Interaction of Phosphorylated FcϵRIγ Immunoglobulin Receptor Tyrosine Activation Motif-based Peptides with Dual and Single SH2 Domains of p72

Chen, Ting; Repetto, Barbara; Chizzonite, R; Pullar, Christine E.; Burghardt, Charles; Dharm, Elizabeth; Zhao, Zhicheng; Carroll, Robert; Nunes, Perla; Basu, Mitali; Danho, Waleed; Visnick, Melean; Kochan, Jarema; Waugh, David A.; Gilfillan, Alasdair M.

doi:10.1074/jbc.271.41.25308

Cited by 61 publications

(53 citation statements)

References 55 publications

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“…Importantly, inhibition was seen at concentrations similar to those used in the present study. The equilibrium binding affinity for association of tandem Syk SH2 domains to the phosphorylated ITAM of the Fc⑀RI receptor has been shown to be 1.4 nM, demonstrating that this is a very high affinity interaction (37). The high binding affinity supports a specific action of the Syk tandem SH2 domain protein.…”

Section: Discussionmentioning

confidence: 86%

Syk and Fyn Are Required by Mouse Megakaryocytes for the Rise in Intracellular Calcium Induced by a Collagen-related Peptide

Melford

Turner

Briddon

et al. 1997

Journal of Biological Chemistry

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Stimulation of platelets by collagen leads to activation of a tyrosine kinase cascade resulting in secretion and aggregation. We have recently shown that this pathway involves rapid tyrosine phosphorylation of an Fc receptor ␥ chain, which contains an immunoreceptor tyrosine-based activation motif (ITAM), enabling interaction with the tandem SH2 domains of the tyrosine kinase Syk. Activation of Syk lies upstream of tyrosine phosphorylation of phospholipase C␥2. In the present study we sought to test directly the role of the ITAM/Syk interaction and the role of the Src-related kinases in collagen receptor signaling using mouse megakaryocytes. We demonstrate that the calcium-mobilizing action of a collagen-related peptide (CRP) is kinase-dependent, inhibited by the microinjection of the tandem SH2 domains of Syk and abolished in Syk-deficient mice. Furthermore, the CRP response is abolished by the Src family kinase inhibitor PP1 and inhibited in Fyn-deficient mice. In contrast, the calcium response to the Gprotein-linked receptor agonist thrombin is not significantly altered under these conditions. These results provide direct evidence of the functional importance of Fyn and Syk in collagen receptor signaling and support the megakaryocyte as a model for the study of proteins involved in this pathway.

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Section: Discussionmentioning

confidence: 86%

Syk and Fyn Are Required by Mouse Megakaryocytes for the Rise in Intracellular Calcium Induced by a Collagen-related Peptide

Melford

Turner

Briddon

et al. 1997

Journal of Biological Chemistry

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“…[24,25] Grucza et al [26] determined binding at different temperatures by fluorescence titration and found a somewhat lower affinity for an ITAM peptide derived from the CD3e-chain of the T cell receptor (35 nM at 25 8C). We performed a van't Hoff analysis of the Grucza data and the outcome compares very well with our results, especially with respect to the DH 0 and DC p values (Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…Binding to the C-terminal SH2 domain is the likely initial step, as this SH2 domain has a much higher affinity for ITAMs than the N-terminal SH2. [25,40] This initial binding step is then rapidly followed by binding to the N-terminal SH2, which locks the tSH2 protein in a closed conformation, with reduced dynamics in the linker region (Scheme 2). Such a sequence of initial association followed by an intramolecular binding process in the complex has also been found to apply in other multivalent interactions.…”

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confidence: 99%

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

et al. 2005

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The Syk tandem Src homology 2 domain (Syk tSH2) constitutes a flexible protein module involved in the regulation of Syk kinase activity. The Syk tSH2 domain is assumed to function by adapting the distance between its two SH2 domains upon bivalent binding to diphosphotyrosine ligands. A thermodynamic and kinetic analysis of ligand binding was performed by using surface plasmon resonance (SPR). Furthermore, the effect of binding on the Syk tSH2 structural dynamics was probed by hydrogen/deuterium exchange and electrospray mass spectrometry (ESI‐MS). Two ligands were studied: 1, a flexible peptide derived from the tSH2 recognition ITAM sequence at the γ chain of the FcεRI‐receptor, and 2, a ligand in which the amino acids between the two SH2 binding motifs in ligand 1 have been replaced by a rigid linker of comparable length. Both ligands display comparable affinity for Syk tSH2 at 25 °C, yet a major difference in thermodynamics is observed. Upon binding of the rigid ligand, 2, the expected entropy advantage is not realized. On the contrary, 2 binds with a considerably higher entropy price of ∼9 kcal mol−1, which is attributed to a further decrease in protein flexibility upon binding to this rigid ligand. The significant reduction in deuterium incorporation in the Syk tSH2 protein upon binding of either 1 or 2, as monitored by ESI‐MS, indicates a major reduction in protein dynamics upon binding. The results are consistent with a two‐step binding model: after an initial binding step, a rapid structural change of the protein occurs, followed by a second binding step. Such a bivalent binding model allows high affinity and fast dissociation kinetics, which are very important in transient signal‐transduction processes.

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“…The SH2 domain-mediated binding of Syk and ZAP-70 to phosphorylated ITAM in subunits of immune receptors is critical for activation of these cells (3,(17)(18)(19)60). In mast cells Syk binds predominantly to the ␥-subunit of Fc⑀RI; the expression in cells of truncated Syk containing just the two SH2 domains has a dominant-negative effect that inhibits binding of native Syk and decreases receptor signaling (52).…”

Section: Discussionmentioning

confidence: 99%

Tyrosines in the Carboxyl Terminus Regulate Syk Kinase Activity and Function

Castro

Zhang

Jamur

et al. 2010

Journal of Biological Chemistry

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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...

show abstract

Interaction of Phosphorylated FcϵRIγ Immunoglobulin Receptor Tyrosine Activation Motif-based Peptides with Dual and Single SH2 Domains of p72

Cited by 61 publications

References 55 publications

Syk and Fyn Are Required by Mouse Megakaryocytes for the Rise in Intracellular Calcium Induced by a Collagen-related Peptide

Syk and Fyn Are Required by Mouse Megakaryocytes for the Rise in Intracellular Calcium Induced by a Collagen-related Peptide

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

Tyrosines in the Carboxyl Terminus Regulate Syk Kinase Activity and Function

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