A Novel Mode of Gleevec Binding Is Revealed by the Structure of Spleen Tyrosine Kinase

Abstract

Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase required for signaling from immunoreceptors in various hematopoietic cells. Phosphorylation of two tyrosine residues in the activation loop of the Syk kinase catalytic domain is necessary for signaling, a phenomenon typical of tyrosine kinase family members. Syk in vitro enzyme activity, however, does not depend on phosphorylation (activation loop tyrosine 3 phenylalanine mutants retain catalytic activity). We have determined the x-ray structure of the unphosphorylated form of the kinase catalytic domain of Syk. The enzyme adopts a conformation of the activation loop typically seen only in activated, phosphorylated tyrosine kinases, explaining why Syk does not require phosphorylation for activation. We also demonstrate that Gleevec (STI-571, Imatinib) inhibits the isolated kinase domains of both unphosphorylated Syk and phosphorylated Abl with comparable potency. Gleevec binds Syk in a novel, compact cis-conformation that differs dramatically from the binding mode observed with unphosphorylated Abl, the more Gleevec-sensitive form of Abl. This finding suggests the existence of two distinct Gleevec binding modes: an extended, trans-conformation characteristic of tight binding to the inactive conformation of a protein kinase and a second compact, cis-conformation characteristic of weaker binding to the active conformation. Finally, the Syk-bound cis-conformation of Gleevec bears a striking resemblance to the rigid structure of the nonspecific, natural product kinase inhibitor staurosporine.Syk family members include two human proteins, Syk (spleen tyrosine kinase) and its closest relative Zap-70 (70-kDa chain-associated protein 1). Syk operates downstream of the B-cell receptor in B-cells, the IgE receptor Fc⑀RI in mast cells, Fc␥R in macrophages (2), and other receptors (3). Zap-70 performs a similar function in T-cell receptor signaling (4). Syk is expressed in a wide range of cell types, although its function is best understood in hematopoietic cells. Knock-out mouse studies have shown that Syk is essential for lymphocyte development (2). Syk has attracted particular interest as a therapeutic target for treatment of asthma, because Syk-deficient mast cells do not degranulate in response to Fc⑀RI aggregation (5, 6). Analyses of three-dimensional structures of the kinase domains of the insulin receptor kinase (7,8), fibroblast growth factor receptor 1 (9), and Lck (10) gave rise to a model of conformational transition upon activation in kinases. In this model, the preactivated conformation is characterized by activation loop occlusion of the ATP-and/or substrate-binding sites ("loop in"), effectively preventing substrate access. Phosphorylation stabilizes an open conformation of the activation loop ("loop out") that does not occlude the substrate-binding sites and is compatible with catalysis (11, 12). In the loop-out conformation, the activation loop also forms a platform for peptide substrate docking (12). In addition to permitting access to the su...

Inhibition of ALK enzymatic activity in T-cell lymphoma cells induces apoptosis and suppresses proliferation and STAT3 phosphorylation independently of Jak3

“…Accordingly, imatinib mesylate (STI-571, gleevec) that very effectively targets BCR/ABL kinase in vivo inhibits at similar concentration several other kinases including wildtype ABL, c-kit, PDGFRb, PDGFRa 27 and, as shown recently, Syk. 31 Even the new generation BCR/ABL inhibitor that seems to bind the activated form of the kinase with the much higher affinity, and is aimed at circumventing resistance to imatinib, displays crossreactivity with at least one other tyrosine kinase, src. 32 The major controversy in the field seems to be the question if NPM/ALK activates STAT3 and other signaling proteins directly or via activation of other kinases, in particular, Jak3.…”

Inhibition of constitutive and BCR-induced Syk activation downregulates Mcl-1 and induces apoptosis in chronic lymphocytic leukemia B cells

“…Phosphorylation at Y352 is considered sufficient to increase Syk activity enough to phosphorylate certain downstream substrates and trans-autophosphorylate tyrosines 525 and 526 in the activation loop. [19][20][21][22][23] The function of the tyrosines in the activation loop has still not been completely resolved, but most studies indicate that their phosphorylation is required to fully convert or stabilize Syk in an active conformation. 22,23 Once activated, Syk propagates the BCR signal by associating with adaptor proteins and phosphorylating important signaling intermediates, including B-cell linker protein (BLNK), PI3K (phosphatidylinositol 3-kinase) and PLCg2 (phospholipase Cg2).…”

Protein Flexibility and Ligand Rigidity: A Thermodynamic and Kinetic Study of ITAM‐Based Ligand Binding to Syk Tandem SH2

“…[5] The b-and g-subunits of FceRI each contain a so-called immunoreceptor tyrosine-based activation motif (ITAM). The consensus sequence for ITAMs is YXX(I/L)-(X) [6][7][8] -YXX(I/L), in which X can be any amino acid. ITAM sequences are bivalent docking…”

“…[7] However, in contrast to many other kinases, this phosphorylation of the activation loop is not necessary for Syk activation. [8] Furthermore, a unique insert in the linker between tSH2 and the kinase domain is necessary for the functioning of Syk in immunoreceptor signaling, and this insert can regulate the ability of Syk to bind ITAMs. [9] An allosteric change induced in tSH2 upon binding of an ITAM sequence may be involved in the regulation of Syk activity.…”