Phosphorylation of Syk activation loop tyrosines is essential Syk function: An in vivo study using a specific anti-Syk activation loop phosphotyrosine antibody

Receptors and Signal Transduction Section, Oral Infection and Immunity Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 12/2000; 275(45):35442-7. DOI: 10.1074/jbc.M004549200
Source: PubMed

ABSTRACT Syk is an important protein-tyrosine kinase in immunoreceptor signaling. FcepsilonRI aggregation in mast cells induces tyrosine phosphorylation and increased enzymatic activity of Syk. The two adjacent tyrosines in the Syk activation loop are thought to be important for the propagation of FcepsilonRI signaling. To evaluate the phosphorylation of these tyrosines in vivo and further understand the relationship of Syk tyrosine phosphorylation with its function, an antibody was developed specific for phosphorylated tyrosines in the activation loop of Syk. FcepsilonRI aggregation on mast cells induced the phosphorylation of both tyrosine residues of the activation loop. The kinase activity of Syk played the major role in phosphorylating its activation loop tyrosines both in vivo and in vitro. In FcepsilonRI-stimulated mast cells, the total Syk tyrosine phosphorylation paralleled the phosphorylation of its activation loop tyrosines and downstream propagation of signals for histamine release. In contrast, the cell surface binding of anti-ganglioside monoclonal antibody AA4 induced only strong general tyrosine phosphorylation of Syk and minimal histamine release and weak phosphorylation of activation loop tyrosines. These results demonstrate that phosphorylation of the activation loop tyrosines is important for mediating receptor signaling and is a better marker of Syk function than is total Syk tyrosine phosphorylation.

    • "SYK is associated with the γ chain of the FcεRI. Binding of SYK to phosphorylated γ chain ITAM through the SYK SH2 domains induces a conformational change in the kinase, leading to its increased enzymatic activity (Zhang et al., 2000; Siraganian et al., 2010). Tec family kinases represent another class of non-receptor protein tyrosine kinases that are implicated in FcεRI-mediated activation. "
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    • "FcεRI signaling relies on Lyn-dependent phosphorylation of ITAMs on the cytoplasmic portion of the β and γ receptor subunits. The protein kinase Syk is recruited to the phosphorylated ITAMs where it becomes activated and autophosphorylated (Zhang et al. 2000; de Castro et al. 2010 "
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    • "Src and Fyn are regulated by phosphorylation on two sites: one at tyrosine 416 which is activatory, and the other at tyrosine 527 which is inhibitory (reviewed by Hunter, 1987). Similar phosphorylation sites are found in other SFK members: activation of phosphorylation are at tyrosines 394 for Lck, at tyrosines 323 and 352 for Syk, and inhibition of phosphorylation are at tyrosines 505 for Lck, and at tyrosines 525 and 526 for Syk (Chow et al., 1993; Law et al., 1996; Deckert et al., 1998; Zhang et al., 2000; Rao et al., 2001). Activating phosphorylation often results from autophosphorylation (Chiang and Sefton, 2000; reviewed by Roskoski, 2004; reviewed by Roskoski, 2005). "
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