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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 FcepsilonRI 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 FcepsilonRI-induced degranulation, nuclear factor for T cell activation and NFkappaB 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.

SUBMITTER: de Castro RO

PROVIDER: S-EPMC2924109 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Tyrosines in the carboxyl terminus regulate Syk kinase activity and function.

de Castro Rodrigo O RO Zhang Juan J Jamur Maria C MC Oliver Constance C Siraganian Reuben P RP

The Journal of biological chemistry 20100616 34

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 FcepsilonRI 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 thes ...[more]

PMID: 20554527

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Project description:Background ? Carboxyl terminus 1 (?CT1) is a 25-amino acid therapeutic peptide incorporating the zonula occludens-1 (ZO-1)-binding domain of connexin 43 (Cx43) that is currently in phase 3 clinical testing on chronic wounds. In mice, we reported that ?CT1 reduced arrhythmias after cardiac injury, accompanied by increases in protein kinase C? phosphorylation of Cx43 at serine 368. Herein, we characterize detailed molecular mode of action of ?CT1 in mitigating cardiac ischemia-reperfusion injury. Methods and Results To study ?CT1-mediated increases in phosphorylation of Cx43 at serine 368, we undertook mass spectrometry of protein kinase C? phosphorylation assay reactants. This indicated potential interaction between negatively charged residues in the ?CT1 Asp-Asp-Leu-Glu-Iso sequence and lysines (Lys345, Lys346) in an ?-helical sequence (helix 2) within the Cx43-CT. In silico modeling provided further support for this interaction, indicating that ?CT1 may interact with both Cx43 and ZO-1. Using surface plasmon resonance, thermal shift, and phosphorylation assays, we characterized a series of ?CT1 variants, identifying peptides that interacted with either ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 or Cx43-CT, but with limited or no ability to bind both molecules. Only peptides competent to interact with Cx43-CT, but not ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 alone, prompted increased pS368 phosphorylation. Moreover, in an ex vivo mouse model of ischemia-reperfusion injury, preischemic infusion only with those peptides competent to bind Cx43 preserved ventricular function after ischemia-reperfusion. Interestingly, a short 9-amino acid variant of ?CT1 (?CT11) demonstrated potent cardioprotective effects when infused either before or after ischemic injury. Conclusions Interaction of ?CT1 with the Cx43, but not ZO-1, is correlated with cardioprotection. Pharmacophores targeting Cx43-CT could provide a translational approach to preserving heart function after ischemic injury.

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Tyrosines in the carboxyl terminus regulate Syk kinase activity and function.

Publications

Tyrosines in the carboxyl terminus regulate Syk kinase activity and function.

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