A chemical genetic approach to the study of Syk and B cell antigen receptor signaling
Syk is an essential protein tyrosine kinase in the BCR signaling pathway. To study the role of Syk in the kinetics of BCR-signaling, we adopted the strategy of engineering an analog-sensitive form of Syk that could be inhibited by an orthogonal inhibitor. We identified Met 442 as a gatekeeper residue in the ATP-binding site of Syk. However, the substitution of M442 to glycine or alanine abrogated Syk’s activity. We identified possible residues for the generation of second-site suppressor mutations to rescue the activity of Syk(M442A) from a structural comparison of Syk and Src. Two of the residues that normally interact with M442 in Syk were replaced by the corresponding residues that interact with T338 in Src. These additional two mutations restored the ability of Syk(M442A) to signal in Syk-deficient cells. The signaling induced by the analog sensitive Syk (Syk-AQL) was specifically inhibited by the treatment with PP1 derivatives. The treatment of cells expressing Syk-AQL with the inhibitor following BCR stimulation dramatically inhibited total tyrosine phosphorylation and the activation of Erk. A brief activation of Syk following BCR stimulation was sufficient to activate NF-κB, but NFAT activation required the sustained activation of Syk. Syk’s activity was required for the retention of receptor complexes on the cell surface, suggesting the importance of Syk in sustained BCR signaling. SH2 domains have important roles in mediating protein-protein interactions in signaling pathways. A phosphopeptidomimetic compound targeting the Lck SH2 domain, RH6-22, that was synthesized as a cell-permeable prodrug that becomes quickly processed within cells into its active form was characterized. RH6-22 inhibited the growth of leukemia cells without inducing any pronounced cell cycle arrest at a specific phase, but delayed cell cycle progression as a whole. Some cell lines treated with RH6-22 exhibited polyploidy and multiple nuclei. RH6-22 also affected the microtubule network. Proteins binding the activated form of RH6-22 were adsorbed to beads conjugated with the compound and investigated via mass spectrometry. Binding proteins included proteins preventing endoreduplication or regulating mitosis. RH6-22 might inhibit the growth of leukemia cells by binding to one or multiple proteins including those important in DNA replication and mitosis. ^
Robert L. Geahlen, Purdue University.
Biology, Molecular|Chemistry, Biochemistry
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