a wt or ITAM-mutated Ig-α (lanes 2–5) were stimulated 3 min prior to lysis with 10 μM pervanadate. The position of a putative Drosophila Scr-family kinase (D-Scr) is indicated by an arrowhead. Molecular Cell 2002 10, 1057-1069DOI: (10.1016/S1097-2765(02)00739-6)

Figure 5 The BCR-Dependent Activation of Syk Requires the Phosphotyrosine Binding Site of the C-Terminal SH2 Domain of Syk (A) Scheme of wt and the analyzed mutants of Syk showing the position of the introduced point mutations or deletions (KN, kinase negative). (B) Western blot analysis of tyrosine phosphorylation (first panel), SLP-65 (second panel), and Syk (third panel) expression in the total lysates of S2 cells coexpressing for 24 hr SLP-65 together with the proteins indicated above. (C) In vitro kinase assay of wt (lane 1), the kinase negative (KN, lane 2), and the mSH2C mutant (lane 3) of Syk. Purified Syk proteins were incubated in the presence of 32P ATP with either GST (left panel) or GST Ig-α (right panel). The amount of phosphorylated proteins GST and Syk (second right panel) was determined by autoradiography, Coomassie blue staining, and Western blotting, respectively. Molecular Cell 2002 10, 1057-1069DOI: (10.1016/S1097-2765(02)00739-6)

Figure 6 Interaction of a GFPSyk(SH2)2 Fusion Protein with Wild-Type or Y/F Mutants of Ig-α or Ig-β (A) Western blot analysis of tyrosine phosphorylation, Ig-α, Syk, and SLP-65 expression (respectively, panel 1, 2, 3, and 4) in the total cell lysates of S2 cells transfected with SLP-65, Syk, and the indicated Ig-α or Ig-β proteins without (-) or with (+) the GFPSyk(SH2)2 fusion protein. (B) Model of the GFPSyk(SH2)2 fusion protein and its interaction with the ITAM sequence of Ig-α. Molecular Cell 2002 10, 1057-1069DOI: (10.1016/S1097-2765(02)00739-6)

Figure 7 The Activity of Syk but Not that of Lyn Is Efficiently Abolished by SHP-1 (A) Western blot analysis of tyrosine phosphorylation (1st and 4th panel) and expression of SLP-65 (2nd panel), Syk (3rd panel), Ig-α (5th panel), and SHP-1 (6th panel) in the total lysates of S2 cells coexpressing for 24 hr SLP-65 + the scBCR and either Syk (lanes 1 and 2) or Lyn (lanes 3 and 4) in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of SHP-1. (B) Redox regulation of PTP and Syk activity. Western blot analysis of tyrosine phosphorylation (1st panel) and expression of Ig-α (2nd panel) and SLP-65 (3rd panel) in the total lysate of S2 cells coexpressing for 24 hr SLP-65 and the indicated proteins. The transfected S2 cells were treated 1 hr prior to lysis with either DMSO (-) or DMSO containing 53 μM of the flavoprotein inhibitor diphenylene iodonium (DPI, +). Molecular Cell 2002 10, 1057-1069DOI: (10.1016/S1097-2765(02)00739-6)

Figure 8 Amplification and Control of BCR Signaling (A) Model of Syk as an allosteric enzyme positively regulated by a product feedback. In the cytosol Syk is mostly an inactive enzyme, as its kinase domain is blocked by an intramolecular complex involving the N-terminal part of Syk. In contact with an ITAM-containing sequence Syk phosphorylates the two ITAM tyrosines, and upon ppITAM binding it is stabilized in an open, active conformation. This results in phosphorylation of neighboring ITAM sequences, further Syk recruitment, and the rapid amplification of the BCR signal. (B) Model of Syk regulation. The positive Syk/ITAM feedback loop at the BCR is negatively controlled by PTPs. The PTP activity is inhibited by H202 and regulated by the redox equilibrium inside the cell. Engagement of the BCR by antigen may result in increased H202 production, thus initiating a double-negative feedback at the BCR. Molecular Cell 2002 10, 1057-1069DOI: (10.1016/S1097-2765(02)00739-6)