title: Generation of a novel system for studying spleen tyrosine kinase function in macrophages and B cells.
abstract: Spleen tyrosine kinase (Syk) is a nonreceptor tyrosine kinase that is expressed primarily in hematopoietic cells. Because this protein has been implicated in processes such as Fc-mediated phagocytosis, BCR signaling, oxidative burst, degranulation, cytokine secretion, and integrin-mediated outside-in signaling, it is hypothesized that Syk may be a viable target in the treatment of a variety of autoimmune and inflammatory diseases. Because efforts to design a small-molecule therapeutic that specifically inhibits Syk have been largely unsuccessful, and genetic studies of Syk have been hampered by the fact that syk-/- mice die in utero, we have taken a chemical genetic approach to study the function of Syk. Specifically, we have created a mutant form of Syk that retains its wild-type function, but is susceptible to inhibition by enlarged derivatives of the tyrosine kinase inhibitor, PP1. We report in this study that Syk M442A S505A reconstituted wild-type function when introduced into murine syk-/- bone marrow-derived macrophages and syk-/- DT40 chicken B cells, as determined by functional and biochemical assays. Furthermore, after screening a series of PP1 derivatives, we identified one compound, namely 2,3-DMB-PP1, that specifically inhibited Syk M442A S505A, but not wild-type Syk. This system provides us with the power to characterize immune functions that are Syk specific, and furthermore, it provides us with a tool to assess how inhibition of Syk may alter an immune response and influence disease pathogenesis and/or progression.
authors.affiliation: Department of Laboratory Medicine, Howard Hughes Medical Institute, University of California, San Francisco, CA 94143, USA.
authors.forename: Allison L
authors.initials: AL
authors.lastname: Miller
authors.forename: Chao
authors.initials: C
authors.lastname: Zhang
authors.forename: Kevan M
authors.initials: KM
authors.lastname: Shokat
authors.forename: Clifford A
authors.initials: CA
authors.lastname: Lowell
mesh_terms: D016475:3T3 Cells; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001402:B-Lymphocytes / Q000201:enzymology*; D001854:Bone Marrow Cells / Q000201:enzymology / Q000276:immunology; D002460:Cell Line; D045744:Cell Line, Tumor; D002645:Chickens; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000502:physiology*; D008264:Macrophages / Q000201:enzymology* / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010587:Phagocytosis / Q000235:genetics / Q000276:immunology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000172:deficiency / Q000502:physiology*; D000072377:Syk Kinase
chemical_list: D007141:Immunoglobulin Fc Fragments; D047908:Intracellular Signaling Peptides and Proteins; D047428:Protein Kinase Inhibitors; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; C000606897:Syk protein, mouse