Tyrosines in the Carboxyl Terminus Regulate Syk Kinase Activity and Function

Abstract

The Syk tyrosine kinase family plays an essential role in immunoreceptor tyrosine-based activation motif (ITAM) signaling. The binding of Syk to tyrosine-phosphorylated ITAM subunits of immunoreceptors, such as Fc⑀RI on mast cells, results in a conformational change, with an increase of enzymatic activity of Syk. This conformational change exposes the COOH-terminal tail of Syk, which has three conserved Tyr residues (Tyr-623, Tyr-624, and Tyr-625 of rat Syk). To understand the role of these residues in signaling, wild-type and mutant Syk with these three Tyr mutated to Phe was expressed in Syk-deficient mast cells. There was decreased Fc⑀RI-induced degranulation, nuclear factor for T cell activation and NFB activation with the mutated Syk together with reduced phosphorylation of MAP kinases p38 and p42/44 ERK. In non-stimulated cells, the mutated Syk was more tyrosine phosphorylated predominantly as a result of autophosphorylation. In vitro, there was reduced binding of mutated Syk to phosphorylated ITAM due to this increased phosphorylation. This mutated Syk from non-stimulated cells had significantly reduced kinase activity toward an exogenous substrate, whereas its autophosphorylation capacity was not affected. However, the kinase activity and the autophosphorylation capacity of this mutated Syk were dramatically decreased when the protein was dephosphorylated before the in vitro kinase reaction. Furthermore, mutation of these tyrosines in the COOH-terminal region of Syk transforms it to an enzyme, similar to its homolog ZAP-70, which depends on other tyrosine kinases for optimal activation. In testing Syk mutated singly at each one of the tyrosines, Tyr-624 but especially Tyr-625 had the major role in these reactions. Therefore, these results indicate that these tyrosines in the tail region play a critical role in regulating the kinase activity and function of Syk.Syk and ZAP-70 are members of a tyrosine kinase family one or both of which are expressed in most hematopoietic cells (1-5). Structurally Syk/ZAP-70 consists of two Src-homology 2 domains (SH2) 3 and a kinase domain followed by a short COOH-terminal extension (tail). The inter-SH2 domain is located between the NH 2 -terminal SH2 (SH2-N) and COOHterminal SH2 (SH2-C), whereas the linker region is between the SH2-C and the kinase domains (6, 7). An alternatively spliced form of Syk, termed SykB, lacks a 23-amino acid sequence in the linker domain (8). Although both isoforms are expressed in immune cells such as the RBL-2H3 rat mast cell line, Syk is more efficient than SykB in immunoreceptor signaling (8,9). Syk is expressed in B cells, mast cells, immature T cells, and platelets, whereas ZAP-70 is in T cells and natural killer cells (3, 10). Syk and ZAP-70 are essential for signal transduction from cell surface immunoreceptors (10 -12). These receptors on many hematopoietic cells such as T, B, and mast cells have subunits, which contain a sequence named the immunoreceptor tyrosine-based activation motif (ITAM) which contains two tyrosine residues (13...

Angiopoietin‐like protein 2 renders colorectal cancer cells resistant to chemotherapy by activating spleen tyrosine kinase–phosphoinositide 3‐kinase‐dependent anti‐apoptotic signaling

“…b). Tyrosine phosphorylation is required to activate Syk kinase activity and function . Thus, we examined Syk activity in SW480/ANGPTL2‐1 and ‐2 versus control cells by Western blotting with a phospho‐Syk antibody.…”

Signaling of the ITK (Interleukin 2-inducible T Cell Kinase)-SYK (Spleen Tyrosine Kinase) Fusion Kinase Is Dependent on Adapter SLP-76 and on the Adapter Function of the Kinases SYK and ZAP70

“…Further studies are warranted to better understand the differential regulation of activation loop tyrosines in ITK-SYK. According to a recent report, loss of the C-terminal tyrosines in SYK led to enhanced phosphorylation in the activation loop tyrosines (30). In contrast, ITK-SYK was equally phosphorylated at the corresponding activation loop tyrosines 385/386 in the absence or presence of functional C-terminal tyrosines (Fig.…”

“…Phenylalanine replacement of tail tyrosines has been reported to result in gain-of-function in SYK family kinases (19). Another recent study showed that loss of C-terminal/tail tyrosines results in enhanced phosphorylation of SYK activation loop tyrosines 525/526, whereas downstream signaling events, such as nuclear factor of activated T cells and NFB activation, were severely hampered (30).…”

Phosphorylated immunoreceptor tyrosine-based activation motifs and integrin cytoplasmic domains activate spleen tyrosine kinase via distinct mechanisms

“…This shows that in addition to the main regulatory Tyrosines Y348 and Y352, there are other residues that can be transphosphorylated by the Syk itself and that activate the kinase. While it has been shown that activation loop tyrosines Y525 and Y526 have little effect on Syk activity, it is well established that the C-terminal tyrosines Y630 and Y631 can stabilize the closed, inactive conformation of the enzyme (12,35), and thus phosphorylation of these could induce the activity of even the Y438F/Y352F mutant. In mouse, Syk residues corresponding to Y630 and Y631 have been found to be autophosphorylated or transphosphorylated (36).…”