Molecular Requirements for Syk-Mediated Intracellular Signaling and B-Cell Transformation

The protein tyrosine kinase Syk is a key mediator of proximal B-cell receptor (BCR) signaling. Following antigen stimulation Syk is recruited to the BCR and becomes activated by sequential phosphorylation at conserved tyrosine (Y) residues. The first event involves phosphorylation at Y352 by Lyn or other Src family kinases, followed by trans-autophosphorylation of YY525/526 in the activation loop. Once activated, Syk further propagates the BCR signal by associating with adaptor proteins and phosphorylating downstream signaling molecules. Recently, we and others have observed that Syk is constitutively phosphorylated on Y352 in primary tumors and cell lines from patients with chronic lymphocytic leukemia (CLL), follicular lymphoma, diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma, indicating that antigen-independent Syk activation may play a role in the pathogenesis of these B-cell malignancies. We have now investigated in more detail the phosphorylation status of Syk in a series of primary CLL B-cells samples and DLBCL cell lines and observed that unlike Y352, the activation loop tyrosines at position 525/526 are not constitutively phosphorylated. However, YY525/526 become efficiently phosphorylated in the same cells following BCR stimulation with anti- IgM or anti-IgG antibodies, indicating that antigen-dependent and antigen-independent Syk activation may differ with respect to the phosphorylation status of the activation loop tyrosines. To determine whether there are differences in the signaling capacity of Syk phosphorylated only on Y352 and Syk phosphorylated on both Y352 and YY525/526, we generated Syk phosphomimetics in which these tyrosines were substituted with aspartic acid (D) or phenylalanine (F), Aspartic acid is commonly used to introduce a negative charge that mimics phosphorylation, whereas phenylalanine is used to mimic an unphosphorylated tyrosine. The various phosphomimetics were transfected into the IL-3 dependent B-cell line BaF3 and their activity was evaluated by analyzing phosphorylation of downstream signaling molecules following IL-3 withdrawal. These experiments revealed that Syk phosphorylated on Y352 but not phosphorylated on YY525/526 is enzymatically active, since a D352-FF525/526 phosphomimetic was capable of inducing efficient phosphorylation of PLCγ2, ERK, Akt, FoxO1/3a, 4E-BP1 and Cbl. To evaluate the activity and substrate specificity of Syk phosphorylated on both Y352 and YY525/526, we first performed experiments with a Syk D352 phosphomimetic. This protein, however, showed only weak phosphorylation at YY525/526, presumably because of inefficient Syk dimerization and trans-autophosphorylation. Therefore, to allow for more effective trans-autophosphorylation at YY525/526 we produced a TEL-Syk fusion protein in which dimerization is induced by the TEL domain. In comparison to the D352-FF525/526 phosphomimetic, the TEL-Syk fusion protein was 7 to 70 fold more potent in inducing the phosphorylation of Akt, ERK and PLCγ2, thus demonstrating that phosphorylation of the activation loop tyrosines substantially increases Syk kinase activity. Moreover, in contrast to the D352 and D352-FF525/526 Syk phosphomimetics, the TEL-Syk protein was capable of sustaining IL-3 independent proliferation of BaF3 cells, indicating that phosphorylation at 525/526 is required for the B-cell transforming capacity of Syk. This possibility was confirmed in subsequent experiments with a mutated TEL-Syk protein in which YY525/526 were substituted with phenylalanines. The TEL-Syk DD525/526 mutant lost the capacity to sustain IL-3 independent growth of BaF3 cells and showed reduced kinase activity that was comparable to the activity of the D352 and D352-FF525/526 Syk phosphomimetics. In conclusion, these data confirm that Syk phosphorylated only on Y352 is enzymatically active and capable of downstream signaling, but also demonstrate that the activity of Syk is greatly enhanced through dimerization and phosphorylation of the activation loop tyrosines, as occurs following BCR crosslinking by antigen.